Transcription of a 5' extended mRNA isoform directs dynamic chromatin changes and interference of a downstream promoter.

Cell differentiation programs require dynamic regulation of gene expression. During meiotic prophase in Saccharomyces cerevisiae, expression of the kinetochore complex subunit Ndc80 is downregulated by a 5' extended long undecoded NDC80 transcript isoform. Here we demonstrate a transcriptional interference mechanism that is responsible for inhibiting expression of the coding NDC80 mRNA isoform. Transcription from a distal NDC80 promoter directs Set1-dependent histone H3K4 dimethylation and Set2-dependent H3K36 trimethylation to establish a repressive chromatin state in the downstream canonical NDC80 promoter. As a consequence, NDC80 expression is repressed during meiotic prophase. The transcriptional mechanism described here is rapidly reversible, adaptable to fine-tune gene expression, and relies on Set2 and the Set3 histone deacetylase complex. Thus, expression of a 5' extended mRNA isoform causes transcriptional interference at the downstream promoter. We demonstrate that this is an effective mechanism to promote dynamic changes in gene expression during cell differentiation.

Introduction

Cell fate programs are driven by underlying gene regulatory networks. The budding yeast gametogenesis program, also known as sporulation, provides an ideal model for understanding principles of cell fate progression. In sporulation a diploid cell exits the mitotic program and produces four haploid gametes packaged as spores. Gametogenesis is driven by a specialized cell division called meiosis (Marston and Amon, 2004). After entering meiosis, diploid cells undergo a single round of DNA replication, and then recombination of homologous chromosomes in a long-lasting prophase. Completion of meiotic prophase is followed by two rounds of chromosome segregation as well as development and packaging of meiotic gametes into spores. In the budding yeast S. cerevisiae, meiosis can be induced synchronously, allowing the study of stage-specific regulation of gene expression.

The yeast meiotic program is dynamically regulated by sequential waves of gene expression (Chu et al., 1998; Primig et al., 2000). Two master transcription factors, Ime1 and Ndt80, control gene expression during meiosis (Kassir et al., 1988; Xu et al., 1995). Ime1 regulates the early genes controlling S phase and prophase, whereas Ndt80 induces sets of genes controlling meiotic chromosome segregation and spore formation (Mitchell et al., 1990; Chu and Herskowitz, 1998). These two transcription factors are critical for stage specific gene expression during meiosis.

Noncoding RNAs (ncRNAs) and alternate mRNA isoforms are expressed throughout yeast meiosis (Brar et al., 2012; Kim Guisbert et al., 2012; Lardenois et al., 2015). In addition, ncRNAs are transcribed from the 3’ end of genes important for meiosis and sporulation (Zhang et al., 2011). However, only a limited number of these ncRNAs have been assigned a biological function. For example, in cells with a single mating type locus (MATa or MATα) transcription of ncRNAs represses IME1 and IME4, two regulators of entry in meiosis (Hongay et al., 2006; van Werven et al., 2012). Throughout meiosis a subset of genes show stage specific expression of mRNA isoforms with often reduced translational capabilities (Brar et al., 2012). However, it is not well understood how ncRNAs and mRNA isoforms contribute to dynamic control of gene expression during yeast meiosis.

One gene with a dynamic expression pattern during yeast meiosis encodes for the kinetochore subunit Ndc80. Ndc80 is downregulated during meiotic prophase and is rapidly induced during meiotic chromosome segregation (Miller et al., 2012; Meyer et al., 2015). In the presence of a spindle, mis-expression of Ndc80 during meiotic prophase causes aberrant meiotic chromosome segregation (Miller et al., 2012). Thus, the dynamic control of Ndc80 expression is critical for meiotic divisions. How Ndc80 is repressed during the early stages of meiosis remained elusive. In the accompanying paper, we show that the transcription of a 5’ extended NDC80 long undecoded transcript isoform (NDC80) is responsible for repressing the coding-competent NDC80 isoform (NDC80) during meiotic S-phase and prophase (Chen et al., 2017). Furthermore, NDC80 cannot be translated into Ndc80 protein due to translation of the upstream open reading frames in this mRNA isoform. Altogether, these results demonstrate that NDC80 functions in a regulatory manner, in which its transcription is both necessary and sufficient to downregulate NDC80 levels during meiotic prophase.

In this work we describe the mechanism by which the 5’ extended NDC80 mRNA isoform represses NDC80 transcription interferes with the downstream NDC80 promoter by establishing a repressive chromatin state. This repression requires both Set3 and Set2. NDC80-mediated repression can be rapidly reversed to suit the physiological needs of the cell. Furthermore, the repression mechanism described here can be adapted to fine-tune gene expression. Thus, transcription of a 5’ extended mRNA isoform mediates transcriptional interference of the downstream promoter, allowing dynamic control of gene expression.

Results

Transcription of a NDC80 mRNA isoform with an extended 5’ leader represses the canonical NDC80 mRNA

Genome wide transcriptome data indicated that there are at least two different mRNA isoforms expressed from the NDC80 locus (Brar et al., 2012; Kim Guisbert et al., 2012; Lardenois et al., 2015). During mitotic growth, an NDC80 mRNA is produced from its core promoter. However, in early meiosis a longer NDC80 mRNA is transcribed from approximately 500 base pairs (bp) upstream of the NDC80 start codon. To investigate the expression pattern of the NDC80 mRNA isoforms more closely in cells undergoing meiosis, we performed northern blot analyses using a probe that recognizes both the ORF and upstream region of NDC80. Cells were first grown in rich medium overnight and then shifted to pre-sporulation medium. After a second overnight growth, cells were transferred to sporulation medium (SPO) and samples were taken at specific intervals throughout meiosis. In wild-type cells entering meiosis, both NDC80 mRNA isoforms were co-expressed (Figure 1A, left panel [control]). After 3 hr in SPO, the expression of the short mitotic isoform decreased, whereas the levels of the longer NDC80 mRNA isoform increased. At 5 hr the short NDC80 form was induced (Figure 1A, left panel [control]), which corresponds to cells entering meiotic divisions (Chen et al., 2017). To control for changes in RNA levels, the expression of unrelated RNA polymerase II and III genes, CIT1 and SCR1 respectively, was measured (Figure 1—figure supplement 1A and B). We observed that CIT1 levels fluctuated throughout the meiotic time course, while SCR1 expression was more constant (Figure 1A and Figure 1—figure supplement 1A and B). Hence, the expression levels of NDC80 isoforms were normalized to SCR1 (Figure 1B and Figure 1—figure supplement 1C). We also measured NDC80 expression in a strain in which the IME1 and IME4 genes were fused to a copper inducible promoter (pCUP-IME1 and pCUP-IME4). The induction of pCUP-IME1 and pCUP-IME4 ensures that cells can enter meiosis synchronously (Berchowitz et al., 2013; Chia and van Werven, 2016). Only the short mitotic isoform was clearly detected in cells arrested before entry into meiosis (Figure 1A, right panel (induced), and Figure 1B). Strikingly, soon after IME1 and IME4 were induced at two hours in SPO, expression of the long NDC80 isoform increased and levels of the mitotic NDC80 isoform decreased (Figure 1A, right panel (induced), and Figure 1B and Figure 1—figure supplement 1C). The mitotic NDC80 isoform was repressed throughout meiotic S-and prophase. Mirroring changes in the levels of the mitotic NDC80 transcript, Ndc80 protein levels also decreased during entry into meiosis, meiotic S-phase and prophase (Figure 1A and Figure 1—figure supplement 1D). As expected, expression of the mitotic NDC80 isoform and Ndc80 protein remained relatively constant when meiosis was not induced (Figure 1A, middle panel (uninduced), Figure 1B and Figure 1—figure supplement 1D). We conclude that during meiotic entry, meiotic S phase and prophase, the expression of a longer NDC80 form is induced and the mitotic form of NDC80 is repressed.

Figure 1.

NDC80 transcription represses the NDC80 promoter.

(A) Expression pattern of two NDC80 mRNA isoforms during starvation and early meiosis. Diploid control cells (FW4644) or cells harboring the CUP1 promoter fused with IME1 and IME4 (pCUP-IME1/pCUP-IME4) (FW1902) were grown overnight in rich medium, shifted to pre-sporulation medium, and subsequently transferred to sporulation medium (SPO). These cells also harbored NDC80 tagged at the carboxy-terminus with three copies of the V5 epitope. After two hours in SPO, IME1 and IME4 were induced with CuSO4 (50 μM) to set in motion synchronous meiosis in pCUP-IME1/pCUP-IME4 cells. Samples were taken at the indicated time points for northern and western blot analyses. To detect the two different NDC80 mRNA isoforms, RNA was extracted, separated by gel electrophoresis, blotted, and hybridized with a probe that spans the NDC80 promoter and the 5’ end of the coding region. As a loading control, we also hybridized the blot with a probe specific for SCR1. Ndc80 protein levels were determined by western blot using anti V5 antibodies. As a loading control, we also detected Hxk1 levels with anti-Hxk1 antibodies. A schematic of the NDC80 locus is shown over the northern blot. The single asterisk denotes the transcription start site of the short mitotic NDC80 mRNA isoform. The double asterisk denotes the transcription start site of the distal 5’ extended long NDC80 mRNA isoform. The distance in base pairs from the NDC80 start codon is also displayed. (B) Quantification of expression of the short mitotic NDC80 mRNA isoform from the proximal transcription start-site (labelled with one asterisk) up till the 4.5 hr time point. The mean of two independent repeats plus the standard error of the mean are displayed. The signal was normalized over SCR1. To control for technical variation between experiments and blots, the 0 hr time point was set to one. (C) NDC80 transcription correlates with reduced TFIIB (Sua7) binding at the NDC80 promoter. The two NDC80 isoforms described in A were defined as long undecoded transcript isoform (NDC80) and Ndc80 coding mRNA isoform (NDC80), respectively. Cells harboring Sua7 tagged with three copies of V5 (Sua7-V5) and the pCUP-IME1/pCUP-IME4 alleles (FW2957) were induced to undergo meiosis synchronously as described in A. Samples for chromatin immunoprecipitation were taken at two hours (2 hr (premeiotic), no NDC80 transcription) and four hours after transfer to sporulation medium (SPO) (4 hr (S + prophase), NDC80 transcription). Cells were fixed with formaldehyde, chromatin extracts were prepared and Sua7-V5 bound DNA fragments were immunoprecipitated using agarose beads coupled with anti-V5 antibodies. The recovered DNA fragments were quantified by qPCR using ten different primer pairs scanning the NDC80 locus, and were normalized over a primer pair directed against the HMR locus. The midpoint position of each primer pair is indicated in the x-axis. The mean normalized signal from three independent experiments plus the standard error of the mean for each primer pair is displayed. ‘rel.’ means relative; ‘occup.”, occupancy. (D) NDC80 transcription is required for inhibiting Sua7 binding in the NDC80 promoter during meiotic prophase. Similar analysis as C except that a mutant strain harboring a deletion upstream in the NDC80 promoter region (ndc80, FW5530) and untagged strains (FW1902 and FW1868) were included in the analysis. A primer pair directed against a NDC80 core promoter was used for the quantification of Sua7 binding at the NDC80 promoter. (E) NDC80 transcription correlates with the establishment of repressive chromatin in the promoter of NDC80 Chromatin structure at the NDC80 locus was determined by ChIP of histone H3 on micrococcal nuclease (MNase) treated extracts in cells that also harbored pCUP-IME1/pCUP-IME4 (FW1902). Samples were taken at two hours (2 hr (premeiotic), no NDC80 transcription) and four hours after transfer to SPO (4 hr (S + prophase), NDC80 transcription), fixed with formaldehyde, and chromatin extracts were treated with micrococcal nuclease. Extracts that predominantly contained mononucleosomes were used for ChIP assays with histone H3 antibodies (see Materials and methods for details). The recovered DNA fragments were quantified by qPCR using ten different primer pairs directed against the NDC80 locus relative to a no MNase input. The signals from each primer pair were then normalized over a primer pair directed against the PHO5 core promoter. The midpoint position of each primer pair is indicated in the x-axis. The mean signal from three independent experiments plus the standard error of the mean for each primer pair is displayed.

(A) The same northern blot and western blot of NDC80 expression during a meiotic time course as described in Figure 1A, except that CIT1 and ribosomal RNA expression levels are also displayed. (B) Quantification of CIT1 and SCR1 northern blot signals from A up till the 4.5 hr time point. The mean of two independent repeats plus the standard error of the mean are displayed. (C) Quantification of expression of the long mRNA NDC80 isoform described in Figure 1A (labelled with two asterisks). The mean of two independent repeats plus the standard error of the mean are displayed. The signal was normalized over SCR1. Since the long isoform is not expressed in premeiotic conditions, the 3 hr time point (as opposed to 0 hr) was set to one to control for technical variation between experiments and blots. (D) Quantification of Ndc80 protein levels as described in Figure 1A. The mean of two independent repeats plus the standard error of the mean are displayed. The signal was normalized over Hxk1. To control for technical variation between experiments and blots, the 0 hr time point was set to one. (E) Example of extract with mononucleosomes prepared from cells (FW1902, 2 hr [premeiotic]). In short, cells were fixed with formaldehyde, treated with zymolase, and subsequently treated with different concentrations of micrococcal nuclease (MNase). To check for the extent of MNase digestion, part of the sample from each extract was reverse crosslinked, purified, and separated by gel electrophoresis. The arrow indicates the extract that was used for subsequent ChIP analysis.

Figure 1—figure supplement 1.

NDC80 transcription represses the NDC80 promoter.

(A) The same northern blot and western blot of NDC80 expression during a meiotic time course as described in Figure 1A, except that CIT1 and ribosomal RNA expression levels are also displayed. (B) Quantification of CIT1 and SCR1 northern blot signals from A up till the 4.5 hr time point. The mean of two independent repeats plus the standard error of the mean are displayed. (C) Quantification of expression of the long mRNA NDC80 isoform described in Figure 1A (labelled with two asterisks). The mean of two independent repeats plus the standard error of the mean are displayed. The signal was normalized over SCR1. Since the long isoform is not expressed in premeiotic conditions, the 3 hr time point (as opposed to 0 hr) was set to one to control for technical variation between experiments and blots. (D) Quantification of Ndc80 protein levels as described in Figure 1A. The mean of two independent repeats plus the standard error of the mean are displayed. The signal was normalized over Hxk1. To control for technical variation between experiments and blots, the 0 hr time point was set to one. (E) Example of extract with mononucleosomes prepared from cells (FW1902, 2 hr [premeiotic]). In short, cells were fixed with formaldehyde, treated with zymolase, and subsequently treated with different concentrations of micrococcal nuclease (MNase). To check for the extent of MNase digestion, part of the sample from each extract was reverse crosslinked, purified, and separated by gel electrophoresis. The arrow indicates the extract that was used for subsequent ChIP analysis.

Our observation that the expression of the mitotic and longer NDC80 mRNA isoforms are inversely correlated during early meiosis, suggests that there may be a direct effect of the longer NDC80 isoform on mitotic NDC80 repression. The accompanying paper by Chen et al. showed by a series of experiments that expression of the longer NDC80 mRNA isoform is responsible for the decline in mitotic NDC80 levels during early meiosis (Chen et al., 2017). Furthermore, Chen et al. showed that nine short upstream open reading frames in the extended 5’ region of the long isoform inhibit translation of Ndc80 protein from this mRNA isoform (Chen et al., 2017). Thus, the long NDC80 mRNA isoform is translationally inert. Hence, this transcript has been defined as the NDC80 long undecoded transcript isoform (NDC80). The short NDC80 protein coding mRNA isoform is called NDC80. This nomenclature is used thereafter.

Transcription of NDC80 correlates with reduced binding of TFIIB and repressive chromatin in the NDC80 promoter

The mechanism by which NDC80 represses the downstream NDC80 promoter might be related to a transcriptional interference mechanism during which intergenic transcription or transcription over promoter regions establishes a repressive chromatin state and prevents transcription factors from binding (Martens et al., 2004; Hainer et al., 2011; van Werven et al., 2012). Similar to transcriptional interference, NDC80-mediated repression of NDC80 is exclusively cis-dominant (Martens et al., 2004; van Werven et al., 2012; Chen et al., 2017). To further investigate whether the mechanism of NDC80-mediated gene repression also shares other features of transcriptional interference, we tested whether NDC80 transcription alters the association of transcription factors with the NDC80 promoter. The binding of the basal transcription factor Sua7 (TFIIB), which is homologous to human TFIIB, changed during meiosis across the NDC80 locus (Figure 1C). Before entry into meiosis, Sua7 was bound to the core promoter of NDC80. However, after IME1 and IME4 induction (four hours in SPO) when NDC80 transcription occurred and cells underwent meiotic S phase, Sua7 binding to the NDC80 core promoter (around −100 bp from AUG) was reduced while binding to the NDC80 promoter (around −600 bp from AUG) increased (Figure 1C). It is worth noting, that the signal for Sua7 binding also showed a peak at −800 bp, which may be due to fluctuation in expression of the adjacent PAN6 gene in the divergent direction. Next, we examined Sua7 binding at the NDC80 promoter in a mutant that does not transcribe NDC80 (ndc80) (Chen et al., 2017). In the ndc80 mutant, no change in Sua7 binding around the NDC80 promoter was observed after induction of IME1 and IME4 (Figure 1D). This result shows that NDC80 prevents TFIIB recruitment at the NDC80 promoter during early meiosis.

The reduction in TFIIB recruitment to the NDC80 promoter could be due to the establishment of a repressive chromatin state. For example, transcription of an intergenic ncRNA across the SER3 promoter directs nucleosome assembly in the promoter, which is essential for SER3 repression in budding yeast (Hainer et al., 2011). Therefore, we examined how the chromatin structure in the NDC80 promoter is modified by NDC80 transcription. To identify where the nucleosomes stably associate with the NDC80 locus, we performed chromatin immunoprecipitation (ChIP) of histone H3 on micrococcal nuclease (MNase) treated chromatin extracts (see material and methods for details) (Figure 1E and Figure 1—figure supplement 1E). In premeiotic cells (labeled ‘2 hr’ in Figure 1E) we detected a relatively low signal around the core promoter of NDC80, which is indicative of a nucleosome free region (NFR) and consistent with active NDC80 transcription. During meiotic prophase (labeled ‘4 hr’ in Figure 1E), when NDC80 was transcribed, the signal around the core promoter increased, indicating that nucleosome occupancy was increased. These findings are consistent with the notion that transcription of NDC80 inhibits TFIIB recruitment and establishes a repressive chromatin state at the NDC80 promoter.

NDC80 transcription promotes Set1-dependent histone H3 lysine 4 dimethylation and Set2-dependent lysine 36 trimethylation in the NDC80 promoter

Co-transcriptional recruitment of chromatin modifying enzymes regulates the chromatin state of genes in the wake of elongating RNA polymerase II. For example, repressive chromatin marks, such as histone three lysine 4 dimethylation (H3K4me2) and lysine 36 trimethylation (H3K36me3) are deposited co-transcriptionally within gene bodies by the Set1 and Set2 methyltransferases (Hampsey and Reinberg, 2003; Kim and Buratowski, 2009). The histone deacetylase complexes Set3C and Rpd3S recognize H3K4me2 and H3K36me3, respectively, and repress cryptic transcription from chromatin carrying these modifications (Carrozza et al., 2005; Keogh et al., 2005; Kim and Buratowski, 2009; Govind et al., 2010). Set1/Set3C and Set2/Rpd3S have also been implicated in transcription-coupled repression of gene promoters (Houseley et al., 2008; Kim et al., 2012; van Werven et al., 2012; Ard and Allshire, 2016a). To investigate whether NDC80 mediated repression of NDC80 also requires Set1/Set3C and Set2/Rpd3S, we measured the distribution of H3K4me2 and H3K36me3 marks at the NDC80 locus (Figure 2A and B). We observed almost no enrichment in the NDC80 promoter of either marks in premeiotic cells (labelled ‘2 hr’ in Figure 2A and B), but H3K36me3 and to a lesser extent, H3K4me2 increased at the NDC80 promoter in meiotic prophase cells (labeled ‘4 hr’ in Figure 2A and B). As expected, the enrichment of H3K4me2 and H3K36me3 depended on Set1 and Set2, respectively (Figure 2A and B, set1∆ and set2∆). In ndc80 cells that do not express NDC80, the deposition of the H3K36me3 and H3K4me2 marks in the NDC80 promoter was reduced (Figure 2C and D). Thus NDC80 transcription promotes the deposition of repressive H3K4me2 and H3K36me3 marks within the NDC80 promoter.

Figure 2.

Transcription of NDC80 promotes H3K4me2 and H3K36me3 in the promoter and 5’ region of NDC80.

(A) NDC80 transcription promotes histone H3 lysine 4 dimethylation (H3K4me2) in the NDC80 promoter. Wild-type (FW1902) and set1Δ (FW3033) cells harboring the pCUP-IME1/pCUP-IME4 were induced to undergo meiosis synchronously (see Materials and methods for details). Samples for chromatin immunoprecipitation were taken at two hours (2 hr (premeiotic), no NDC80 transcription) and four hours in sporulation medium (SPO) (4 hr (S + prophase), NDC80 transcription). Cells were fixed with formaldehyde, chromatin extracts were prepared and H3K4me2 or H3 enriched fragments were immunoprecipitated using magnetic Prot A beads coupled with anti-H3K4me2 or anti-H3 antibodies, respectively. The recovered DNA fragments were quantified by qPCR using ten different primer pairs scanning the NDC80 locus. The midpoint position of each primer pair is indicated in the x-axis. The mean enrichment from three independent experiments plus the standard error of the mean for each primer pair is displayed. The H3K4me2 signal was normalized over histone H3. (B) Similar to A, except that histone H3 lysine 36 trimethylation (H3K36me3) abundance was determined by ChIP. Wild-type (FW1902) and set2Δ (FW1472) cells harboring the pCUP-IME1/pCUP-IME4 alleles were used for the analysis. (C) Similar to A except that the ChIP for H3K4me2 was performed in control cells (FW1902) and cells harboring a deletion upstream in the NDC80 promoter region (ndc80, FW1868). For the analyses we used primer pairs directed against the NDC80 promoter (A), and the 5’ region of the NDC80 gene (B). The mean fold enrichment from three independent experiments plus the standard error of the mean for each primer pair are displayed. The signals were normalized to the levels of H3. (D) Similar to C except that H3K36me3 levels were determined by ChIP. (E) Transcription of NDC80 during mitotic growth leads to increased H3K4me2 levels in the NDC80 promoter. Control cells (UB91) and cells harboring NDC80 driven by the GAL1-10 promoter (pGAL-NDC80) (UB3338) were grown to exponential phase in rich medium with raffinose and galactose (YP-RG). Subsequently, cells were treated with β-estradiol, which induces translocation of the Gal4-ER chimeric transcription factor to the nucleus to activate the GAL1-10 promoter. Samples were taken at 0 and 3 hr after induction of NDC80 for ChIP. H3K4me2 and histone H3 levels were determined as described in C, except using a primer pair directed against the NDC80 core promoter. The mean enrichment from three independent experiments plus the standard error of the mean are displayed. (F) Similar to E except that H3K36me3 levels were determined by ChIP.

Next, we examined whether deposition of the H3K36me3 and H3K4me2 marks was dependent on the identity of the promoter that makes NDC80 mRNA. We replaced the NDC80 promoter with an inducible GAL1-10 promoter, and expressed NDC80 in rich nutrient conditions in cells harboring the Gal4-ER chimeric transcription factor, which responds to β-estradiol. We observed a moderate increase of H3K4me2 in cells that harbored NDC80 compared to control cells, which was independent of NDC80 transcription (Figure 2E). One explanation is that the GAL1-10 promoter is leaky and can increase H3K4me2 levels without induction with β-estradiol. In contrast to H3K4me2, H3K36me3 levels were strongly enriched in the NDC80 promoter when NDC80 was induced (Figure 2F). Control cells harboring the wild-type NDC80 promoter did not show increased H3K36me3 levels. We conclude that the deposition of H3K36me3, but not H3K4me2, is independent of the identity of the promoter that directs NDC80 transcription. Taken together, deposition of repressive chromatin marks in the NDC80 promoter requires NDC80 transcription.

Set2 and Set3 contribute to NDC80 mediated repression of NDC80

Because H3K36me3 and H3K4me2 marks localize to the NDC80 promoter when NDC80 is transcribed, we examined whether Set1/Set3C and Set2/Rpd3S contribute to NDC80 repression. Since Set1 also plays an important role in meiotic recombination, we deleted SET3 to test how the Set1/Set3C pathway regulates the NDC80 locus (Borde et al., 2009; Acquaviva et al., 2013; Sommermeyer et al., 2013). In the set2∆ set3∆ double mutant, but not the single mutants, both NDC80 and NDC80 transcripts were detected throughout multiple time points in early meiosis, and the steady-state level of Ndc80 protein remained high (Figure 3A and Figure 3—figure supplement 1A, compare the time points from two to five hours between control and mutant cells). The set2∆ set3∆ double mutant cells entered and underwent meiosis with delayed kinetics (Figure 3—figure supplement 1B). Thus, it is possible that a population of cells never entered meiosis and continued to express the mitotic NDC80 mRNA isoform. We improved the kinetics of meiosis by adopting a different meiotic synchronization protocol (Figure 3B). Instead of growing cells in pre-sporulation medium, we shifted them directly to sporulation medium after they reached saturation in nutrient rich conditions. We then induced IME1 and IME4. This synchronization procedure reduced the delay in meiotic divisions (compare Figure 3—figure supplement 1B and C). In addition, meiotic S phase was completed in more than 75 percent of cells after 6 hr, indicating that the majority of cells had entered meiosis (Figure 3C). Importantly, NDC80 mediated repression was still compromised in set2∆ set3∆ double mutant cells despite improved synchrony of meiosis (Figure 3D and Figure 3—figure supplement 1D, compare the time points from three-to five hours for the control with three-to six hours for the mutant cells). Further analyses of selective time-points (3.5 and 4.5 hr) confirmed that there were significant differences in NDC80 levels between the control and the set2∆ set3∆ double mutant, but not the single mutants (Figure 3E).

Figure 3.

Set2 and Set3 mediate NDC80 induced gene repression of NDC80.

(A) Set2 and Set3 are required for repression of NDC80 expression during early meiosis. Control (FW1902), set2Δ (FW2929), set3Δ (FW2928) and set2Δset3Δ (FW1922) cells harboring pCUP-IME1/pCUP-IME4 and NDC80-V5 were grown in rich medium, transferred to pre-sporulation medium, and then shifted to SPO medium. After 2 hr, IME1 and IME4 expression were induced, and samples for northern and western blot analyses were taken at the indicated time points. Northern blot membranes were prepared and hybridized with a probe that detects both NDC80 and NDC80 transcripts. As a loading control, membranes were also hybridized with SCR1. Ndc80 protein was detected with anti-V5 antibodies and Hxk1 levels were determined with anti-hexokinase antibodies. (B) Scheme of the synchronous meiosis protocol in which cells were shifted directly from rich medium to SPO medium. Cells were grown in rich medium (YPD) to OD600 of 1–2, shifted to reduced glucose medium (YPD, 1% glucose) grown overnight to saturation, and then transferred to SPO. After 2 hr, IME1 and IME4 were induced. (C) Flow cytometry analysis of DNA content in control (FW1902) and set2Δset3Δ (FW1922) strains. Synchronous meiosis was induced as described in B. Samples were taken at the indicated time points after transfer to SPO and were stained with propidium iodide. (D) Similar to A except that meiosis was induced as described in B. (E) Strains described in A were grown to undergo a synchronous meiosis as described in B, and selective time points were taken for northern blot analysis of NDC80 and NDC80 transcripts on the same membrane. As a loading control, the northern membranes were hybridized with SCR1. The NDC80 levels were quantified (right panel) and data from three independent experiments plus the standard error of the mean (SEM) is displayed. One-tailed, unpaired t-tests were conducted to test if the differences in NDC80 levels were statistically significant. A single asterisk * denotes p-value<0.05. A double asterisk ** denotes p-value<0.01. ‘n.s.’ means ‘not significant’. To control for technical variation between different northern blots, the NDC80 signal from the two hour time point from the control strain of each blot was set to one. (F) NDC80 transcription requires Set2 and Set3 to establish a repressive chromatin state at the promoter of NDC80 Chromatin structure at the NDC80 locus was determined by ChIP of histone H3 on micrococcal nuclease (MNase) treated extracts in control (FW1902) and set2Δ set3Δ (FW1922) cells as described in A. Samples were taken prior to IME1/IME4 induction at 2 hr in SPO (2 hr, premeiotic) and after induction at 4 hr in SPO (4 hr, S + prophase), fixed with formaldehyde, and mononucleosome fragments were isolated. The recovered DNA fragments were quantified by qPCR using ten different primer pairs directed against the NDC80 locus relative to a no MNase input. The signals from each primer pair were then normalized over a primer pair directed against the PHO5 core promoter. The midpoint position of each primer pair is indicated in the x-axis. The mean signal from three independent experiments plus the standard error of the mean for each primer pair is displayed. (G) Ectopic expression of NDC80 is lethal in mitosis, but is rescued in a set2Δ set3Δ mutant. Spot assays of control cells, which harbor a wild-type NDC80 locus, with SET2 SET3 (UB1252), set2Δ (UB3545), set3Δ (UB3547), and set2Δ set3Δ (UB3549); as well as cells expressing NDC80 from the heterologous GAL promoter (pGAL-NDC80) with SET2 SET3 (UB1218), set2Δ (UB1236), set3Δ (UB1237), and set2Δ set3Δ (UB1235). These cells also expressed the Gal4 fused to estrogen receptor (Gal4-ER), which translocates to the nucleus in the presence of β-estradiol to activate the GAL1-10 promoter. Cells were grown overnight on YP-glycerol plates, diluted in sterile water, and spotted on YP +raffinose + galactose (YP-RG) plates in the absence or presence of β-estradiol. (H) Ectopic expression of NDC80 fails to downregulate Ndc80 in the set2Δ set3Δ mutant. Cells expressing NDC80 from the GAL promoter with SET2 SET3 (UB1217) or set2Δ set3Δ (UB8114) were grown to exponential phase in YP-RG, and they were induced to express NDC80 with β-estradiol. Samples were taken at the indicated time points. Ndc80 protein levels were determined by western blot using anti-V5 antibodies. Hxk1 levels were detected with anti-hexokinase antibodies. Ndc80 and Hxk1 were quantified and the relative expression (Ndc80/Hxk1) with respect to the 0 hr time point is displayed.

(A) Quantification of NDC80 and NDC80 levels in the experiment described in Figure 3A. Signals are normalized to SCR1. To control for variation in overall signal between different northern blots, the NDC80 signal at the 0 hr time point was set to one, and the NDC80 signal at the 3 hr time point was set to one. (B) set2Δ set3Δ mutants undergo meiosis with delayed kinetics. Kinetics of meiotic divisions (MI + MII) in control (FW1902), set2Δ (FW2929), set3Δ (FW2928) and set2Δset3Δ (FW1922) cells harboring pCUP-IME1/pCUP-IME4 and NDC80-V5. Cells were grown in rich medium, transferred to pre-sporulation medium, and then shifted to sporulation medium. After 2 hr, IME1 and IME4 expression were induced, and samples were taken at the indicated time points, fixed, and stained with DAPI. The percentage of cells with one, two or more DAPI masses was determined for at least 200 cells per time point. (C) Similar to B, except that cells were directly shifted from rich medium to SPO. (D) Quantification of NDC80 and NDC80 levels in the experiment described in Figure 3D. Signals are normalized to SCR1. The relative expression (NDC80) with respect to the 0 hr time point is displayed (left panel). The relative expression (NDC80/SCR1) with respect to the 3 hr time point is displayed (right panel). (E) Same experiment as Figure 3E, except that the NDC80 levels on northern blots were quantified, first normalized to SCR1 and then normalized to the intensity of NDC80 at 3.5 hr in control cells. The mean from three independent experiments plus the standard error of the mean are displayed. (F) Example of extract with mononucleosomes from set2Δset3Δ cells (FW1922, 2 hr [premeiotic]) prepared as described in Materials and methods. In short, cells were fixed with formaldehyde, treated with zymolase, and subsequently treated with different concentrations of micrococcal nuclease (MNase). Part of the sample from each extract was reverse crosslinked, purified, and separated by gel electrophoresis. The arrow indicates the extract that was used for subsequent ChIP analysis. (G) Levels of NDC80 are not affected in the set2Δ set3Δ double mutant. Control (UB1217) and set2Δ set3Δ (UB8114) cells harboring NDC80 driven by the GAL1-10 promoter were grown in YP-RG medium overnight, diluted and treated with β-estradiol during exponential growth. Samples were taken at the indicated time points. RNA was extracted, reverse transcribed, and the NDC80 mRNA levels were determined by qPCR. Signals were normalized to ACT1. The mean from three independent experiments plus the standard error of the mean are displayed. (H) Ectopic expression of NDC80 fails to downregulate Ndc80 in the set2Δ set3Δ mutant. Western blot of Ndc80 for SET2 SET3 cells harboring an NDC80-V5 allele (UB1240) or harboring a pGAL-NDC80 allele (UB1217), as well as set2Δset3Δ cells with NDC80-V5 (UB8110) or pGAL-NDC80 (UB8114). Cells were grown as described in Figure 3H, treated with β-estradiol, and samples were taken at the indicated time points. Ndc80 protein levels were determined with anti-V5 antibodies. Hxk1 levels were detected with anti-hexokinase antibodies. Ndc80 and Hxk1 were quantified, and the relative expression (Ndc80/Hxk1) with respect to the 0 hr time point is indicated. The western blot is from the same experiment described in Figure 3H, except that the data for the strains harboring the NDC80-V5 allele are included. (I) Quantification of Figure 3—figure supplement 1H. Ndc80 signals were first normalized to Hxk1 and were then normalized with respect to the 0 hr time point. The mean from three independent experiments plus the standard error of the mean are displayed.

Figure 3—figure supplement 1.

The Set2 and the Set3 mediate NDC80 induced gene repression of NDC80.

(A) Quantification of NDC80 and NDC80 levels in the experiment described in Figure 3A. Signals are normalized to SCR1. To control for variation in overall signal between different northern blots, the NDC80 signal at the 0 hr time point was set to one, and the NDC80 signal at the 3 hr time point was set to one. (B) set2Δ set3Δ mutants undergo meiosis with delayed kinetics. Kinetics of meiotic divisions (MI + MII) in control (FW1902), set2Δ (FW2929), set3Δ (FW2928) and set2Δset3Δ (FW1922) cells harboring pCUP-IME1/pCUP-IME4 and NDC80-V5. Cells were grown in rich medium, transferred to pre-sporulation medium, and then shifted to sporulation medium. After 2 hr, IME1 and IME4 expression were induced, and samples were taken at the indicated time points, fixed, and stained with DAPI. The percentage of cells with one, two or more DAPI masses was determined for at least 200 cells per time point. (C) Similar to B, except that cells were directly shifted from rich medium to SPO. (D) Quantification of NDC80 and NDC80 levels in the experiment described in Figure 3D. Signals are normalized to SCR1. The relative expression (NDC80) with respect to the 0 hr time point is displayed (left panel). The relative expression (NDC80/SCR1) with respect to the 3 hr time point is displayed (right panel). (E) Same experiment as Figure 3E, except that the NDC80 levels on northern blots were quantified, first normalized to SCR1 and then normalized to the intensity of NDC80 at 3.5 hr in control cells. The mean from three independent experiments plus the standard error of the mean are displayed. (F) Example of extract with mononucleosomes from set2Δset3Δ cells (FW1922, 2 hr [premeiotic]) prepared as described in Materials and methods. In short, cells were fixed with formaldehyde, treated with zymolase, and subsequently treated with different concentrations of micrococcal nuclease (MNase). Part of the sample from each extract was reverse crosslinked, purified, and separated by gel electrophoresis. The arrow indicates the extract that was used for subsequent ChIP analysis. (G) Levels of NDC80 are not affected in the set2Δ set3Δ double mutant. Control (UB1217) and set2Δ set3Δ (UB8114) cells harboring NDC80 driven by the GAL1-10 promoter were grown in YP-RG medium overnight, diluted and treated with β-estradiol during exponential growth. Samples were taken at the indicated time points. RNA was extracted, reverse transcribed, and the NDC80 mRNA levels were determined by qPCR. Signals were normalized to ACT1. The mean from three independent experiments plus the standard error of the mean are displayed. (H) Ectopic expression of NDC80 fails to downregulate Ndc80 in the set2Δ set3Δ mutant. Western blot of Ndc80 for SET2 SET3 cells harboring an NDC80-V5 allele (UB1240) or harboring a pGAL-NDC80 allele (UB1217), as well as set2Δset3Δ cells with NDC80-V5 (UB8110) or pGAL-NDC80 (UB8114). Cells were grown as described in Figure 3H, treated with β-estradiol, and samples were taken at the indicated time points. Ndc80 protein levels were determined with anti-V5 antibodies. Hxk1 levels were detected with anti-hexokinase antibodies. Ndc80 and Hxk1 were quantified, and the relative expression (Ndc80/Hxk1) with respect to the 0 hr time point is indicated. The western blot is from the same experiment described in Figure 3H, except that the data for the strains harboring the NDC80-V5 allele are included. (I) Quantification of Figure 3—figure supplement 1H. Ndc80 signals were first normalized to Hxk1 and were then normalized with respect to the 0 hr time point. The mean from three independent experiments plus the standard error of the mean are displayed.

Previous work showed that the set2∆ mutant exhibits increased nucleosome dynamics leading to de-repression of cryptic promoters (Venkatesh et al., 2012). In addition, the set3∆ mutant displays reduced histone H3 density in the 5’ region of transcribed genes (Kim and Buratowski, 2009). Set2 and Set3 are also required for transcription coupled chromatin changes in the IME1 promoter by the long noncoding RNA IRT1 (van Werven et al., 2012). These findings prompted us to examine whether Set2 and Set3 are necessary for NDC80 mediated nucleosome assembly in the NDC80 promoter. We found that even though NDC80 was efficiently transcribed in set2∆ set3∆ cells during early meiosis (Figure 3E, Figure 3—figure supplement 1D, right panel, and Figure 1E), repressive chromatin was not established at the NDC80 promoter (Figure 3F and Figure 3—figure supplement 1F).

We also tested whether Set2 and Set3 are required for repression of NDC80 when NDC80 is expressed ectopically during vegetative growth. Although NDC80 transcription was lethal in wild-type control cells, there was a partial growth rescue in either set2∆ or set3∆ cells. Importantly, growth was restored almost completely in set2∆ set3∆ cells (Figure 3G), due to de-repression of Ndc80 protein expression despite elevated transcription of the NDC80 mRNA (Figure 3H, Figure 3—figure supplement 1G–I). Altogether, these results show that both Set2 and Set3 are necessary for efficient repression of the NDC80 promoter by NDC80 transcription.

Gene repression by NDC80 is reversible

Ndc80 is an essential kinetochore protein required for chromosome segregation in both mitosis and meiosis (Ciferri et al., 2007). Thus, Ndc80 levels must be restored when cells either transit from prophase to meiotic divisions or when cells re-enter the mitotic cycle before meiotic commitment. This transition necessitates that the repression by NDC80 transcription be rapidly reversible so that cells can resume cell divisions in a timely manner. Indeed, NDC80 levels swiftly increase prior to meiotic divisions to facilitate chromosome segregation (Chen et al., 2017). To examine how gene repression by NDC80 transcription can adapt to changes in cell fate, we measured NDC80 and NDC80 expression kinetics in meiotic prophase cells that re-entered the mitotic cell cycle (Figure 4A). Cells carrying the ndt80∆ mutation were arrested in meiotic prophase, and returned to growth by providing them with rich medium. Strikingly, almost no NDC80 mRNA could be detected 15 min after return to growth (Figure 4B–C and Figure 4—figure supplement 1A). Concomitantly, NDC80 was almost fully re-expressed at the same time and Ndc80 protein levels increased after 30 min (Figure 4B–D). Therefore, NDC80 mediated repression of NDC80 is rapidly reversible, allowing for adaptation to physiological needs and re-entry into the mitotic cell cycle.

Figure 4.

NDC80 mediated repression is reversible.

(A) Schematic overview of the experimental set-up. (B) Cells repress NDC80 and induce NDC80 when returned to a nutrient rich environment (YPD). Cells harboring ndt80Δ and pCUP-IME1/pCUP-IME4 (FW3856) were grown in rich medium, shifted and grown in pre-sporulation medium, and subsequently transferred to SPO. Samples were taken prior to induction of IME1 and IME4 at 0 hr and 2 hr (premeiotic [pre]), and after induction at 4 hr (S-phase) and 6 hr (prophase) in SPO. After 6 hr, cells were either transferred to rich medium or kept in SPO medium. Samples for RNA and protein were taken at the indicated time points. To detect NDC80 and NDC80 expression, RNA was extracted, separated by gel electrophoresis, blotted, and hybridized with a probe that spans the NDC80 promoter and coding region. As a loading control for northern blots, we also probed membranes for SCR1. Ndc80 protein levels were determined by western blot using anti-V5 antibodies. As a loading control we also detected Hxk1 levels with anti-Hxk1 antibodies. (C) Quantification of NDC80 and NDC80 levels as described in B. The signals were normalized over SCR1. The error bars represent the standard error of the mean from two independent experiments. To control for technical variation between experiments the 0 hr and 4 hr time points were set to one for NDC80 and NDC80, respectively. (D) Quantification of Ndc80 protein levels during return to rich medium as described in B. The Ndc80 protein levels were normalized to Hxk1 protein abundance. The relative levels with respect to the 6 hr time point are displayed. The mean from two independent experiments plus the standard error of the mean is displayed. (E) H3K36me3 is rapidly lost from the NDC80 promoter and 5’ region after return to growth. Growth conditions were similar to B, except that histone H3 lysine 36 trimethylation (H3K36me3) levels were quantified at the NDC80 promoter during return to growth in a nutrient rich environment. Samples for chromatin immunoprecipitation were taken at the indicated time points. Cells were fixed with formaldehyde, chromatin extracts were prepared and H3K36me3 and histone H3 enriched fragments were immunoprecipitated with anti-H3K36me3 or anti-H3 antibodies, respectively. The recovered DNA fragments were quantified by qPCR using a primer directed against the NDC80 promoter (primer A) and a primer directed against the 5’ region of the NDC80 gene (primer B). The mean enrichment from three independent experiments plus the standard error of the mean for each primer pair is displayed. We also analyzed the signal at the 3’ end of the ACT1 open reading frame. The H3K36me3 signals were normalized to the histone H3 signal.

(A) Quantification of NDC80 during return to rich medium as described in Figure 4B. RNA was extracted, reverse transcribed, and NDC80 mRNA levels were determined by quantitative PCR. Signals were normalized to ACT1. The mean from two independent experiments plus the standard error of the mean is displayed. (B) Ime1 protein is unstable. Cells harboring Hemagglutinin-tagged IME1 under the control of the CUP1 promoter were grown in rich medium (UB1026), shifted to pre-sporulation medium, and subsequently transferred to SPO. After 2 hr, Ime1 was induced. After 4 hr in SPO, cells were split, and either treated with ethanol (EtOH) or cycloheximide (CHX, 0.2 mg/ml). Samples were taken at the indicated time points. Ime1 protein levels were determined by western blot using anti-HA antibodies. As a loading control, we also detected Hxk1 levels. (C) Bulk histone H3K36me3 levels remain constant during return to a nutrient rich environment. Cells harboring ndt80Δ (FW3856) were grown in rich medium, shifted and grown in pre-sporulation medium, and subsequently transferred to SPO. After 6 hr, cells were either transferred to rich medium or continued in SPO medium. Samples were taken at the indicated time point. H3K36me3 and histone H3 levels were determined by western blot using anti-H3K36me3 and anti-histone H3 antibodies, respectively. The relative abundance of H3K36me3 normalized to histone H3 is indicated.

Figure 4—figure supplement 1.

NDC80 mediated repression is reversible.

(A) Quantification of NDC80 during return to rich medium as described in Figure 4B. RNA was extracted, reverse transcribed, and NDC80 mRNA levels were determined by quantitative PCR. Signals were normalized to ACT1. The mean from two independent experiments plus the standard error of the mean is displayed. (B) Ime1 protein is unstable. Cells harboring Hemagglutinin-tagged IME1 under the control of the CUP1 promoter were grown in rich medium (UB1026), shifted to pre-sporulation medium, and subsequently transferred to SPO. After 2 hr, Ime1 was induced. After 4 hr in SPO, cells were split, and either treated with ethanol (EtOH) or cycloheximide (CHX, 0.2 mg/ml). Samples were taken at the indicated time points. Ime1 protein levels were determined by western blot using anti-HA antibodies. As a loading control, we also detected Hxk1 levels. (C) Bulk histone H3K36me3 levels remain constant during return to a nutrient rich environment. Cells harboring ndt80Δ (FW3856) were grown in rich medium, shifted and grown in pre-sporulation medium, and subsequently transferred to SPO. After 6 hr, cells were either transferred to rich medium or continued in SPO medium. Samples were taken at the indicated time point. H3K36me3 and histone H3 levels were determined by western blot using anti-H3K36me3 and anti-histone H3 antibodies, respectively. The relative abundance of H3K36me3 normalized to histone H3 is indicated.

One explanation for the swift shut down of NDC80 transcription is that its transcriptional activator is degraded or depleted. The accompanying paper showed that Ime1 is the major transcriptional activator of NDC80 transcription (Chen et al., 2017). Under nutrient rich conditions, IME1 transcription shuts down and Ime1 protein translocates to the cytoplasm (Colomina et al., 2003; van Werven and Amon, 2011). In addition, we found that Ime1 has a half-life of only a few minutes, facilitating rapid clearance of Ime1 protein during return-to-growth (Figure 4—figure supplement 1B). We propose that during return-to-growth, Ime1 is rapidly inactivated, resulting in a quick decrease in NDC80 transcription.

We hypothesized that the dynamic changes of NDC80 mediated repression during return to growth would be reflected in the chromatin state of the NDC80 promoter. We found that H3K36me3 at the 5’ end of NDC80 was strongly reduced within 15 min and almost completely lost within 30 min after cells returned to a nutrient rich environment (Figure 4E). The loss was specific to the NDC80 locus because the levels at the 3’ end of the ACT1 gene increased slightly while bulk H3K36me3 levels did not change (Figure 4E and Figure 4—figure supplement 1C). Thus NDC80-mediated gene repression is reversible, allowing for rapid and dynamic changes in gene expression and chromatin state.

Gene repression by NDC80 transcription is tunable

Work from Escherichia coli showed that gene regulation by transcriptional interference is not binary with an on or off state, but can be utilized to fine-tune gene expression levels (Bordoy et al., 2016; Hao et al., 2016). The work prompted us to investigate whether transcriptional interference by NDC80 could also be tunable, thus enabling incremental changes in NDC80 expression levels. To scale the level of NDC80 expression, we used a tightly controlled, inducible system. The system utilizes a heterologous, chimeric transcriptional activator (LexA-ER-AD) whose activity is induced in a concentration-dependent manner by β-estradiol (Ottoz et al., 2014). Varying the number of LexA-binding sites (lexO) in the NDC80 promoter and titrating the concentration of β-estradiol, enabled scalable transcriptional induction of NDC80 (Figure 5—figure supplement 1A). The growth defect caused by NDC80 expression in mitosis was more severe with elevated concentrations of β-estradiol and higher number of lexO sites in the NDC80 promoter (Figure 5A). The higher the luti transcription, the greater the inhibition of NDC80 expression. Thus, modulating NDC80 transcription levels allows scalable transcriptional repression of NDC80 in a population of cells.

Figure 5—figure supplement 1.

Gene repression by NDC80 is tunable.

(A) NDC80 levels in the presence of variable number of lexO sites in the NDC80 promoter. Cells harboring 0, 1, 2, 3, or 8 lexO and LexA-ER-AD (UB8374, UB8358, UB8362, UB8366, and UB8370) were grown in YPD overnight. Subsequently, cells were diluted and exponentially growing cells were treated with 10 or 20 nM β-estradiol for 3 hr. RNA was extracted, reverse transcribed, and NDC80 mRNA levels were determined by quantitative PCR. Signals were normalized to ACT1. The mean from three independent experiments plus the standard error of the mean are displayed. (B) Ndc80 protein level in SET2 SET3 cells harboring none (UB12945) or 8 lexO sites (UB12949), or set2∆ set3∆ cells with none (UB12947) or 8 lexO sites (UB12951). All four strains carry LexA-ER-AD. Ndc80 protein was detected by anti-V5 immunoblot. Hxk1 levels were used as a loading control. ‘Pre’ denotes pre-induction. Exponentially growing cells were treated with ethanol (0 nM), 15 nM, or 20 nM β-estradiol. Samples were taken at 2 hr or 4 hr after β-estradiol induction. Ndc80 level was normalized to Hxk1 level, and the number under each lane shows the Ndc80/Hxk1 ratio normalized to that in the pre-induction condition. (C) Same as Figure 5—figure supplement 1B, except that the mean of two independent experiments plus standard error of the mean are displayed. (D) Increased promoter NDC80 activity bypasses NDC80 mediated repression. Exponentially growing cells were treated with ethanol or 1 μM β-estradiol. Samples were taken at 3 hr or 6 hr after β-estradiol induction. The amount of samples loaded corresponded to the same OD across all the cultures. Ndc80 level was normalized to Hxk1 level, and the number under each lane shows the Ndc80/Hxk1 ratio normalized to that in the pre-induction condition. For the analyses, we used the strains described in Figure 5C. (E) Quantification of Ndc80 protein levels from Figure 5—figure supplement 1D. The Ndc80 protein levels were normalized to Hxk1 protein abundance. The relative levels with respect to the 0 hr time point are displayed. The mean from three independent experiments plus the standard error of the mean are displayed.

Figure 5.

Gene repression by NDC80 is tunable.

(A) Adjustable expression of NDC80 using the LexA-lexO system. Spot assay of cells harboring 0, 1, 2, 3, or 8 lexO binding sites in the NDC80 promoter (UB8374, UB8358, UB8362, UB8366, UB8370) in the presence of different concentrations of β-estradiol. These cells also expressed LexA fused to an activation domain (AD) and the human estrogen receptor (ER) (LexA-ER-AD). Cells were grown overnight, diluted in sterile water, and spotted on YPD plates in the absence or presence of different concentrations of β-estradiol. (B) Similar to A, except that set2Δset3Δ mutant cells (UB8691, UB8686 and UB8693) were included for the analysis. (C) Increased NDC80 promoter activity bypasses NDC80 mediated repression. Cells were grown overnight, diluted in sterile water, and spotted on YP +raffinose + galactose plates in the absence or presence of β-estradiol (1 μM). For the analyses, we used three sets of strains: (1) Cells with a wild-type NDC80 and with a functional MSE site (+MSE), but with either a wild-type NDT80 (UB3351) or a pGAL1-10 driven NDT80 (pGAL-NDT80, UB3370); (2) cells with pGAL-NDC80 and with a functional MSE site, along with either a wild-type NDT80 (UB5154) or pGAL-NDT80 (UB9181); (3) cells with pGAL-NDC80 and a non-functional MSE site, along with either pGAL-NDT80 (UB9921) or wild-type NDT80 (UB9923). These cells also expressed Gal4-ER to activate pGAL driven expression.

(A) NDC80 levels in the presence of variable number of lexO sites in the NDC80 promoter. Cells harboring 0, 1, 2, 3, or 8 lexO and LexA-ER-AD (UB8374, UB8358, UB8362, UB8366, and UB8370) were grown in YPD overnight. Subsequently, cells were diluted and exponentially growing cells were treated with 10 or 20 nM β-estradiol for 3 hr. RNA was extracted, reverse transcribed, and NDC80 mRNA levels were determined by quantitative PCR. Signals were normalized to ACT1. The mean from three independent experiments plus the standard error of the mean are displayed. (B) Ndc80 protein level in SET2 SET3 cells harboring none (UB12945) or 8 lexO sites (UB12949), or set2∆ set3∆ cells with none (UB12947) or 8 lexO sites (UB12951). All four strains carry LexA-ER-AD. Ndc80 protein was detected by anti-V5 immunoblot. Hxk1 levels were used as a loading control. ‘Pre’ denotes pre-induction. Exponentially growing cells were treated with ethanol (0 nM), 15 nM, or 20 nM β-estradiol. Samples were taken at 2 hr or 4 hr after β-estradiol induction. Ndc80 level was normalized to Hxk1 level, and the number under each lane shows the Ndc80/Hxk1 ratio normalized to that in the pre-induction condition. (C) Same as Figure 5—figure supplement 1B, except that the mean of two independent experiments plus standard error of the mean are displayed. (D) Increased promoter NDC80 activity bypasses NDC80 mediated repression. Exponentially growing cells were treated with ethanol or 1 μM β-estradiol. Samples were taken at 3 hr or 6 hr after β-estradiol induction. The amount of samples loaded corresponded to the same OD across all the cultures. Ndc80 level was normalized to Hxk1 level, and the number under each lane shows the Ndc80/Hxk1 ratio normalized to that in the pre-induction condition. For the analyses, we used the strains described in Figure 5C. (E) Quantification of Ndc80 protein levels from Figure 5—figure supplement 1D. The Ndc80 protein levels were normalized to Hxk1 protein abundance. The relative levels with respect to the 0 hr time point are displayed. The mean from three independent experiments plus the standard error of the mean are displayed.

During transcription nucleosomes are disassembled and reassembled by histone chaperones that associate with RNA polymerase (Venkatesh and Workman, 2015). Therefore, higher levels of NDC80 transcription could lead to an increased rate of nucleosome deposition in the NDC80 promoter and thus scalable NDC80 repression. If so, then sufficiently high levels of NDC80 transcription should be sufficient for repressing NDC80 without requiring Set1/Set3C and Set2/Rpd3S to maintain repressive chromatin. Cells with both pathways compromised (set2∆set3∆) and harboring three or eight lexO sites did not show a growth defect when exposed to intermediate levels of β-estradiol (15 nM), whereas control cells did (Figure 5B). This result was expected because in the set2∆set3∆ mutant background NDC80 mediated repression is impaired (also see Figure 3). Surprisingly at higher concentrations of β-estradiol (25 nM), set2∆set3∆ mutant cells harboring three lexO sites exhibited a moderate growth defect while cells with eight lexO sites exhibited a severe growth defect. We also measured the Ndc80 protein levels in control and set2∆set3∆ mutant cells harboring 0 or 8 copies of lexO sites. The growth defects observed in Figure 5B were reflected in the Ndc80 protein levels (Figure 5—figure supplement 1B and C). These data suggest that high levels of NDC80 transcription could bypass the requirement for Set2 and Set3 in NDC80 repression.

Since increased expression of NDC80 leads to stronger repression of NDC80, we tested whether the strength of the NDC80 promoter influenced the effectiveness of NDC80 mediated repression. To examine this, we increased the levels of NDC80 by ectopically expressing the meiotic transcription factor Ndt80 in mitotic cells. Ndt80 induces the expression of NDC80 via the middle sporulation element (MSE) in the NDC80 promoter (Chen et al., 2017). In the presence of Ndt80 expression, the growth defect caused by NDC80 transcription was suppressed (Figure 5C). This suppression is dependent on the presence of the MSE site in the NDC80 promoter (Figure 5C, compare MSE positive versus negative in the presence of pGAL-NDT80 and pGAL-NDC80). In addition, the growth changes as observed in the spot assays correlated well with Ndc80 protein levels (Figure 5—figure supplement 1D and E). Thus, increased transcription from the NDC80 promoter can bypass NDC80 mediated repression. Taken together, transcriptional interference by expression of a 5’ extended transcript can be tuned by adjusting the relative strengths of the distal and proximal promoters. Hence, this mechanism can be adapted as a regulatory module to generate a range of gene expression outputs.

Discussion

Eukaryotic cells have evolved various mechanisms to achieve dynamic control of gene expression during cell fate progression. Here we demonstrate how Ndc80 levels are temporally regulated during the budding yeast meiotic program. Together with an accompanying paper, we show that NDC80 transcription is repressed by an unusual mechanism during the prophase stage of meiosis (Chen et al., 2017). Transcription of a 5’ extended mRNA isoform interferes with transcription of the downstream canonical NDC80 mRNA isoform. The mechanism of gene repression described here is tunable, rapidly reversible, and an effective way to generate changes in gene expression.

The transcriptional mechanism of NDC80 repression

Repression of Ndc80 protein production during meiotic prophase is critical for setting up faithful chromosome segregation during meiosis (Miller et al., 2012). Two NDC80 mRNA isoforms were identified: the Ndc80 coding isoform (NDC80), and the 5’ extended long undecoded transcript isoform (NDC80), which cannot be translated into Ndc80 protein (Chen et al., 2017). We showed that NDC80 transcription interferes with the downstream NDC80 promoter. First, the expression of NDC80 and NDC80 are anti-correlated in cells undergoing meiosis synchronously. Second, mutants defective in NDC80 transcription cannot repress NDC80 (Chen et al., 2017). In particular transcription of truncated NDC80 fails to repress NDC80, thus excluding promoter competition as a mechanism for repression. Third, transcription of NDC80 inhibits TFIIB binding to the NDC80 promoter (Figure 1). Fourth, repression by NDC80 only works in cis and NDC80 RNA does not localize to a discrete region in the nucleus, making it unlikely that the NDC80 RNA itself is important to the repression mechanism (Chen et al., 2017). However, we cannot rule out that the nascent NDC80 RNA contributes to repression of NDC80 expression.

How does NDC80 interfere with NDC80 transcription? Our data show that Set2 and Set3 is essential for establishing a repressive chromatin state and for inhibiting NDC80 transcription. We propose a two-step mechanism (Figure 6). First, transcription of NDC80 deposits Set1 mediated H3K4me2 and Set2 mediated H3K36me3 in the NDC80 promoter. Second, co-transcriptional deposition of these marks facilitates the recruitment of the histone deacetylase complexes Set3C and Rpd3S (Carrozza et al., 2005; Keogh et al., 2005; Kim and Buratowski, 2009). Notably, the two marks localize to the same area of the NDC80 promoter, perhaps indicating that there is redundancy between the two pathways (see following section in the discussion). Previous work established a role for Set2 in suppressing histone exchange and promoting nucleosome stability through chromatin remodelers (Venkatesh et al., 2012; Smolle et al., 2012). Hypo-acetylated histones are also associated with increased nucleosome stability (Venkatesh and Workman, 2015). Hence, cells lacking both Set2 and Set3 show reduced nucleosome occupancy in the NDC80 promoter, and NDC80 transcription is unimpeded despite active NDC80 transcription (Figure 3). Taken together, NDC80 repression of NDC80 is mediated by co-transcriptional chromatin reorganization of the NDC80 promoter.

Figure 6.

Model for NDC80 mediated repression of NDC80.

During mitosis the NDC80 promoter has an open chromatin structure and NDC80 is transcribed. During meiotic S-phase and meiotic prophase, transcription of NDC80 deposits Set1-dependent H3K4me2 and Set2-dependent H3K36me3 in the same area of the NDC80 promoter. Co-transcriptional deposition of these marks facilitates the recruitment of the histone deacetylase complexes Set3C and Rpd3S. This leads to increased nucleosome occupancy in the NDC80 promoter and NDC80 transcription is repressed. The set of reversing arrows indicates that NDC80 mediated repression of NDC80 is reversible.

The Set1/Set3C and Set2/Rpd3S pathways have well characterized roles in preventing cryptic transcription and regulating gene expression via long noncoding RNA transcription (Carrozza et al., 2005; Keogh et al., 2005; Kim and Buratowski, 2009; Kim et al., 2012; van Werven et al., 2012; Ard and Allshire, 2016b ; Venkatesh et al., 2016). It has been reported that Set2 and Set3 modulate the expression of different genes based on the length of adjacent ncRNAs, which overlap with their promoters. (Kim et al., 2012; Kim et al., 2016). Kim et al. demonstrate that during a series of carbon source shifts, genes whose promoters overlap with longer transcripts (~2.0 kb) are repressed by Set2/Rpd3S whereas those with shorter overlapping transcripts (~0.9 kb), are repressed by Set1/Set3C (Kim et al., 2016). According to their classification, NDC80 is a short overlapping transcript. Strikingly, NDC80 mediated repression of NDC80 was compromised in the set2∆set3∆ double mutant cells, but not in the single mutants. (Figure 3). We propose that Set1/Set3C and Set2/Rpd3S act redundantly during NDC80 mediated repression of NDC80.

The mechanism that we have described here has clear similarities with transcriptional interference mediated by intergenic or promoter transcription. Like NDC80, transcription of the intergenic/promoter ncRNA, SRG1, leads to increased nucleosome occupancy and lower binding of activators in the downstream SER3 promoter (Martens et al., 2004; Hainer et al., 2011). Transcriptional interference is also important for mating-type control of sporulation. In cells with a single mating type, transcription of a long noncoding RNA IRT1, in the promoter of the master regulatory transcription factor Ime1, is critical for IME1 repression (van Werven et al., 2012). Similar to NDC80, IRT1 transcription establishes repressive chromatin throughout the IME1 promoter via a mechanism requiring both Set2 and Set3. Interestingly, while Set1 and Set2 act on different parts of the IME1 promoter, the H3K4me2 and H3K36me3 marks overlap in the NDC80 promoter suggesting that both modifications control the same promoter region. Perhaps, H3K4me2 and H3K36me3 occur on the same nucleosome as part of a repressive combinatorial histone code.

NDC80 mediated repression of NDC80 is dynamic

Our synchronous meiosis and return to growth experiments have shed light on the dynamics of NDC80 mediated repression of NDC80. During meiotic prophase Ndc80 levels decline, but as soon as cells enter meiotic divisions, Ndc80 levels increase. Chen et al. showed that the transcription factor Ndt80 activates the meiotic wave of NDC80 expression (Chen et al., 2017). Expression of Ndt80 can bypass NDC80 induced repression suggesting that repression is not refractory to increasing levels of NDC80 transcription (Figure 5). This ensures that NDC80 can be rapidly produced after meiotic prophase, when Ndc80 becomes essential for proper meiotic divisions. Interestingly, the MSE site (approximately −200 bp upstream from the AUG) in the NDC80 promoter is not protected by nucleosomes even during NDC80 transcription (Figure 1E), which may explain the ability of Ndt80 to activate NDC80 even while NDC80 is expressed.

The dynamic nature of the regulation is also illustrated by our finding that NDC80 mediated repression can be rapidly reversed (Figure 4). We propose that the reversibility of the repression may provide two main benefits. First, it allows for temporal control of NDC80 expression during meiosis as NDC80 mediated repression of NDC80 specifically occurs during meiotic S-phase and meiotic prophase. Cells that progressed into meiotic divisions were able to bypass NDC80 mediated repression and re-expressed NDC80 (Chen et al., 2017). Second, rapid adjustments to NDC80 expression in response to changing environmental cues can be directly integrated at the NDC80 promoter. When we exposed cells arrested in meiotic prophase to a nutrient rich environment and allowed cells to re-enter the mitotic cell cycle, NDC80 expression was lost and NDC80 was rapidly induced (Figure 4). The mechanism for the reversibility of NDC80 mediated repression is not fully understood. We speculate that transcriptional activators and chromatin remodelers stimulate nucleosome eviction in the NDC80 promoter during its activation. In line with this idea, levels of the repressive H3K36me3 in the NDC80 promoter are rapidly lost when the repression is reversed (Figure 4E). This is despite the purported stability of the H3K36me3 mark due to its role in reducing histone turnover (Smolle et al., 2012; Venkatesh et al., 2012; Sein et al., 2015). More work is needed to fully characterize the molecular mechanism of reversing NDC80 mediated repression.

In addition to its reversibility, NDC80-mediated repression can be adapted to fine-tune gene expression. Using a scalable expression system, we showed that modulating the levels of NDC80 affects the efficiency of NDC80 repression (Figure 5). The higher the levels of NDC80 transcription, the better the repression of NDC80 becomes. Notably, Set2 and Set3 are no longer required for repressing NDC80 when NDC80 is highly expressed. One possible explanation is that the rate of nucleosome deposition at the NDC80 promoter is increased during higher levels of NDC80 transcription. In this situation, the requirement for histone deacetylase complexes to stabilize nucleosomes becomes obsolete. Alternatively, elongating RNA polymerase might physically interfere with the NDC80 promoter when NDC80 is highly expressed.

Whereas most studies have reported a binary switch for transcription interference mechanisms (Martens et al., 2004; Hongay et al., 2006; Camblong et al., 2007; Bumgarner et al., 2009; van Werven et al., 2012), we propose that transcriptional interference by expression of a 5’ extended transcript is tunable. This principle could be further adapted and used in synthetic genetic circuits to modulate gene expression levels. Indeed, mechanisms of transcriptional interference have been applied to coordinate activities of adjacent genes in both E. coli and budding yeast (Buetti-Dinh et al., 2009; Bordoy et al., 2016; Hao et al., 2016; Hoffmann et al., 2016).

Concluding remarks

Transcriptional interference by 5’ extended isoforms might be wide-spread in yeast and across species. Transcript isoform sequencing in yeast revealed that the 5’ and 3’ ends of mRNAs are extremely heterogeneous (Pelechano et al., 2013). During budding yeast meiosis, more than 190 genes express an extended 5’ leader sequence (Brar et al., 2012). Some examples have been further confirmed and show a clear inhibitory effect on expression from the downstream promoter (Liu et al., 2015; Xie et al., 2016). However, how the expression of different mRNA isoforms regulate gene expression remains unexplored at the genome-wide level. In higher eukaryotes including human cells, a wide range of 5’ extended mRNA isoforms are also expressed often in a cell type-specific manner (Wang et al., 2008; Aanes et al., 2013; Brown et al., 2014). Understanding the principles underlying gene regulation by 5’ extended mRNA isoforms during yeast meiosis will deepen our understanding of how complex differentiation programs in higher eukaryotes are regulated.

Materials and methods

Yeast strains

Yeast strains used in this paper were derived from the sporulation proficient SK1 strain background, except for the strains that harbored the LexA/lexO system for which the W303 strain background was used. The genotypes are listed in Supplementary file 1. Gene or promoter deletions were generated using the one-step deletion protocol as described previously (Longtine et al., 1998). Tagging Sua7 with three copies of V5 epitope at C-terminus was performed using a one-step integration protocol using a V5-tagging cassette. The LexA/lexO system was described previously (Ottoz et al., 2014).

Growth and conditions

The synchronous meiosis procedure using pCUP-IME1 and pCUP-IME4 was described, previously (Berchowitz et al., 2013; Chia and van Werven, 2016). In short, cells were grown to saturation overnight in YPD (1.0% (w/v) yeast extract, 2.0% (w/v) peptone, 2.0% (w/v) glucose, and supplemented with tryptophan (9.6 mg/l), uracil (2.4 mg/l) and adenine (1.2 mg/l)). These cells were then shifted to pre-sporulation medium (BYTA) (1.0% (w/v) yeast extract, 2.0% (w/v) tryptone, 1.0% (w/v) potassium acetate, 50 mM potassium phthalate) where they were grown for 16–18 hr. Subsequently, cells were centrifuged, washed with sterile milliQ water, centrifuged again and suspended in sporulation medium (SPO) (0.3% (w/v) potassium acetate and 0.02% (w/v) raffinose]) at OD600 = 2.5. After two hours, copper (II) sulphate (50 µM) was added to induce IME1 and IME4 expression from the CUP1 promoter and initiate meiosis synchronously. For the synchronous experiments described in Figure 3B–3E, we shifted cells directly from saturated YPD culture to SPO medium, and induced pCUP-IME1/pCUP-IME4 after two hours in SPO (Chia and van Werven, 2016).

For meiotic time-courses not synchronized by pCUP-IME1/pCUP-IME4 (Figure 1A, left panel), cells were grown and treated as described previously (Falk et al., 2010). In short, cells were grown to saturation overnight in YPD. These cells were then shifted to BYTA where they were grown for another 16–18 hr. Subsequently, cells were washed with sterile milliQ water, and transferred to SPO medium (0.3% (w/v) potassium acetate and 0.02% (w/v) raffinose)).

For the return to growth experiments, ndt80∆ cells were left for six hours in SPO to arrest them in meiotic prophase. Cells were then transferred to an equivalent volume of pre-warmed YPD. All experiments were performed at 30°C in a shaker incubator at 300 rpm.

For Figure 2E–F, cells were grown for 6–8 hr in YPD at 30°C, diluted to an OD600 of 0.002, transferred to YP (1.0% (w/v) yeast extract and 2.0% (w/v) peptone)+2% raffinose+2% galactose (YP-RG) and grown for another 16–18 hr. The cells were diluted to an OD600 of 0.2, grown for another 2.5–3 hr, diluted back to OD 0.2 and induced to express NDC80 by the addition of 1 μM β-estradiol. 25–30 OD600 units of cells were collected for ChIP analyses at 0 hr and at 3 hr after induction.

For the time courses in Figure 3H, Figure 3—figure supplement 1H and I, cells were grown for 6–8 hr in YPD at 30°C, diluted to an OD600 of 0.002, transferred to YP-RG and grown for another 16–18 hr. Cells were diluted to an OD600 of 0.2 in YP-RG and NDC80 expression was induced by the addition of 1 μM β-estradiol. Samples were taken at 0, 3, 4.5, and 6 hr after β-estradiol addition.

For Figure 4—figure supplement 1B, cells were grown to saturation overnight in YPD. These cells were then shifted to BYTA, in which they were grown for another 16–18 hr. Subsequently, cells were washed with sterile milliQ water, and transferred to SPO medium. After two hours in SPO, copper (II) sulphate (50 µM) was added to induce IME1 and IME4 from the CUP1 promoter. At four hours in SPO, cells were either treated with ethanol or cycloheximide (0.2 mg/ml). Samples for western blotting were taken at 15, 30, 45, 60 and 90 min after adding ethanol or cycloheximide.

For Figure 5—figure supplement 1A, cells were grown for 16–18 hr in YPD at 30°C and then diluted to an OD600 of 0.2. Subsequently, the cells were grown for 2.5–3 hr to reach exponential phase. The cells were diluted again to an OD600 of 0.2 and induced to express NDC80 with either 10 or 20 nM of β-estradiol. Cells were collected for qPCR analysis at 0 hr and at 3 hr after induction.

For Figure 5—figure supplement 1B and C, cells were grown in YPD at 30 ˚C overnight to saturation, diluted to OD600 of 0.1, and then grown to OD600 of 0.3–0.5 at 30 °C. Three OD600 units of cells were taken as the pre-induction samples. Cells were then diluted to an OD600 of 0.1 in YPD and split into three flasks. Subsequently, β–estradiol was added to the cells with a final concentration of either 15 nM or 25 nM. Ethanol was added as to the uninduced cells. 2 hr after β–estradiol induction,~3 OD600 units of cells were taken for western analysis, and at 4 hr, another ~3 OD600 units of cells were collected. All the samples were processed according to the western blotting protocol. The OD600 of each culture was also measured when the samples were taken. Equal OD600 units of samples was loaded during the gel electrophoresis.

Nuclei/DAPI counting

DAPI staining was used to monitor meiotic divisions throughout meiotic time courses. Cells were fixed in 80% (v/v) ethanol, pelleted by centrifugation and re-suspended in PBS with DAPI (1 µg/ml). Cells were sonicated for a few seconds and left in the dark at room temperature for at least 5 min. The proportion of cells containing one, two, three, or four DAPI masses was counted using a fluorescence microscope. At least two independent biological experiments were performed for each meiotic time-course experiment.

Flow cytometry analysis

Flow cytometry was used to monitor meiotic DNA replication as described previously (Chia and van Werven, 2016). Cells were fixed in 80% (v/v) ethanol and re-suspended in 50 mM Tris-HCl pH 7.5. Cells were sonicated for a few seconds and were treated with 0.2 mg/ml ribonuclease A in 50 mM Tris-HCl pH 7.5 at 37°C overnight. Cells were stained with 50 µg/ml propidium iodide in FACS buffer (200 mM Tris-HCl pH 7.5, 211 mM NaCl and 78 mM MgCl2) for one hour at room temperature before flow cytometry analysis (BD LSRFortessa, BD Biosciences). Propidium iodide stained cells were excited with a 561 nm yellow-green laser and signals were detected using a 610/20 yellow filter. Pulse shape analysis (pulse height against pulse area) was used to exclude clumps and doublets. DNA content from single cells was estimated with a histogram of counts against pulse area. At least 50,000 cells were used for the analysis.

Western blotting

Western blotting was used to determine Ndc80 protein levels. Protein extracts were prepared by using trichloroacetic acid (TCA) extraction protocol. In short, cells were pelleted by centrifugation (~2400 g, 1 min, room temperature) and re-suspended in 5% w/v TCA for at least 10 min. For vegetative samples, pellets were washed with TE50 buffer (50 mM Tris pH 7.5, 1 mM EDTA), then with acetone, and completely dried. For meiotic samples, the pellets were only washed with acetone and dried. Furthermore, pellets were mixed with lysis buffer (50 mM Tris pH 7.5, 1 mM EDTA, 2.75 mM dithiothreitol (DTT)) and cells were disrupted using a mini beadbeater (BioSpec). Lysates were mixed with SDS loading buffer (187.5 mM Tris pH 6.8, 6.0 % v/v β-mercaptoethanol, 30% v/v Glycerol, 9.0 % w/v SDS, 0.05% w/v Bromophenol Blue) and boiled for 5 min for denaturation. After polyacrylamide gel electrophoresis, proteins were transferred onto PVDF or nitrocellulose membranes using the Mini Trans-Blot Cell (Bio-Rad (CA, USA)). The membranes were blocked for 60 min in blocking buffer (1% w/v BSA, 1% w/v milk) before incubation with mouse anti-V5 (R96025, Sigma-Aldrich (MO, USA)) at a 1:2000 dilution overnight at 4°C. Hxk1 loading control was detected using a rabbit anti-hexokinase antibody (H2035, Stratech (Newmarket, UK)) at a 1:8000 dilution overnight at 4°C. Membranes were then washed in PBST (phosphate buffered saline with 0.01% (v/v) Tween-20) and incubated with IRDye 800CW goat anti-mouse and IRDye 680RD donkey anti-rabbit secondary antibodies (LI-COR (NE, USA)) at a 1:15000 dilution. Protein levels were detected using an Odyssey Imager (LI-COR). To detect HA tagged Ime1, a mouse anti-HA antibody was used at a 1:2000 dilution (901501, Biolegend (CA, USA)). All other steps were performed as above. At least two independent biological experiments were performed for each western blot experiment.

To measure bulk histone H3 levels, membranes were blocked for 60 min in blocking buffer (5% w/v milk) before incubation with rabbit anti-H3, C terminus (07–690, Millipore (MA, USA)) at a 1:3000 dilution overnight at 4°C. To measure bulk H3K36me3 levels, membranes were blocked for 60 min in blocking buffer (1% w/v BSA, 1% w/v milk) before incubation with rabbit anti-H3K36me3 (Ab9050, Abcam (Cambridge, UK)) at a 1:1000 dilution overnight at 4°C. Membranes were then washed in PBST and incubated with HRP conjugated ECL donkey anti-rabbit secondary antibodies (GE Healthcare (IL, USA)) at a 1:8000 dilution. After addition of ECL substrate (GE Healthcare), protein levels were detected using an ImageQuant RGB 600 machine (GE Healthcare). At least two independent biological experiments were performed for each western blot experiment.

RT-qPCR

To quantify NDC80 mRNA levels as described in Figure 4—figure supplement 1A, we used a reverse transcription combined with quantitative PCR (RT-qPCR) protocol. Total RNA was isolated, purified and treated with DNAse (Macherey-Nagel (Düren, Germany)). 750 ng of total RNA was reverse-transcribed using random primers and Protoscript II (NEB (MA, USA)), and single-stranded cDNA was quantified by real-time PCR using SYBR green mix (Life Technologies). The signals were normalized to ACT1 transcript levels. The oligonucleotide sequences used for RT-PCR experiments are displayed in Supplementary file 2.

For the RT-qPCR in Figure 5—figure supplement 1A, RNA was isolated by acid phenol-chloroform extraction, treated with DNase (TURBO DNA-free kit, Thermo Fisher (MA, USA)), and reverse transcribed into cDNA (Superscript III Supermix, Thermo Fisher). The cDNA was quantified using the Absolute Blue qPCR Mix (Thermo Fisher). The NDC80 signals were normalized to ACT1 transcript levels. The oligonucleotide sequences used for RT-qPCR experiments are displayed in Supplementary file 2.

Chromatin immunoprecipiation

Chromatin immunoprecipitation (ChIP) experiments were performed as described previously. Cells were fixed in 1.0% v/v of formaldehyde for 15–20 min at room temperature and quenched with 100 mM glycine. Cells were broken using a mini beadbeater (BioSpec) and crosslinked chromatin was sheared by sonication using a Bioruptor (Diagenode (Seraing, Belgium), 7 cycles of 30 s on/off). Extracts were incubated for 2 hr or overnight at 4 ˚C with magnetic Prot A beads (Sigma) coupled with a polyclonal antibody against Histone H3 trimethyl lysine 36 (Ab9050, Abcam), Histone H3 dimethyl lysine 4 (Ab32356, Abcam) or Histone H3 (Ab1791, Abcam). Subsequently, reverse cross-linking was done in Tris-EDTA buffer (100 mM Tris pH 8.0, 10 mM EDTA, 1.0% v/v SDS) at 65°C overnight. After 2 hr of proteinase K treatment, samples were cleaned up and H3K36me3 enrichment was measured by real-time PCR using SYBR green mix (Life Technologies (CA, USA)) and primers corresponding to the NDC80 promoter and the 5’ region of the NDC80 open reading frame. Sua7-V5 binding was measured using similar procedures, except that anti-V5 agarose beads (Sigma-Aldrich) were used instead. The oligonucleotide sequences used for ChIP experiments are displayed in Supplementary file 2.

ChIP on micrococcal nuclease (MNase) treated chromatin extracts

To determine the chromatin structure at the NDC80 locus, we extracted mononucleosomes using a MNase digestion protocol that was described previously followed by ChIP for histone H3 (Rando, 2010, 2011). Approximately 250 OD600 units of cells were crosslinked for 15 min with formaldehyde (1% v/v) and the reaction was quenched with glycine (125 mM). Subsequently, cells were resuspended in 20 ml of buffer Z (1 M sorbitol, 50 mM Tris-HCl pH 7.4) plus β-mercaptoethanol (10 mM) and treated with 250 μg of T100 Zymolase (MP Biomedicals (CA, USA)) for 60 min. Next, cells were resuspended in 2.5 ml NP buffer (0.5 mM spermidine, 1 mM β-mercaptoethanol (β -ME), 0.075% (w/v) Tergitol solution-type NP-40 detergent (NP-40), 50 mM NaCl, 10 mM Tris-HCl pH 7.4, 5 mM MgCl2, 1 mM CaCl2), and extract was treated with 5, 0.625, 0.2 or 0.04 μl of MNase (2 mg/ml, NEB) for 30 min at 37°C, the reaction was quenched with EDTA (10 mM). The extract was adjusted to 0.1 M HEPES-KOH pH 7.5, 150 mM NaCl, 0.1% w/v sodium deoxycholate, and 1% w/v Triton X-100. To check for the extent of MNase digestion, 60 μl of MNase treated and untreated extracts were reverse crosslinked overnight in SDS-TE (1% (w/v) SDS, 10 mM Tris pH 8, 1 mM EDTA), treated with RNase A and purified DNA fragments were separated by gel electrophoresis. The extracts which the showed a mono-nucleosome pattern were used for ChIP with histone H3 antibodies. The ChIP was performed with 600 μl of extract as described in the chromatin immunoprecipitation section of the Materials and methods. ChIP samples were quantified by qPCR on a 7500 FAST Real-Time PCR machine (Applied Biosystems (CA, USA)). Scanning primer pairs covering the NDC80 locus and upstream region were used for the analysis. Signals were quantified relative to untreated genomic DNA, and normalized over a primer pair directed against the PHO5 core promoter (Chang and Vancura, 2012). The oligonucleotide sequences are available in Supplementary file 2.

Northern blotting

We adapted a northern blot protocol that was described previously (Koster et al., 2014). In short, RNA was extracted with Acid Phenol:chloroform:IAA (125:24:1) and precipitated in ethanol with 0.3M sodium acetate. RNA samples were denatured in a glyoxal/DMSO mix (1M deionized glyoxal, 50% v/v DMSO, 10 mM sodium phosphate (NaPi) buffer pH 6.5–6.8) at 70°C for 10 min. Denatured samples were mixed with loading buffer (10% v/v glycerol, 2 mM NaPi buffer pH 6.5–6.8, 0.4% w/v bromophenol blue) and separated on an agarose gel (1.1% w/v agarose, 0.01M NaPi buffer) for at least 3 hr at 80 V. RNAs were then transferred onto nylon membranes overnight by capillary transfer. rRNA bands were visualized by methylene blue staining. The membranes were blocked for at least 3 hr at 42°C in ULTRAhyb Ultrasensitive Hybridization Buffer (Thermo Fisher) before hybridization. The radioactive probes specific to NDC80, SCR1 and CIT1 were synthesized using a Prime-It II Random Primer Labeling Kit (Agilent (CA, USA)), a DNA template and ATP (α−32P) (Perkin-Elmer (MA, USA)). The oligo-nucleotide sequences used for amplifying the NDC80, CIT1 or SCR1 templates are displayed in Supplementary file 2. At least two independent biological experiments were performed for each northern blot experiment.

Quantification of northern and western blots

NDC80 and SCR1 levels were estimated from northern blots using ImageJ (Schneider et al., 2012). The net intensity of each band of interest was determined by subtracting the mean background intensity of the areas immediately above and below the band. Signals were first normalized to SCR1 levels and further normalized to a specific band on the same membrane (usually the first time point when either NDC80 or NDC80 appeared) For Figure 3E, one-tailed, unpaired t tests were conducted to determine if the difference in NDC80 levels between mutant and control strains were statistically significant.

Intensities of Ndc80 and Hxk1 bands on western blots were quantified using Image Studio Lite (LI-COR). Ndc80 levels were first normalized to Hxk1 levels and further normalized to that of the first time point on the same membrane.

Spot growth assay

For strains harboring NDC80 under control of the GAL1-10 promoter, cells were first grown on YP plus 2% glycerol (YPG) plates overnight, and then re-suspended in milliQ H2O to an OD600 of 0.2. Next, 5-fold serial dilutions were performed and diluted cells were spotted onto either YP-RG plates with no β-estradiol or YP-RG plates supplemented with1 μM β-estradiol. The cells were incubated at 30°C for 1–2 days. Note that the GAL1-10 promoter in the SK1 strain background does not directly respond to galactose. At least two independent biological experiments were performed for each spot assay experiment.

For strains harboring constructs in which NDC80 expression is driven by LexA/lexO, cells were grown on YPD plates, re-suspended in milliQ water to an OD600 of 0.2, serially diluted as above, and then spotted onto either YPD plates with no β-estradiol or YPD plates with different concentrations of β-estradiol (10, 15, 20, 25, or 30 nM). The cells were incubated at 30°C for 1 day before imaging. At least two independent biological experiments were performed for each spot assay experiment.

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Transcription of a 5' extended mRNA isoform directs dynamic chromatin changes and interference of a downstream promoter" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Jessica Tyler as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. As you will see from the comments below, there was enthusiasm for this manuscript as a companion to the co-submitted manuscript from Chen et al. However, there were also several important weaknesses that need to be addressed before the paper can be published in eLife. We realize that we are requesting a substantial amount of work to strengthen the experiments and their conclusions, but given that your manuscript is a co-submission, we considered that it was fairer to give you the opportunity to revise the paper rather than to reject it and keep the door open to a resubmission at a later date.

Summary:

In this manuscript, the authors provide mechanistic insight into how an mRNA that initiates ~500bp upstream of the NDC80 gene represses transcription from the NDC80ORF promoter. In the accompanying manuscript, basic features of the NDC80 regulatory system were described, including demonstration that the long mRNA, termed NDC80luti, and the ORF mRNA appear anti-correlated and that blocking transcription from the NDC80luti promoter derepresses the ORF transcript. In this manuscript, the authors find support for a transcriptional interference mechanism in which transcription from the upstream start inhibits PIC assembly at the ORF promoter and establishes a histone modification pattern at the ORF promoter (enrichment of H3K4me2 and H3K36me3) that has been previously associated with recruitment of two well characterized yeast HDACs, Set3C and Rpd3S. The authors extend their work by showing that the NDC80 regulatory system is reversible, when cells are switched from sporulation to growth media, and tunable, when the levels of the NDC80luti transcript are systematically elevated through a lexA-ER system.

Demonstration of the molecular basis of the repression mechanism is a valuable complement to the accompanying paper by Chen et al. However, as the authors acknowledge, involvement of cis-repression of a promoter by an overlapping non-coding transcript via transcription-coupled histone methylation and establishment of repressive chromatin is not a novel mechanism, being established at several other genes in yeast. In addition to the examples for this cited by the authors, there is the case of the upstream ncRNA that represses SER3 by promoting nucleosome assembly over the SER3 promoter, and antisense transcripts that mediate histone methylation of the promoter for the sense transcript at FLO11. Other studies, including recent work from the Buratowski lab on a genome-wide scale, showed that noncoding transcription across promoters leads to enrichment of H3K4me2 and H3K36me3 and repression of the promoters. With that said, the study does provide insight into an interesting example of regulated transcriptional interference.

In addition to the need for a more thorough coverage of relevant precedents for similar examples of gene regulation (see above), there are some important control experiments lacking, notably a failure to measure by ChIP the total H3 levels in the NDC80 promoter – to distinguish nucleosomes from non-histone chromatin protein occupancy, and to normalize changes in methylation for changes in H3 occupancy – and lack of quantification of changes in the NDC80 transcript, which is required to firmly establish the role of Set2/Set3 in repressing this promoter. In addition, there are a number of instances where direct experimental evidence is insufficient to support mechanistic claims made; these instances need to be remedied by inclusion of more data or by significant rewriting of text to soften conclusions and make them more nuanced. Statistical analysis of replicate data is missing for measurements of mRNA levels by northern analysis. Loading and normalization controls are lacking in multiple figures.

Essential revisions:

1) The increase in MNase-resistance in the NDC80 promoter region is not necessarily due to nucleosomes as it has been shown that non-histone protein binding in NDRs can also confer MNase-resistance (PMID: 28157509). For this reason, a ChIP of the MNase-resistant chromatin with histone antibodies is required to establish nucleosome versus non-histone protein occupancy.

2) In Figure 2C changes in nucleosome occupancy are inferred upon repression of the downstream promoter (at -100), but there is no evidence for loss of nucleosomes accompanying activation of the upstream promoter. Is this because the upstream promoter is nucleosome free even when repressed or is there another explanation?

3) Figure 2. Some information on the sequences of the promoters should be presented. Does the Sua7 occupancy profile make sense with respect to positions of core promoter elements? Why is the highest Sua7 signal detected 400bp upstream of the NDC80 TSS?

4) Figure 2. How were the nucleosome positioning data normalized? Typically these data are normalized to a known, highly occupied nucleosome position (for example, Sekinger et al. Mol Cell 18: 735). Also control gels should be included to show the MNase digestion profile.

5) ChIP with histone antibodies is also required to normalize the H3-K36me3 and H3-K4me2 ChIP signals in Figure 3A-D and Figure 3—figure supplement 1 in order to establish that the amount of methylation per nucleosome is increased over the NDC80 promoter in a manner dependent on the luti promoter. K4me2 levels should be included in Figures 3C and 3D.

6) Figure 4A: RNA loading controls are needed in the northern blots. Quantification of the mRNA from replicates with a loading control is needed to establish that the NCD80 transcript is elevated reduced in prophase in the double mutant relative to WT, as this seems to be limited to the 3hr timepoint, and even this increase is not evident in Figure 4D. Perhaps the only meaningful increase in terms of Ndc80p expression occurs at 4-5hr.

7) Figure 4G. How many repetitions were done to calculate the relative Ndc80 levels? Also, the levels of the Hxk1 loading control seem to be changing both in this panel and in Figure 4—Figure supplement 1. This is perhaps not surprising given the change in carbon source in this experiment and the known connections of Hxk1 to carbon metabolism.

8) The accompanying Chen et al. paper provides evidence that Ndt80 is required for induction of the short NDC80 mRNA. If so, how can it be induced in the experiment of Figure 5A conducted with an ndt80 mutant?

9) Figure 5. Panel B lacks an RNA loading control and quantitation. The data in panel E need to be normalized to total H3 levels.

10) Figure 6B needs to be accompanied by measurements of Ndc80 protein to confirm that the requirement for Set2/Set3 in Ndc80p repression have been bypassed. Similarly for Figure 6C.

11) Figure 7. The figure does not add very much to the discussion. The vertical red lines are not defined. The nucleosomes look more like Pol II molecules with modified CTDs. Please modify accordingly.

12) In the Discussion, the authors conclude that their data show the "co-transcriptional recruitment of Set1/Set3C and Set2/Rpd3S is essential for establishing a repressive chromatin state and inhibiting NDC80 transcription". Recruitment of none of these factors was tested and therefore the text needs to be modified accordingly.

Non-essential revisions:

1) Figure 2 provides the main data supporting a transcriptional interference mechanism. As presented, the data are insufficient to support the stated conclusions. First, additional factors should be assayed by ChIP, beyond Sua7. In particular, how does NDC80 expression influence Ndt80 occupancy in the NDC80 promoter region? Second, what is the effect of blocking NDC80 transcription on Sua7 and Ndt80 occupancy? What is the effect of blocking NDC80 on nucleosome occupancy? Note: the additional ChIP experiments suggested in this point are encouraged, but are not considered essential for publication. However, if the authors choose not to strengthen this part of the study, it is essential that they substantially modulate the strength of their statements in the text to be in line with what the data actually demonstrate.

2) Figure 4. The authors use set2∆ as a proxy for removing Rpd3S. The HDAC could be directly tested by performing similar northern and western blots with an rco1∆ strain. The authors argue that loss of the two HDAC systems alleviates transcriptional interference. This could be tested by ChIP of Sua7 in the HDAC mutant strains. Surprisingly, the authors show effects of deleting SET2 and SET3 on nucleosome occupancy but not histone acetylation levels at the ORF promoter. Their model should be tested more directly with better choices of mutants and ChIPs. Note: the additional ChIP experiments suggested in this point are encouraged, but are not considered essential for publication. However, if the authors choose not to strengthen this part of the study, they need to substantially modulate the strength of their statements in the text to be in line with what the data actually demonstrate. For example, without directly testing recruitment of HDACs, which can be tricky to do, they cannot say that their data show recruitment of HDACs via H3K36me3 and H3K4me2. They can merely describe this as a likely scenario. However, they could provide more support for this model by measuring histone acetylation levels, which is not hard to do.

3) Figure 1 essentially recapitulates the same or similar experiments described in the accompanying paper by Chen et al., with somewhat different promoter replacements or termination insertions for the NDC80 transcript. Given this redundancy, the authors should consider omitting this figure. If they prefer to retain the figure, substantial improvements are needed. Here, the corresponding Ndc80 protein data are lacking for these promoter and termination constructs; although the quality of the northern data is better here for the terminator construct. However, mRNA loading controls and quantification of the northern data from replicates is lacking and should be provided.

4) It's surprising in Figure 2C that the nucleosome peak at ~-500, which is probably the +1 nucleosome for the luti mRNA promoter doesn't decline in prophase on induction of this transcript. This deserves some comment.

5) Figure 2B: It's surprising that TFIIB binding increased the most during prophase at -800, >300 bp upstream of the TSS for the luti transcript, raising questions about the location of the luti promoter. This deserves some comment.

6) Integrate Figure 3—figure supplement 1 data into Figure 3, and add premeiotic and S-prophase labels to data key.

7) Subsection “Gene repression by NDC80 transcription is tunable”, last paragraph: "repression" should probably be "expression".

8) Subsection “NDC80 mediated repression of NDC80 is dynamic”, end of first paragraph: it would be helpful to cite explicitly the data that supports this statement.

9) It would be nice to see a northern analysis to examine both long and short NDC80 mRNAs in Figure 6A. This would provide support for the last two sentences in the first paragraph of the subsection “Gene repression by NDC80 transcription is tunable”.

Essential revisions:

1) The increase in MNase-resistance in the NDC80ORF promoter region is not necessarily due to nucleosomes as it has been shown that non-histone protein binding in NDRs can also confer MNase-resistance (PMID: 28157509). For this reason, a ChIP of the MNase-resistant chromatin with histone antibodies is required to establish nucleosome versus non-histone protein occupancy.

We now performed the experiment with histone H3 ChIP on MNase treated chromatin extracts. The results are similar to our original observation. We concluded that nucleosomes bind the NDR region of the NDC80 locus when NDC80 is transcribed.

2) In Figure 2C changes in nucleosome occupancy are inferred upon repression of the downstream promoter (at -100), but there is no evidence for loss of nucleosomes accompanying activation of the upstream promoter. Is this because the upstream promoter is nucleosome free even when repressed or is there another explanation?

We have two potential explanations:

1) The transcriptional repressor of NDC80, Ume6, binds to the upstream promoter (see Chen et al), which could exclude nucleosomes from binding to the same sequence. When NDC80 is activated by Ime1-Ume6, the region remains relatively unchanged, because Ume6 binding to the promoter occurs prior to Ime1-dependent activation. Hence, the upstream promoter remains free of nucleosomes prior and during activation of NDC80 transcription.

2) Alternatively, the Ume6 repressor keeps the +1 nucleosome in place. When Ime1 is expressed together with Ume6, it activates NDC80 and its transcription causes the +1 nucleosome to remain positioned.

3) Figure 2. Some information on the sequences of the promoters should be presented. Does the Sua7 occupancy profile make sense with respect to positions of core promoter elements? Why is the highest Sua7 signal detected 400bp upstream of the NDC80luti TSS?

We have attempted to look for core promoter elements, which in yeast are not so well characterized, except for the TATA element. We were not able to identify an obvious TATA element in the NDC80 promoter, which is consistent with its previous classification as a TATA-less promoter (PMID: 15006352). Sua7 binding is approximately at -100 bp from AUG, which is within the region where core promoter elements are expected in yeast (PMID: 15006352).

The Sua7 signal in the upstream promoter region can be explained by expression of the neighbouring gene, PAN6, which is transcribed in the opposite direction compared to NDC80. We observed that PAN6 expression increases during entry into meiosis, which may explain the reason for the broad peak of Sua7 binding.

4) Figure 2. How were the nucleosome positioning data normalized? Typically these data are normalized to a known, highly occupied nucleosome position (for example, Sekinger et al. Mol Cell 18: 735). Also control gels should be included to show the MNase digestion profile.

We have now included examples of how we prepared the MNase treated extracts, and show the MNase digestion pattern on a gel (Figure 1—figure supplement 1E and Figure 3—figure supplement 1G). We have performed ChIP with histone H3 on extracts with predominantly mononucleosomal DNA fragments. We have normalized our data to PHO5 sequences. This control gave consistent results.

5) ChIP with histone antibodies is also required to normalize the H3-K36me3 and H3-K4me2 ChIP signals in Figure 3A-D and Figure 3—figure supplement 1 in order to establish that the amount of methylation per nucleosome is increased over the NDC80ORF promoter in a manner dependent on the luti promoter. K4me2 levels should be included in Figures 3C and 3D.

We have now performed histone H3 ChIPs for Figure 2 (old Figure 3) and used this to normalize our H3K4me2 or H3K36me3 signals. We have also included K4me2 ChIPs in a mutant that does not express NDC80_luti (ndc80) in premeiotic cells and S-prophase cells (Figure 2C). It is worth noting that in the old version we used a different mutant for the K36me3 experiment (ndc80). To keep it consistent in the revised manuscript, we repeated the experiment with the ndc80mutant (Figure 2D). The results are comparable between the two mutants. Finally, we checked K4me2 by ChIP when NDC80 is expressed from the GAL promoter in Figure 2E.

6) Figure 4A: RNA loading controls are needed in the northern blots. Quantification of the mRNA from replicates with a loading control is needed to establish that the NCD80ORF transcript is elevated reduced in prophase in the double mutant relative to WT, as this seems to be limited to the 3hr timepoint, and even this increase is not evident in Figure 4D. Perhaps the only meaningful increase in terms of Ndc80p expression occurs at 4-5hr.

We have now performed loading controls for the northern blots in Figure 3A and quantified the experiment. Different time-course experiments (biological replicates) always give the same trends, but do not always show the same level of changes. One explanation is that time course experiments have inherent variability with regards to meiotic kinetics between biological replicates. This makes it tricky to take the average of multiple experiments, because the variation in mRNA level is due to both the biological variation as well as the variation in meiotic synchrony. Furthermore, in experiments where the wild type and mutant samples were run on different gels, they were transferred to different blots and hybridized separately, which could also cause technical variability.

Therefore, we only show representative experiments for Figure 3A and 3D (old Figure 4A and 4D). To address this issue, we further examined the NDC80 levels of selective time-points from three independent experiments in Figure 3E, for the earlier meiotic time points, where the pCUP-IME1/IME4 system yields better synchrony. For each experiment, the wildtype and mutant samples were run on the same gel/blot/hybridization. We observed significant differences in NDC80expression at 3.5 and 4.5 hours between the wild-type and set2 set3 double mutant, but not the single mutants.

Please note that we replaced the experiment in Figure 3D with another repeat because hybridization with the SCR1 loading failed.

7) Figure 4G. How many repetitions were done to calculate the relative Ndc80 levels? Also, the levels of the Hxk1 loading control seem to be changing both in this panel and in Figure 4—figure supplement 1. This is perhaps not surprising given the change in carbon source in this experiment and the known connections of Hxk1 to carbon metabolism.

The Figure 3H (formerly Figure 4G) shows the quantification for that particular repeat. We have now included the quantification from two independent experiments in Figure 3—figure supplement 1E. There was no change in carbon source in this experiment. The samples were treated with β-estradiol that induces the GAL promoter using Gal4-ER fusion.

8) The accompanying Chen et al. paper provides evidence that Ndt80 is required for induction of the short NDC80ORF mRNA. If so, how can it be induced in the experiment of Figure 5A conducted with an ndt80 mutant?

Our data suggest that there at least two ways to activate NDC80 transcription. One is during regular mitotic cell cycle of which we do not know the corresponding transcription factors. This is also seen from the fact that NDC80is expressed before entry into meiosis. The second way is during meiosis by the transcription factor Ndt80. In Figure 4 (old Figure 5), we examined how NDC80 transcription is reactivated when cells are switched from meiotic entry back to mitotic growth. We propose that the transcription factors important for NDC80 expression in mitosis controls the re-activation during return to mitotic cell cycle. The accompanying paper by Chen et al. proposes that Ndt80 bypasses NDC80 mediated repression of NDC80. Therefore we performed the experiment in an ndt80 mutant background, the induction of NDC80in an ndt80 mutant is consistent with mitotic mediated activation of NDC80.

9) Figure 5. Panel B lacks an RNA loading control and quantitation. The data in panel E need to be normalized to total H3 levels.

We have now included a loading control and normalized the ChIP signals to histone H3. We have quantified the levels of both NDC80 and NDC80 from two independent experiments in Figure 4C.

10) Figure 6B needs to be accompanied by measurements of Ndc80 protein to confirm that the requirement for Set2/Set3 in Ndc80p repression have been bypassed. Similarly for Figure 6C.

We now included Ndc80 protein levels for Figure 5B and 5C (old Figure 6B and 6C) and in Figure 5—figure supplement 1B-E. Example western blots and the mean of at least two independent experiments are shown in these figures.

11) Figure 7. The figure does not add very much to the discussion. The vertical red lines are not defined. The nucleosomes look more like Pol II molecules with modified CTDs. Please modify accordingly.

We have now changed the figure, accordingly.

12) In the Discussion, the authors conclude that their data show the "co-transcriptional recruitment of Set1/Set3C and Set2/Rpd3S is essential for establishing a repressive chromatin state and inhibiting NDC80ORF transcription". Recruitment of none of these factors was tested and therefore the text needs to be modified accordingly.

We have removed the terms Set1/Set3C and Set2/Rpd3S from most parts of the manuscript, and mention Set2 and Set3 only. We discuss Set1/Set3C and Set2/Rpd3S only in the Discussion and Introduction.

Non-essential revisions:

1) Figure 2 provides the main data supporting a transcriptional interference mechanism. As presented, the data are insufficient to support the stated conclusions. First, additional factors should be assayed by ChIP, beyond Sua7. In particular, how does NDC80luti expression influence Ndt80 occupancy in the NDC80 promoter region? Second, what is the effect of blocking NDC80luti transcription on Sua7 and Ndt80 occupancy? What is the effect of blocking NDC80luti on nucleosome occupancy? Note: the additional ChIP experiments suggested in this point are encouraged, but are not considered essential for publication. However, if the authors choose not to strengthen this part of the study, it is essential that they substantially modulate the strength of their statements in the text to be in line with what the data actually demonstrate.

We agree that ChIP for more factors is desirable. We have tried to ChIP for several factors at the NDC80 promoter. We tested sequence specific transcription factors that have been implicated in regulating NDC80 expression by genome-wide studies: Hcm1, Swi4, and Fkh1. In addition, we have tried to ChIP for basal transcription factors such as: Taf1 and TBP. Unfortunately, none the factors gave a reproducible ChIP signal. One explanation is that NDC80 expression quite low during mitosis (only a few mRNA copies per cell). This makes it is challenging to obtain robust ChIP signals. We have modulated our statements and only talk about TFIIB (as this is only factor that worked) and not preinitiation complex.

2) Figure 4. The authors use set2∆ as a proxy for removing Rpd3S. The HDAC could be directly tested by performing similar northern and western blots with an rco1∆ strain. The authors argue that loss of the two HDAC systems alleviates transcriptional interference. This could be tested by ChIP of Sua7 in the HDAC mutant strains. Surprisingly, the authors show effects of deleting SET2 and SET3 on nucleosome occupancy but not histone acetylation levels at the ORF promoter. Their model should be tested more directly with better choices of mutants and ChIPs. Note: the additional ChIP experiments suggested in this point are encouraged, but are not considered essential for publication. However, if the authors choose not to strengthen this part of the study, they need to substantially modulate the strength of their statements in the text to be in line with what the data actually demonstrate. For example, without directly testing recruitment of HDACs, which can be tricky to do, they cannot say that their data show recruitment of HDACs via H3K36me3 and H3K4me2. They can merely describe this as a likely scenario. However, they could provide more support for this model by measuring histone acetylation levels, which is not hard to do.

We agree that our model predicts an increase in histone acetylation. However, given that we had put a lot of effort and time in completing the other essential and non-essential comments, we decided to test this further in a follow up study. We have modulated our conclusions in the Results section and refer the Set2 and/or Set3. We only refer to the Set1/Set3C and Set2/Rpd3S pathways in the Discussion.

3) Figure 1 essentially recapitulates the same or similar experiments described in the accompanying paper by Chen et al., with somewhat different promoter replacements or termination insertions for the NDC80luti transcript. Given this redundancy, the authors should consider omitting this figure. If they prefer to retain the figure, substantial improvements are needed. Here, the corresponding Ndc80 protein data are lacking for these promoter and termination constructs; although the quality of the northern data is better here for the terminator construct. However, mRNA loading controls and quantification of the northern data from replicates is lacking and should be provided.

We have removed Figure 1 from the manuscript, and made a new Figure 1 consisting of parts of the old Figure 1 and the complete Figure 2. We included the first data panel of the old Figure 1 in the new Figure 1, for which we added a loading control and quantified the signals from the northern and western blot.

4) It's surprising in Figure 2C that the nucleosome peak at ~-500, which is probably the +1 nucleosome for the luti mRNA promoter doesn't decline in prophase on induction of this transcript. This deserves some comment.

We agree that it is somewhat surprising that there was no shift in nucleosome occupancy around that +1 positon. Given that this a PCR based assay, we cannot exclude that there are subtle changes that we missed. Given that Ume6 binds the region prior to its activation, perhaps the Ume6 repressor keeps the +1 nucleosome in place. When Ime1 is expressed together with Ume6, it activates NDC80 and transcription causes the +1 nucleosome to remain positioned.

5) Figure 2B: It's surprising that TFIIB binding increased the most during prophase at -800, >300 bp upstream of the TSS for the luti transcript, raising questions about the location of the luti promoter. This deserves some comment.

PAN6 transcribed in the divergent direction is directly adjacent NDC80. Its expression increases upon meiotic entry, which may explain the shift in peak. See also point 3 of Essential revisions.

6) Integrate Figure 3—figure supplement 1 data into Figure 3, and add premeiotic and S-prophase labels to data key.

Has been added, accordingly.

7) Subsection “Gene repression by NDC80luti transcription is tunable”, last paragraph: "repression" should probably be "expression".

Has been changed, accordingly.

8) Subsection “NDC80luti mediated repression of NDC80ORF is dynamic”, end of first paragraph: it would be helpful to cite explicitly the data that supports this statement.

We now changed the section:

“Interestingly, the MSE site (approximately -200 bp upstream from the AUG) in the NDC80 promoter is not protected by nucleosomes even during NDC80 transcription (Figure 1E), which may explain the ability of Ndt80 to activate NDC80 while NDC80luti is expressed.

The dynamic nature of the regulation is also illustrated by our finding that NDC80 mediated repression can be rapidly reversed (Figure 4).”

9) It would be nice to see a northern analysis to examine both long and short NDC80 mRNAs in Figure 6A. This would provide support for the last two sentences in the first paragraph of the subsection “Gene repression by NDC80luti transcription is tunable”.

We agree that this is would further support our data. However, the experiment is not trivial. The northern blot can be tricky to quantify because the background signal around the relatively weak NDC80 mRNA band will most likely increase when NDC80 mRNA is highly expressed, owing to the close proximity of the transcripts. Therefore we have measured Ndc80 protein levels in cells harbouring no or 8 copies of lexO sites in the NDC80 promoter instead. We used different concentrations of β-estradiol to measure the effect on Ndc80 protein levels. The differences in growth inhibition observed in the spot assay were also reflected at the Ndc80 protein level (see Figure 5—figure supplement 1B and C).