Polycomb- and REST-associated histone deacetylases are independent pathways toward a mature neuronal phenotype.

The bivalent hypothesis posits that genes encoding developmental regulators required for early lineage decisions are poised in stem/progenitor cells by the balance between a repressor histone modification (H3K27me3), mediated by the Polycomb Repressor Complex 2 (PRC2), and an activator modification (H3K4me3). In this study, we test whether this mechanism applies equally to genes that are not required until terminal differentiation. We focus on the RE1 Silencing Transcription Factor (REST) because it is expressed highly in stem cells and is an established global repressor of terminal neuronal genes. Elucidation of the REST complex, and comparison of chromatin marks and gene expression levels in control and REST-deficient stem cells, shows that REST target genes are poised by a mechanism independent of Polycomb, even at promoters which bear the H3K27me3 mark. Specifically, genes under REST control are actively repressed in stem cells by a balance of the H3K4me3 mark and a repressor complex that relies on histone deacetylase activity. Thus, chromatin distinctions between pro-neural and terminal neuronal genes are established at the embryonic stem cell stage by two parallel, but distinct, repressor pathways.

Introduction

Undifferentiated pluripotent cells present a unique dilemma with regard to gene regulation; genes that promote differentiation must be repressed to maintain pluripotency, yet this repression must be reversible to allow for rapid response to developmental cues. The repressed status, often referred to as poised, is conferred by epigenetic modifications established at loci encoding developmental regulators. Specifically, global histone modification patterns in embryonic stem cells (ESCs) have revealed the coexistence of trimethylation of histone H3 at lysines 4 and 27 (H3K4me3 and H3K27me3) at promoters of genes encoding key lineage-determining factors (Bernstein et al., 2006). This dual chromatin status has been termed bivalence to reflect the juxtaposition of modifications typically associated with functionally active and transcriptionally repressed promoters, respectively. The H3K27me3 mark is established by the methyltransferase EZH2 within the Polycomb Repressor Complex 2 (PRC2) (Pengelly et al., 2013), which effectively provides a counterbalance to factors that promote H3K4me3 and active expression (Bernstein et al., 2006). This PRC2-dependent state has been proposed as a universal mechanism to confer pluripotency by controlling all developmental lineages, but its application to the neuronal lineage has not been tested rigorously. This is of interest because for most lineages the key developmental regulators are activators, while in the neuronal lineage a key regulator is the transcriptional repressor REST (NRSF).

Specifically, REST is a master developmental regulator that controls a large suite of genes that encode proteins critical for neuronal maturation, such as cellular migration, axonal pathfinding, and synaptic transmission (Johnson et al., 2007, 2008; Otto et al., 2007). Further, REST is expressed at very high levels in embryonic stem cells, contrary to other developmental regulators. A global REST knockout results in embryonic lethality, pointing to an essential function for REST in general embryonic development following the ESC stage (Chong et al., 1995; Schoenherr and Anderson, 1995). In neural progenitors, REST levels decrease until it completely leaves the chromatin at terminal differentiation of most neurons. Preventing its dismissal from chromatin delays greatly neuronal maturation in vivo (Mandel et al., 2011) and alters neural progenitor pool identities in vitro (Covey et al., 2012). In stem/progenitor cells, developmental genes required for neuronal lineage decisions are repressed, including pro-neural and REST-regulated genes (Buckley et al., 2009, Ballas et al., 2005), but whether the mechanisms that regulate these classes of genes are the same or different remains an open question.

Prior studies have shown an important role for PRC2 repression on poised genes of multiple lineages. On the one hand, although the terminal neuronal genes regulated by REST are poised in stem cells, REST is itself a repressor and may not require the additional repression mechanism of Polycomb. On the other hand, the non-coding RNA (ncRNA) HOTAIR has been shown to act as an adapter between the core PRC2 component EZH2 and the REST co-factor Kdm1a (Tsai et al., 2010), suggesting a connection between PRC2 and REST in ESCs. In addition, other groups have observed biochemical interaction between REST and PRC2 members (Dietrich et al., 2012; Mozzetta et al., 2014) and recruitment of H2K27me3 to RE1 sites (Arnold et al., 2013). Therefore, we performed three studies to test directly for the existence of a functional relationship between PRC2 and REST in ESCs. First, we performed a mass spectrometric analysis of REST complexes to identify ESC-specific co-factors in an unbiased manner. Second, we asked whether REST-occupied neuronal genes were marked by H3K27me3, and furthermore, whether PRC2 activity was compromised in Rest ESCs. Finally, exploiting a Rest ESC line, we examined the consequences of the loss of REST on chromatin marks and gene expression.

Results

REST complexes purified from ESCs

Previous studies of REST-interacting proteins in ESCs used a candidate approach and focused on co-factors characterized in differentiated cells (Ballas et al., 2005; Yu et al., 2011). In the current study, we considered the possibility that ESC-specific co-factors might be involved in regulatory mechanisms of REST that were unique to pluripotent cells. To test this idea we performed a mass spectrometric analysis of REST complexes using a mouse ESC line that stably expressed both the biotin conjugating enzyme, BirA (Kim et al., 2009), and REST tagged with a biotin acceptor sequence. The stable line expressed approximately five-fold higher levels of REST than normal ESCs with no differences in pluripotency markers compared to WT cells (not shown). Multidimensional Protein Identification Technology (MudPIT) analysis was performed on three independent streptavidin purifications. Proteins that were co-purified with REST in at least two of three pull-downs and were weakly represented, if at all, in the BirA control pull-downs are shown in Table 1. None of the known epigenetic regulators identified as co-factors by mass spectrometry were specific to ESCs. However, we did identify almost all known REST co-factors including CoREST1 and Sin3a as well as the chromatin modifying enzymes, HDAC1 and 2, Kdm1a and G9a/Glp, and the G9a-associated adaptors CDYL and WIZ1, all of which have been shown biochemically to be present within REST complexes in terminally differentiated cell types (Andres et al., 1999; Grimes et al., 2000; Hakimi et al., 2002; Roopra et al., 2004; Mulligan et al., 2008), thus validating our approach. We noted that an additional CoREST family member, CoREST2, was also present in the pull-downs. We confirmed the presence of CoREST2, as well as a subset of other co-factors, at RE1 sites in ESCs by chromatin immunoprecipitation (ChIP, Figure 1—figure supplement 1). We also identified several new factors, some with known functions (Smarca5, Mdc1) and some with no known function (D1Pas1, Table 1). In contrast to these factors, components of the Polycomb repressor complexes were not identified according to our criteria. It was possible that the specific conditions used to generate the whole-cell extracts used in the MudPIT analysis precluded identification of Polycomb proteins. Therefore, we repeated mass spectrometry analysis on streptavidin pull-downs from nuclear extracts (Abmayr et al., 2006). Under these conditions, we did identify the PRC2 complex members Suz12 (3 and 4 peptides in BioT REST pull-down replicates, 0 and 0 peptides in Control) and Ezh2 (3 and 5 peptides in BioT REST, 0 and 0 peptides in Control). Co-immunoprecipitation analysis using nuclear extract confirmed only the Suz12 interaction, as well as the interactions with known REST co-repressors (Figure 1—figure supplement 2A). Importantly, however, the members of the PRC2 complex required for the methyltransferase activity, Ezh2, and for complex formation, Eed (Montgomery et al., 2005), were both absent from the co-immunoprecipitation (Figure 1—figure supplement 2A). These results indicate that REST protein does not interact with an enzymatically active PRC2 complex in ESCs. To supplement this proteomic approach, and as an independent test for the role of PRC2 members in REST regulation, we used a genome-wide ChIP-seq approach.

Table 1.

REST-bound genomic regions with repeated consensus RE1 motifs.

Co-factors identified within REST complexes were purified from ESCs

DOI: http://dx.doi.org/10.7554/eLife.04235.003

Columns list the chromosome and base pair coordinates (Region Start & Region End) of the REST-binding domain identified by PeakRanger analysis of ChIP-Seq read distribution. RE1 Start and RE1 End columns give the coordinates corresponding to the positions of individual RE1 motifs found by FIMO within the corresponding region. Orientation column lists whether the RE1 motif is on the forward (+) or reverse (−) DNA strand, and the p-value column gives the calculated log-odds score from the comparison of a discovered motif to a position weighted matrix corresponding to the full consensus RE1 motif.

DOI: http://dx.doi.org/10.7554/eLife.04235.004

Functional		Experiment 1		Experiment 2		Experiment 3
category	Gene	BioT REST	Control	BioT REST	Control	BioT REST	Control
Bait	REST	41		37		27
Corepressor	Rcori	4		8		4
	Rcor2	8	2	19	3	8
	Sin3a	8		11		6
Histone tail	HDAC1	11		12		7	3
modifying	HDAC2	7		9	2	4
enzyme	LSD1	18		26		21	3
	Prmt5	2		2		2
	Wdr5	3		2
	Ehmt2/G9a	5		18		8
	Ehmti			4		6
	Wiz			4		6
Adaptor	Cdyl	5		5		3
	Cdyl2	3		3
Chromatin	Smarca5	3		4		5	2
remodeler	Supt16h	3		4	4	6
	Ssrpi	3				2
Other	Gata2b	3				2
repressor	MBD3			2		4
F-box	Fbxwi 1	2		9		4
protein	Btrc	2		6
Transposase	Lin28A			4		2
	Trim71	2		2
DNA	Mdd	2				2
binding	Bclafi	2				2
	Utf1	2				3
Unclear	Bxdc2	2		2		3
	D1Pas1	8		4		11
	Gcdh	3		6		2
	Pdcd11	2		4		2
	Pop1	2		2		2
	Wwox	2		4
	Pura			3		2
	Dimti	2		2

Proteins are listed were identified in all streptavidin purifications of biotin-tagged REST (3 out of 3) but not represented in more than one of the negative control samples. Columns list the functional category, protein symbol, and the number of unique peptides detected in REST and negative control purifications.

Figure 1—figure Supplement 1.

REST is required for recruitment of co-factors to RE1 sites in ESCs.

ChIP assays were performed in WT and Rest ESCs using antibody against Sin3a, Cdyl, CoREST1, CoREST2, Hdac2, G9a, and Kdm1a to compare recruitment of endogenous co-factors at the RE1 sites near Igsf21, Kcnk9, Erich1, Npas4, and Rad51. ChIP assays done in parallel with normal rabbit IgG are included as a negative control.

DOI: http://dx.doi.org/10.7554/eLife.04235.006

Figure 1—figure Supplement 2.

Detection of REST binding to PRC2 members is biochemically possible, but a true interaction is unlikely.

(A) Immunoprecipitation was performed with streptavidin beads from biotin-tagged REST ESCs or BirA control ESCs and the demarcated proteins were assayed by Western blot. (B) Previously published ChIP-seq data sets were interrogated over H3K27me3 peaks and REST peaks normalized to the signal found at the H3K27me3 peaks.

DOI: http://dx.doi.org/10.7554/eLife.04235.007

The majority of REST-occupied sites, including promoters, are regulated independently of PRC2

A Polycomb complex was not represented in our analysis of REST complexes, but it was possible that the streptavidin pull-downs might not co-purify ncRNA-mediated associations. Therefore, we compared the genomic distributions of REST and H3K27me3 enrichment to determine whether PRC2 is recruited to REST-bound sites in ESCs. 2136 genomic regions targeted by REST were identified by our ChIP-seq in mouse ESCs, which is comparable to prior REST ChIP-seq studies in human T cells (Johnson et al., 2007) and a different mouse ESC line (Johnson et al., 2008). The DNA sequence of REST-bound regions was analyzed and 96.6% (2064 REST-bound sites) contained either the complete or partial consensus RE1 sequence motif (Otto et al., 2007) (Figure 1—figure supplement 3B). This sequence analysis also showed that the RE1 sequence is the central determinant for REST recruitment because regions with the highest enrichment contained multiple repeats of complete RE1 sites aligned with the same strand orientation (Zhang et al., 2006; Jothi et al., 2008) (Figure 1—figure supplement 3A). Conversely, regions that contained a single right half of the RE1 motif were associated with low levels of enrichment (Otto et al., 2007) (Figure 1—figure supplement 3A). REST-binding and relative enrichment indicated by ChIP-seq were confirmed by ChIP-quantitative PCR analysis for a subset of loci (Figure 1—figure supplement 3C). Although ncRNA-mediated interactions linking REST to PRC2-bound chromatin have been proposed (Tsai et al., 2010), the strong correlation between REST-binding and RE1 DNA sequences suggests that any alternative mechanisms of stable recruitment to chromatin were not prevalent in ESCs. This did not preclude, however, the inverse possibility that REST could recruit PRC2 to chromatin adjacent to RE1 sites. However, assessment of H3K27me3 domains showed that only a small minority (∼3%) of REST-bound sites were associated with significant enrichment of H3K27me3 relative to input, and only 0.5% of H3K27me3-enriched domains were associated with REST binding (Figure 1A). Even if the effective footprint of REST sites was extended 1 kb in both directions, the proportion overlapping with H3K27me3 peaks was only 12.6% of REST peaks and 2.3% of H3K27me3 peaks. Furthermore, our own analysis of ChIP-seq results published previously show that the PRC2 factors Suz12 and Ezh2 bind at extremely low levels, if at all, at REST sites relative to sites of H3K27me3 enrichment (Figure 1—figure supplement 2B). None of these data sets supports a strong functional connection between these distinct complexes, or between RE1 sites and PRC2, in ESCs.

Figure 1—figure Supplement 3.

Characteristics of REST-bound loci.

(A) Regions with multiple RE1 motifs show a strong association with REST. REST ChIP-Seq data displayed as sequence tags aligned to the mouse genome assembly (mm9) viewed in the UCSC genome browser. Green and red hash marks represent sequences matching the forward and reverse strands, respectively. Consensus RE1 sequence motifs are indicated by black hash marks. All views are shown at an equal size relative to the scale bar. (B) FIMO definition of the RE1 motif was derived from ChIP-seq peaks. (C) Relative enrichment of REST measured by ChIP-Seq was confirmed by ChIP-qPCR. ChIP assays were performed using antibody raised against a peptide corresponding to a fragment of mouse REST in WT (red bars) and Rest (blue bars) in ESCs at regions associated with RE1 motifs and regions lacking an RE1 site (Oct4 and MageA8). (D) REST targets associated with trimethylation of histone H3K4 or H3K27 enrichment are preferentially localized near gene promoters. Graph shows the percentage of REST-bound regions that overlap with domains of the bivalent histone modification pattern consisting of H3K4me3 and H3K27me3 enrichment (red bars), H3K4me3 enrichment alone (green bars), or without overlap to either modification (tan bars).

DOI: http://dx.doi.org/10.7554/eLife.04235.008

Figure 1.

PRC2 establishes H3K27me3 in ESCs independent of REST repression.

(A) A limited number of REST-occupied sites are associated with domains of H3K27me3 enrichment in ESCs, even if defined more broadly (+/− 1 kb). (B) H3K27me3 levels are stable in Rest ESCs in the majority of regions targeted by PRC2. The scatter-plot shows the relative enrichment of H3K27me3 ChIP-Seq signal in wild type (WT, x-axis) and Rest ESCs (y-axis) at regions targeted by PRC2 in WT ESCs. (C) As in (B), but at identified REST-binding sites. (D) Chromatin immunoprecipitation analysis showing H3K27me3-enrichment changes at RE1 sites near PRC2-targeted regions in WT and Rest ESCs (* indicates p < 0.05), normalized for H3 density.

DOI: http://dx.doi.org/10.7554/eLife.04235.005

ChIP assays were performed in WT and Rest ESCs using antibody against Sin3a, Cdyl, CoREST1, CoREST2, Hdac2, G9a, and Kdm1a to compare recruitment of endogenous co-factors at the RE1 sites near Igsf21, Kcnk9, Erich1, Npas4, and Rad51. ChIP assays done in parallel with normal rabbit IgG are included as a negative control.

DOI: http://dx.doi.org/10.7554/eLife.04235.006

(A) Immunoprecipitation was performed with streptavidin beads from biotin-tagged REST ESCs or BirA control ESCs and the demarcated proteins were assayed by Western blot. (B) Previously published ChIP-seq data sets were interrogated over H3K27me3 peaks and REST peaks normalized to the signal found at the H3K27me3 peaks.

DOI: http://dx.doi.org/10.7554/eLife.04235.007

(A) Regions with multiple RE1 motifs show a strong association with REST. REST ChIP-Seq data displayed as sequence tags aligned to the mouse genome assembly (mm9) viewed in the UCSC genome browser. Green and red hash marks represent sequences matching the forward and reverse strands, respectively. Consensus RE1 sequence motifs are indicated by black hash marks. All views are shown at an equal size relative to the scale bar. (B) FIMO definition of the RE1 motif was derived from ChIP-seq peaks. (C) Relative enrichment of REST measured by ChIP-Seq was confirmed by ChIP-qPCR. ChIP assays were performed using antibody raised against a peptide corresponding to a fragment of mouse REST in WT (red bars) and Rest (blue bars) in ESCs at regions associated with RE1 motifs and regions lacking an RE1 site (Oct4 and MageA8). (D) REST targets associated with trimethylation of histone H3K4 or H3K27 enrichment are preferentially localized near gene promoters. Graph shows the percentage of REST-bound regions that overlap with domains of the bivalent histone modification pattern consisting of H3K4me3 and H3K27me3 enrichment (red bars), H3K4me3 enrichment alone (green bars), or without overlap to either modification (tan bars).

DOI: http://dx.doi.org/10.7554/eLife.04235.008

(A) Normalized enrichment values for H3K27me3 at defined H3K27me3 peaks were derived from several previously published data sets and plotted against one another. Numbers indicate Pearson coefficient.

DOI: http://dx.doi.org/10.7554/eLife.04235.009

(A) Ezh2 occupancy was increased at RE1 sites within H3K27me3 domains that gained H3K27me3 in the absence of REST. ChIP assays were performed with anti-Ezh2 to compare enrichment at RE1 sites between WT (light orange) and Rest (dark orange) ESCs.

DOI: http://dx.doi.org/10.7554/eLife.04235.010

Despite the limited association between H3K27me3 enrichment and REST binding, we asked whether promoters targeted by both repressive mechanisms (REST and PRC2) represented specific gene classes, because H3K27me3 marks several key developmental factors in ESCs. The dual PRC2/REST-occupied genes were primarily canonical REST-regulated mature neuronal genes rather than pro-neural or developmental genes per se, and therefore showed no ontological category enrichment (data not shown). Developmental regulators of multiple lineages, such as Cdx4 and Runx1, which are associated with the bivalent marks H3K27me3 and H3K4me3 (Mikkelsen et al., 2007), similarly showed no gene ontology differences between those occupied by REST and those that were not. These results suggest that there exists no specific functional class of genes that is regulated by REST and Polycomb in tandem.

To determine whether PRC2 activity at REST-occupied sites, when it did occur, was dependent on REST binding, we asked whether H3K27me3 was lost from these regions in Rest ESCs. We integrated the ChIP-seq signal across both narrow REST-binding domains and across a continuous broad domain to avoid nucleosome occupancy fluctuations due to loss of REST binding. By this analysis, we found that levels of H3K27me3 at defined H3K27me3 sites were largely maintained (Figure 1B, Pearson's coefficient = 0.53). The changes observed between WT and Rest ESCs were very similar to those observed between WT data sets published previously (Figure 1—figure supplement 4). Specifically, we found that >95% of H3K27me3-enriched regions associated with REST-bound sites in WT cells were also enriched for H3K27me3 in Rest ESCs (Figure 1A and C, Pearson's coefficient = 0.85). The number of H3K27me3-overlapping domains was also essentially the same between wild-type and REST knockout ESCs (Figure 1A). Similar results were observed when only REST sites within 5 kb of gene promoters were analyzed (data not shown). In the small number of instances where H3K27me3 levels did change, some genes lost H3K27me3 in Rest ESCs, including Scn8a, Galnt9, and Vgf (Figure 1D and Table 2), while other genes, including Dner, Otop3, and Cacng2, gained H3K27me3 (Figure 1D and Table 2). The losses and gains were validated by quantitative ChIP-PCR (Figure 1D). The HoxA11 and Oct4 genes, which are not bound by REST in ESCs, represent positive and negative controls for the H3K27me3 mark, respectively. The occupancy of EZH2 at these same regions was altered similar to H3K27me3 (Figure 1—figure supplement 5). These results taken together indicated that REST was not required for establishment or maintenance of H3K27me3, throughout the genome generally or at loci targeted by REST specifically. However, in a very limited number of cases, H3K27me3 was lost in Rest ESCs, reflecting either direct or indirect influence of REST on PRC2-binding. The increase in H3K27me3 at select sites may reflect block of PRC2 binding by REST due to close proximity of their binding sites. Why certain dually occupied genes lost or gained H3K27me3 in response to loss of REST was not obvious based on the function of the encoded proteins, but could be related to the timing of expression in vivo.

Figure 1—figure Supplement 4.

H3K27me3 levels from WT and Rest ESCs are as similar as H3K27me3 levels from different published reports.

(A) Normalized enrichment values for H3K27me3 at defined H3K27me3 peaks were derived from several previously published data sets and plotted against one another. Numbers indicate Pearson coefficient.

DOI: http://dx.doi.org/10.7554/eLife.04235.009

Table 2.

REST-associated genes with significant changes in H3K27me3 levels were measured in Rest ESCs

DOI: http://dx.doi.org/10.7554/eLife.04235.011

Gene Symbol	Gene Name	Change in Rest−/− ESC
Prune2	Prune homolog 2	2.1
Fosb	FBJ osteosarcoma oncogene B	2.0
Mast1	Microtubule associated serine/threonine kinase 1	1.8
Celf4	Bruno-like 4, RNA binding protein	1.7
Kiaa1152	Uncharacterized protein C14orf118 homolog	1.7
Dner	Delta/notch-like EGF-related receptor	1.6
Cacng2	Stargazin	1.6
Bdnf	Brain derived neurotrophic factor	1.6
Hes3	Hairy and enhancer of split 3	1.6
Otop3	Otopetrin 3	1.5
A330050F15Rik	Uncharacterized protein LOC320722	1.5
Skor2	SKI family transcriptional corepressor 2	1.4
Nmnat2	Nicotinamide nucleotide adenylyltransferase 2	−1.3
Cnnm1	Cyclin M1	−1.3
Cabp1	Calcium binding protein 1	−1.5
Kcnb1	K+ voltage gated channel, Shab-related subfamily	−1.6
Celsr3	Flamingo homolog 1	−1.7
Mapt	Microtubule-associated protein tau isoform a	−1.7
Bsn	Bassoon	−2.2
Vgf	VGF nerve growth factor inducible	−4.4
Sarm1	Sterile alpha and TIR motif containing 1	−12.1
Galnt9	Polypeptide Gal NAc transferase 9	Loss
Smpd3	Sphingomyelin phosphodiesterase 3	Loss
Scn8a	Na2+ voltage-gated channel, type VIM, alpha	Loss

List of genes located near REST bound regions that were associated with PRC2 in WT ESCs and showed a significant difference in H3K27me3 enrichment relative to Rest ESCs.

Figure 1—figure Supplement 5.

Ezh2-enrichment at REST-bound loci.

(A) Ezh2 occupancy was increased at RE1 sites within H3K27me3 domains that gained H3K27me3 in the absence of REST. ChIP assays were performed with anti-Ezh2 to compare enrichment at RE1 sites between WT (light orange) and Rest (dark orange) ESCs.

DOI: http://dx.doi.org/10.7554/eLife.04235.010

Chromatin marks at the REST-binding site provide a signature for neuronal genes in ESCs

Although the above experiments ruled out a major role for PRC2 in REST-regulated repression, our mass spectrometry results using whole-cell extracts revealed three histone modifying enzymes in the purified REST complex that are often associated with repression: the histone H3K9 methyltransferase, G9a, histone deacetylases (HDACs) 1 and 2, and the histone H3K4me1/2 demethylase, Kdm1a. Chromatin immunoprecipitation analysis showed that G9a recruitment was lost at RE1 sites in Rest ESCs (Figure 1—figure supplement 1), and we observed a significant reduction in the levels of H3K9me2 in the region of the RE1 sites in 12/16 genes (Figure 2A). Two control genes expressed in ESCs but lacking RE1s did not show any change (Figure 2A). Consistent with the above findings, there was no correlation between changes in H3K9me2 in WT and Rest ESCs and changes in H3K27me3 (R2 = 0.002), again underscoring the independence of REST and PRC2 in chromatin remodeling.

Figure 2.

Chromatin modification changes due to loss of REST.

(A) REST-dependent establishment of 2Me-H3K9, measured by ChIP, is impaired at RE1 sites in Rest ESCs irrespective of changes in H3K27me3 levels. Oct4 and MageA8 are genes expressed in ESCs that lack RE1 sites. (B) Increased histone acetylation is detected at most RE1-associated promoters in the absence of REST, irrespective of changes in H3K27me3 levels. Oct4 and Gapdh promoter regions are expressed in ESCs and lack RE1 sites. (C) H3K4me3 enrichment is increased at most RE1-associated promoters in Rest ESCs, independent of H3K27me3 levels (* indicates p < 0.05).

DOI: http://dx.doi.org/10.7554/eLife.04235.012

The presence of HDACs in the REST complex predicted increased H3K9ac enrichment at RE1 sites in Rest ESCs, which we observed in 11/16 analyzed genes, with no change in the controls that lacked RE1 sites (Figure 2B). Importantly, there was no correlation between enrichment of H3K9ac and gain or loss of H3K27me3 (R2 value = 0.056) due to the loss of REST.

Although MLL proteins were not present in the REST immuno-complex, we tested for the presence of the H3K4me3 mark because it is associated with active or ‘poised’ promoters in ESCs in opposition to H3K27me3 (Bernstein et al., 2006) or REST (Ballas et al., 2005). Of 16 genes containing RE1 sites, H3K4me3 was increased significantly in eight of them in Rest ESCs, independent of the presence of the H3K27me3 mark and whether it was altered by the loss of REST (Figure 2C; R2 = 0.029). Thus, in the context of the bivalent hypothesis, although Polycomb is not an active component for REST-regulated genes, the presence or absence of the H3K4me3 mark may be an important aspect of the chromatin signature orchestrated by REST. As further evidence for this, we found that 37% (441/1202) of REST sites within 20 kb of target genes overlapped with H3K4me3 peaks, a number that increased to 62% for those REST sites within 5 kb of the TSS (417/617).

REST-dependent H3K4me3 changes in ESCs coincide with changes in neuronal gene expression

To determine the functional consequences of chromatin modification changes due to the loss of REST repression, we performed RNA-seq on transcripts from WT and Rest ESCs. As expected, numerous REST target genes (binding site identified within 20 kb of the transcription start site) show an expected increase in expression levels in Rest ESCs. However, the expression data show no correlation with changes in H3K27me3 levels, either at the REST-binding site (Figure 3A) or at the TSS (Figure 3B). From this analysis we conclude that even the small H3K27me3 changes observed due to the loss of REST have little effect on the expression of REST target genes, further supporting the functional independence of REST from Polycomb. When REST target genes are further categorized according to their promoter status regarding H3K27me3 and H3K4me3 (Young et al., 2011) in WT ESCs, it is evident that all classes of REST target genes are de-repressed, irrespective of other marks (Figure 3C). In comparison, REST target genes are not de-repressed in Eed ESCs (Ferrari et al., 2014) (Figure 3D), which show drastic decreases in the H3K27me3 mark. Combined, these results support the conclusion that REST is the primary repressor of its target genes and the roles of Polycomb and the H3K27me3 mark are functionally dispensable for its activity.

Figure 3.

REST-dependent changes in expression of REST targets are correlated significantly with REST-dependent changes in H3K4me3, not H3K27me3.

(A) RNA-seq log2(Fold Change) results for Rest ESCs are not correlated with changes in H3K27me3 levels at REST sites or (B) REST target transcriptional start sites (TSS). (C) All REST target genes are de-repressed in Rest ESCs regardless of H3K27me3 or H3K4me3 status. (D) In contrast, REST targets show no transcriptional changes in Eed ESCs, which have highly reduced levels of H3K27me3, and genes with this mark show significant increases in expression (p < 0.005). (E) Changes in H3K4me3 enrichment in Rest ESCs strongly correlate with REST target gene expression changes (p < 0.01). (F) Expression levels of H3K4me3-marked REST target genes are significantly reduced relative to H3K4me3-marked genes and de-repressed in Rest ESCs (*p < 0.05, **p < 0.001).

DOI: http://dx.doi.org/10.7554/eLife.04235.013

Bivalent developmental genes that become activated during differentiation are proposed to lose the repressive H3K27me3 mark but maintain the active H3K4me3 mark. Having shown that the REST and Polycomb pathways were largely independent, we asked which REST-dependent chromatin marks at the ESC stage might influence transcript levels of these targets. To this end, we performed a multiple regression analysis to determine which chromatin changes due to the loss of REST at the ESC stage were most likely associated with the observed expression changes. From this analysis, only the chromatin mark H3K4me3 correlated significantly with the expression changes observed in Rest ESCs (p <0 0.02, Figure 3E). Further support for the importance of the H3K4me3 mark at REST targets is provided by the absolute levels of expression of REST target genes when categorized as above. Specifically, genes occupied by REST, marked either by just H3K4me3 or by H3K4me3 together with H3K27me3, show significantly lower expression levels than non-REST target genes (Figure 3F, p < 0.001 and p < 0.05, respectively). When REST is deleted from ESCs, the expression levels of these H3K4me3-marked REST target genes are increased and approaches that of non-target H3K4me3-marked genes. This REST-dependent repression of H3K4me3-enriched promoters suggests that one of the primary functions of REST in ESCs is to counter RNA Pol II recruitment and maintenance of this activating mark.

REST antagonizes H3K4me3 through the activity of histone deacetylases

Despite functioning independently, Polycomb and REST repressor complexes in ESCs can generate similar downstream molecular effects by blunting H3K4me3 signaling at transcriptionally poised genes required for differentiation to proceed. To identify the mechanism for the increases in H3K4me3 after the loss of the REST complex, we monitored H3K4me3 levels at REST sites in ESCs that were mutant for the co-repressors G9a, Kdm1a, or HDACs (Figure 4A). We used the histone deacetylase inhibitor trichostatin-A (TSA) as a proxy for HDAC loss, due to the redundancy of HDAC family members (Montgomery et al., 2007). Only TSA treatment correlated significantly with increased H3K4me3 (p < 0.01, Figure 4B), consistent with previous studies that have indicated a negative interaction between de-acetylation at lysine 9 by HDAC activity and trimethylation at lysine 4 (Lee at al., 2006b). As expected, we also observed elevated acetylated H3K9 levels at RE1 sites after TSA treatment (Figure 4—figure supplement 1). Additionally, we utilized gene expression data sets published previously to analyze the transcriptional consequences of co-repressor removal. This analysis revealed that REST targets are de-repressed only in the absence of HDAC activity, but not when G9a and Kdm1a are mutated (Figure 4C). By focusing on those genes for which we observed H3K4me3 effects, we also found a significant correlation between the magnitude of the change in RNA levels when REST is deleted and that when histone deacetylase activity is strongly inhibited by TSA (p < 0.005, Figure 4D). These results suggest that REST repression in ESCs is mediated primarily by recruited HDACs that serve as a counterbalance to H3K4me3 levels and basal RNA polymerase II activity, although the nature of the cross-talk between HDACs and H3K4 trimethylation in this context awaits future investigation.

Figure 4.

REST antagonizes H3K4me3 in ESCs through histone deacetylase activity.

(A) Only treatment with the histone deacetylase inhibitor trichostatin-A (TSA) results in the increased H3K4me3 enrichment seen in Rest ESCs. The active Oct4 and GAPDH promoter regions that lack RE1 sites were included as control regions enriched for H3K4me3. (B) Changes in H3K4me3 enrichment at RE1 sites due to the loss of REST are significantly correlated with those due to TSA treatment (p < 0.01). (C) Microarray analysis reveals that HDAC inhibition by trichostatin-A (TSA) preferentially de-represses REST targets, unlike the loss of G9a or Kdm1a. (D) Changes in expression of select REST target genes due to REST loss significantly correlate with changes in expression due to HDAC inhibition with TSA (p < 0.001).

DOI: http://dx.doi.org/10.7554/eLife.04235.014

(A)H3K9ac enrichment is increased after treatment with histone deacetylase inhibitor trichostatin-A (TSA) but not when other co-factors are eliminated from the REST complex.

DOI: http://dx.doi.org/10.7554/eLife.04235.015

Figure 4—figure Supplement 1.

H3K9ac levels increase after TSA treatment.

(A)H3K9ac enrichment is increased after treatment with histone deacetylase inhibitor trichostatin-A (TSA) but not when other co-factors are eliminated from the REST complex.

DOI: http://dx.doi.org/10.7554/eLife.04235.015

Discussion

The Polycomb-mediated bivalent pattern of histone modifications, consisting of H3K4me3 and H3K27me3, has been proposed as a central mechanism for maintenance of a poised transcriptional status in undifferentiated stem cells. However, a study on early zebra fish embryos showed that only 36% of inactive gene promoters were associated with a bivalent histone modification pattern, while 28% exhibited enrichment of H3K4me3 alone, yet remained inactive (Vastenhouw et al., 2010). This suggests that in addition to PRC2, alternative repressor mechanisms exist for recruiting chromatin modifiers to poised genes, although such proteins have not been identified. We propose that the REST/HDAC repressor mechanism represents one such alternative mechanism for genes in the neuronal lineage. By extension, our results indicate that both neuronal cell fate determining genes and neuronal genes expressed later in the differentiation program are poised in ESCs, albeit by different mechanisms.

We identified three classes of REST-bound sites: 1) sites that lacked trimethylation at either H3K4 or H3K27, 2) sites that exhibited the bivalent modification pattern H3K4me3 and H3K27me3, and 3) sites marked by H3K4me3 only (Figure 1—figure supplement 3D). The first class, lacking trimethylation, was preferentially located distal to promoter regions. Preliminary studies on these distal sites show that some overlap with the enhancer mark H3K4me1 and/or H3K27ac (data not shown), potentially indicating a role for REST-directed repression at specified distal enhancer regions, an intriguing hypothesis that awaits future analysis. However, because it is currently unclear which promoters/genes these distal sites regulate, we focused on the other two subclasses, which were located within 20 kb of annotated TSSs. The majority of these REST-associated regions were enriched for H3K4me3, consistent with our idea that the balance of REST-recruited HDACs and H3K4me3 was sufficient to poise neuronal genes independent of Polycomb.

The REST-bound promoters with H3K27me3 raised the possibility of a functional link between REST and PRC2 in ESCs at these sites. Additionally, a previous report that the ncRNA HOTAIR can link REST and PRC2 suggested that ncRNA-mediated interactions in ESCs could result in PRC2 recruitment to REST-bound RE1 sites, and conversely, that REST complexes could be recruited to PRC2 bound regions independent from recognition of the RE1 motif (Tsai et al., 2010). Our results are not consistent with either of these scenarios. First, REST-binding sites within the ESC genome appeared to be dependent exclusively on the underlying DNA sequence, because REST binding was correlated strictly with the presence of RE1 motifs. Second, the Polycomb complex member Eed, which is required for H3K27me3 deposition (Montgomery et al., 2005), was absent from the REST complexes characterized by mass spectrometry and co-immunoprecipitation, undermining the likelihood of either repressive complex directly targeting the other. Third, only a minority of REST-bound sites was associated with H3K27me3 enrichment and PRC2 localization (∼3%). Moreover, H3K27me3 was not preferentially enriched at regions with multiple RE1 sites and thus did not show strong association with REST. Finally, more than 97% of the RE1 sites associated with PRC2 in wild-type cells also showed H3K27me3 enrichment in Rest ESCs, including at gene promoters, indicating that REST was not required for PRC2 recruitment at these regions. Taken together, we conclude that REST and PRC2 act largely independently, even at shared target genes, in ESCs.

The term ‘developmental regulators’ has been used to describe Polycomb targets in ESCs (Boyer et al., 2006; Lee et al., 2006a). Therefore, we considered the possibility that the small subclass of REST/PRC2 targets might represent a specialized set of genes for promoting neural development. Gene ontology analysis, however, revealed no apparent distinction between biological functions in this subclass and the biological functions associated with the REST pattern alone. Both subclasses contained genes known to influence neurodevelopment, many of which persist in the adult nervous system, as well as other categories considered to be late neuronal genes involved in mature neuronal function, such as synaptic components and voltage-gated channels. Thus, PRC2 does not specifically target regulators of neurodevelopment within the REST-regulated network of genes.

Different criteria used to define and quantify H3K27me3 domains may explain the discrepancies between our conclusions and those of others suggesting that REST mediates PRC2 recruitment in ESCs (Dietrich et al., 2012). A critical distinction is that our analysis defined H3K27me3-enriched regions before comparing the computed H3K27me3 signals between WT and Rest ESCs. By applying this initial binary condition, our analysis avoids the contribution of fluctuations in background signal. We argue that comparing the computed H3K27me3 ChIP-seq signals at all REST sites without considering initial H3K27me3 background signal would always find some level of relationship between REST and PRC2 and therefore eliminate the null hypothesis a priori. Therefore, we limited our comparisons of H3K27me3 domains to regions that also showed the clear presence of PRC2 activity in WT ESCs. Additionally, the loss of REST may generate small but reproducible effects in measured histone modifications due to local changes in nucleosome density, rather than actual changes in a specific modification (Zheng et al., 2009). Similar to a recent study that showed the presence of REST evicts nucleosomes at RE1 DNA sequence motifs (Valouev et al., 2011), we evaluated in vivo nucleosome positioning in ESCs and found that phasing of nucleosomes centered at RE1 motifs was displaced in Rest cells (data not shown). Therefore, although it can appear that PRC2 activity is increased specifically at RE1 sites by the loss of REST (Dietrich et al., 2012); this likely reflects a secondary consequence of the gain of a nucleosome at the RE1 site, due to the loss of the REST protein and subsequent fill-in of its footprint by a single histone octamer. How REST-associated nucleosome positioning generally affects gene expression is not yet known, but there are well-documented examples in lower eukaryotes of dynamic nucleosome positioning as a mechanism of gene regulation (Bai and Morozov, 2010).

In addition to maintaining nucleosome-depleted regions, our results indicate that REST likely counterbalances RNA Pol II activity primarily through recruitment of histone deacetylase activity in undifferentiated cells, echoing results observed in human T cells (Zheng et al., 2009). Acetylated histone tails have been shown to interact with bromodomains of transcription factors, such as Brd4, which promotes recruitment of Mediator complexes or positive transcription elongation factor b (P-TEFb) and release of paused RNA Pol II (Jang et al., 2005; Yang et al., 2005; Wu and Chiang, 2007). These interactions may explain the observed dependence of H3K4me3 on TSA and H3K9 acetylation.

The net transcriptional effect on genes in Rest ESCs was variable and depended on the locus (Johnson et al., 2008; Jorgensen et al., 2009), which is likely due to specific activators being present or absent in ESCs, as well as additional repressive mechanisms that may be acting at the same target. However, changes in the H3K4me3 mark (and histone acetylation) due to the loss of REST were significantly correlated with changes in gene expression, while the other histone modifications we analyzed were not. This suggests that REST-directed repression of H3K4 methyltransferases or activation of H3K4me3 demethylases is important to restrict the amount of expression from these genes. A potential candidate demethylase is SMCX (Jarid1C), which binds REST in HeLa cells and can regulate promoter H3K4me3 levels (Tahiliani et al., 2007), although we found no evidence of SMCX binding in our mass spectrometry results. In addition, there is evidence that H3K4me3 ‘primes’ non-expressed genes for acetylation and increased gene expression after histone deacetylase loss (Wang et al., 2009; Lopez-Atalaya et al., 2013). Thus, as neuronal differentiation proceeds and REST/HDAC levels on target chromatin decrease dramatically, those genes previously marked with H3K4me3 increase this mark simultaneously with H3K9 acetylation in a rapid feed-forward mechanism.

Based on our results, we propose that the loss of the REST or Polycomb repressor complexes from different sets of genes, in conjunction with the recruitment of transcriptional activators, allows for finely tuned, graded expression changes over the course of differentiation. In stem/progenitor cells, REST is a key repressor of genes crucial to the terminally differentiated neuron, while PRC2 is a repressor of a REST independent pathway regulating pro-neural genes that are required at earlier differentiation stages (Mohn et al., 2008). Finally, ‘terminal selector’ genes, which are transcriptional activators in mature neurons, also drive their own expression to maintain the terminally differentiated phenotype (Hobert, 2011). In a similar but reversed case, the REST gene, which itself contains a REST-binding site, may function to reduce its own expression so that differentiation can proceed unidirectionally.

It will be interesting in the future to see whether repressors in other cell lineages play similar roles in poising terminal genes in stem/progenitor cells. A recent study has suggested that structural genes encoding mature cardiac cell functions are regulated primarily by transcriptional activators rather than by H3K27me3 (Paige et al., 2012). An alternative possibility, based on our study, is that these temporally delayed cardiac genes are repressed by factors, still to be identified, which recruit chromatin modifiers other than Ezh2 in order to balance the activation mark in stem cells. In neurons, direct reprogramming can occur by introducing pro-neural (Ascl1) along with terminal genes (e.g. Myt1l) into somatic cells (Vierbuchen et al., 2010) perhaps because they represent distinct regulatory pathways. Better knowledge of the factors regulating terminally differentiated gene chromatin could provide insight into the mechanisms underlying direct reprogramming of fibroblasts into different types of cells (Nam et al., 2013).

Materials and methods

Construction of Flag-BioT-mREST (pFBmR)

Mouse REST (mREST) CDS, lacking the start and stop codons and flanked by BamH1 sequences, was amplified from pcDNA3.1A(−)-mREST-Myc-His (Mandel, unpublished) using the following PCR primers: 5′- GCG CGG ATC CCC ACC CAG GTG ATG GGG CA -3′ (JL70112a) and 5′- GCG CGG ATC CCT ACT CCT GCT CCT CCC GC -3′ (JL70705a) (underlined are BamHI sites). The fragment was cloned into a TOPO-TA vector, released by BamH1, and then cloned in frame into the BamH I site in pEFrFLAG-BIOpGKpuropAv1 (pFL-Big) (Wang et al., 2006), kindly provided by Jianlong Wang and Stuart Orkin (Harvard Medical School).

Generation of a Flag-BioT-REST-expressing ESC cell line

The pFL-Big plasmid was kindly provided by Jianlong Wang and Stuart Orkin (Harvard Medical School), the N6 and N8 ESC lines were provided by Zhou-Feng Chen, (Washington University in St. Louis). Plasmid pFBmR was linearized with Sca I and transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) into BirA-J1 ES cells that stably express the Escherichia coli Bir A ligase (Wang et al., 2006) (kindly provided by Jianlong Wang and Stuart Orkin) and maintained in 15% FBS in DMEM (#11965; Gibco) supplemented with penicillin/streptomycin, 2 mM L-glutamine, non-essential amino acids (#M7145; Sigma), 0.1 mM 2-mercaptoethanol, 8 mg/l adenosine, 8.5 mg/l guanosine, 7.3 mg/l uridine, 7.3 mg/l cytidine, 2.4 mg/l thymidine, and 10 U/ml LIF (# ESG1107; Chemicon/Millipore) on tissue culture plates coated with 0.1% gelatin (Sigma). Stable BioT-REST expressing cells were selected in 2 µg/ml puromycin and individual clones were hand picked under a microscope. The clones were then screened by Western blot analysis for REST protein level, using an antibody raised against the C-terminus of hREST (Ballas et al., 2005). One clone expressing REST at levels ∼5 fold that of endogenous REST (clone #60) was used in the streptavidin pull-down experiment for mass spectrometric analysis.

REST complex purification

ES cells expressing BioT-REST (clone #60) from 10, 15 cm dishes were used for each pull down. The cells were harvested and pelleted, then lysed in 2.5 ml cold lysis/binding buffer (0.5 mM EDTA, 150 mM NaCl, 0.5% Triton X-100, 10% glycerol, 1 mM NaF, 1 mM Na3VO4, 0.5 mM DTT in pH 7.5, 50 mM Tris–Cl with 1× Roche complete protease inhibitors cocktail) with the help of sonication on ice (4 rounds of 20 strokes, output 4, 40% duty cycle, Sonifier). The cell lysate was cleared by centrifuge at 4°C and incubated with buffer-exchanged 200 µl streptavidin M-280 magnetic beads (Dynal beads/Invitrogen) at 4°C for 3 hr in lysis/binding buffer. After the incubation, the beads were pelleted and washed three times with 1 ml cold lysis/binding buffer and three times with 1 ml cold PBS. The beads were then eluted twice with 200 µl and 100 µl elution buffer (1:1 (vol/vol) acetonitrile/H2O in 0.1% trifluoroacetic acid) at 65°C for 10 min. The two eluates were combined and SpeedVac dried under no heat and subjected to MudPIT analysis as powder. The parental BirA-J1 ES cells, which express E. coli BirA ligase but no BioT-tagged REST, were processed and analyzed in parallel as the negative control pull down.

Proteomic analysis of REST complex

The eluted REST complex was solubilized in 8 M urea containing 10 mM dithiothreitol and incubated at 60°C for 30 min. The solution was cooled to room temperature and iodoacetamide was added to a final concentration of 15 mM and incubated at room temperature for 20 min in dark. The solution was then diluted to a final urea concentration of 2 M with 100 mM Tris–HCl. The proteins were digested with 1 μg of trypsin at 37°C overnight. The digestion was terminated by adding formic acid to 5%, and centrifuged. Half of the peptides containing supernatant were used for liquid chromatography coupled with mass spectrometry analysis to identify proteins. Peptides from each pull-down sample were pressure-loaded onto a 250 µm i.d. fused silica capillary column packed with a 3 cm, 5 µm Partisphere strong cation exchanger (SCX, Whatman, Clifton, NJ) and a 3 cm, 10 µm Aqua reversed-phase C18 material (Phenomenex, Ventura, CA), with the SCX end fritted with immobilized Kasil 1624 (PQ Corperation, Valley forge, PA). After desalting, a 100 µm i.d. capillary with a 5 µm pulled tip packed with a 10 cm, 54 µm Aqua C18 material was attached to a ZDV union, and the entire split-column was placed inline with an Agilent 1100 quaternary HPLC (Agilent, Palo Alto, CA) and analyzed using a modified, six-step multi-dimensional protein identification technology (MudPIT) described previously (Washburn et al., 2001). As the peptides were eluted from the microcapillary column, they were electrosprayed directly into an LTQ linear ion trap mass spectrometer (ThermoFinnigan, San Jose, CA) with the application of a distal 2.5 kV spray voltage. A cycle of one full-scan mass spectrum (400–1400 m/z) followed by 5 data dependent MS/MS scan at a 35% normalized collision energy was repeated continuously throughout each step of the multidimensional separation. The resulting MS/MS spectra were searched with the SEQUEST algorithm (Griffin et al., 1995) against a mouse IPI database (version 3.30, released at 28 June 2007) that was concatenated to a decoy database in which the sequence for each entry in the original database was reversed. The search parameters include a static cysteine modification of 57 amu and no trypsin specificity. The database search results were assembled and filtered using the DTASelect program (Tabb et al., 2002) requiring a protein level false discovery rate less than 1%, all peptides identified are required to be tryptic, and at least two peptides are required for a protein to be identified. Under such filtering conditions, no peptide hit from the reverse database was found.

Cell culture

Mouse ESCs cultures, N6 (WT) and N8 (Rest) (Jorgensen et al., 2009), were cultured in DMEM medium described above. ESCs were cultured on feeder layers of irradiated mouse embryonic fibroblasts and passaged three times on plates coated with 0.1% gelatin to eliminate MEFs before harvesting cells for RNA or chromatin purification.

RNA-seq sample preparation and analysis

Total RNA was extracted using TRIzol (Invitrogen) followed by on-column DNAse treatment with RNase-free DNase and RNesay mini kit (Qiagen). 2 μg total RNA was used to make one sequencing library. Two biological replicates were made for each condition: WT ESC and REST ESC. Indexed libraries were prepared using the Illumina TruSeq RNA Sample Preparation Kit v2 (San Diego, CA). Four libraries were mixed at equal concentration and sequenced by an Illumina HiSeq 2000 sequencer at the OHSU Massively Parallel Sequencing Shared Resource (MPSSR). Reads were mapped using Subread (Liao et al., 2013) and gene counts assigned using FeatureCounts (Liao et al., 2014). Differential expression analysis was performed using edgeR (Robinson et al., 2010) with p-values assessed by both tag-wise and common dispersion analysis. Primary reads and mapped gene counts can be found at GSE59442.

mRNA expression analysis

Total RNA was isolated by disrupting cultured cells with Trizol reagent (Invitrogen) followed by chloroform extraction and ethanol precipitation/wash according to the manufacturer's instructions. For each sample, 1 μg of purified RNA was used as template for first strand cDNA synthesis with random hexamer primers and SuperScript III reverse transcriptase (Invitrogen) following the standard manufacturer’s protocol. cDNA quantities were evaluated by quantitative real-time PCR measuring SYBR Green fluorescence on an ABI 7900HT. Following activation of the hot start polymerase at 95°C for 10 min, reactions were cycled 40 times at 95°C for 15 s and 60°C for 1 min. Experimental cDNA samples were run in triplicate. Primer sequences used for amplification are listed in Table 1—Source data 1. Relative gene expression for genes of interest (GOI) was calculated using the ΔΔCt method and normalized to Gapdh levels to control for variation in reaction inputs. Standard deviation of the normalized expression was calculated as; SD = (normalized value) × ln(2) × √((SDGapdh)2 + (SDGOI)2).

Chromatin Immunoprecipitation (ChIP) analysis

ChIP analyses were performed as described previously (Ballas et al., 2005). Briefly, cells were treated with 1% formaldehyde for 10 min at RT to form protein–DNA crosslinks. Crosslinking reaction was quenched by addition of glycine to a final concentration of 0.125 M and incubating for 5 min at RT, followed by two washes with PBS. Harvested cells were resuspended in nuclei isolation buffer (5 mM HEPES, pH 8.0, 85 mM KCl, and 0.5% Triton X-100) and incubated for 10 min on ice. Pelleted nuclei were resuspended in nuclei lysis buffer (50 mM Tris–HCl, pH 8.0, 10 mM EDTA, and 1% SDS) at an approximate concentration of 107 cells per ml prior to shearing chromatin by sonication to a final size range of ∼100–750 bp. Chromatin lysate was diluted 1:10 with ChIP dilution buffer and specific antibodies were added for overnight incubation at 4°C. The following antibodies were used for immunoprecipitations: anti-H3K4me3 (07-473; Millipore), anti-Ac-H3K9 (H9286; Sigma), anti-2Me-H3K9 (ab1220; Abcam), anti-H3K27me3 (9733; Cell Signaling), anti-H3 (2650; Cell Signaling), anti-REST-C (Ballas et al., 2005; Otto et al., 2007), anti-Ezh2 (5246; Cell Signaling), LSD1 (Kdm1a) antibody from Yang Shi (Harvard Medical School). Protein A conjugated magnetic beads that had been blocked with BSA were used to purify immunocomplexed chromatin fragments by incubating with sample lysates for 3 hr at 4°C. Beads were sequentially resuspended in low salt, high salt, and LiCl wash buffers followed by two final washes in TE buffer. Immunoprecipitated chromatin was eluted from the beads resuspended in elution buffer (50 mM Tris HCl, pH 8.0, 100 mM NaHCO3, 1% SDS, and 200 mM NaCl) during reversal of formaldehyde crosslinks by overnight incubation at 65°C. Elutions were treated with RNase A (1 hr at 37°C) and proteinase K (2 hr at 55°C) prior to a final purification of DNA by column chromatography (Qiagen PCR Purification). Quantities of immunoprecipitated DNA were measured relative to signal from input samples by real-time PCR and analyzed using the ΔΔCt method. Primer sequences used for ChIP analysis are listed in Table 1—source data 1.

Statistical analysis

Data were analyzed using linear regression analysis (Figures 3E, Figure 4B,D), or Student's t-test (all other figures). A threshold of p < 0.05 was interpreted as significant.

ChIP Seq analysis

ChIP-isolated DNA was pooled (three technical replicates done in parallel from each of the two independent biological replicates) and fragments were processed to blunt ends followed by A-tailing to facilitate ligation of Illumina oligo adapters. PCR amplification was run for 12–14 cycles with primers complementary to adapter sequence to amplify the pool of ChIP DNA with addition of the adapter sequence. PCR products in the range of 200–300 bp were isolated by agarose gel electrophoresis followed by gel extraction. 5 ng of sheared DNA purified from chromatin samples without immunoprecipitation was also processed in this manner as an input control. DNA fragments were sequenced using the Illumina Genome Analyzer II platform. The number of unique reads aligned to the mm9 assembly for each ChIP-Seq was: REST (WT ESC) 11,954,736, H3K27me3 (WT ESC) 17,893,323, H3K27me3 (Rest ESC) 14,102,052, and Input (WT ESC) 12,092,824. Raw reads were aligned with Bowtie and only uniquely mapped reads were kept. After alignment, PeakRanger (Feng et al., 2011) and MACS (Zhang et al., 2008) were used to call peaks and the overlap peak set was retained. Overlapped regions may have different boundaries. To identify H3K27me3 peaks conserved between cell lines and called by independent means, we used only those MACS-called peaks that overlapped between our H3K27me3 peak set and those identified by the Encode project (Grant et al., 2011). To find the genomic regions with increased levels of H3K27me3 after REST knock out, the H3K27me3 ChIP in REST knock-out is used as the ‘treatment’ and H3K27me3 ChIP in wild-type as the ‘control’ for both of the two software programs and the same significance threshold was set for both. The data sets were then swapped and analyzed for regions with decreased levels of H3K27me3. To get the reads for histograms shown in Figure 1—figure supplement 3A, the ‘wig’ module of PeakRanger parsed all aligned reads and counted reads within the specified regions. To find the REST-binding motif (RE1), REST peak coordinates were used as input for FIMO (Grant et al., 2011). Previously published ChIP-seq data sets used in the analysis of H3K27me3 were GSE51006 (Ferrari et al., 2014), GSE48172 (Hu et al., 2013), GSE27341 (Arnold et al., 2013), and GSE49431 (Kaneko et al., 2013). Previously published ChIP-seq data set used in the analysis of PRC2 components was GSE49431 (Kaneko et al., 2013). Previously published ChIP-seq data sets used in the analysis of REST complex components were GSE27841 (Whyte et al., 2012) and GSE24841 (Williams et al., 2011). Previously published ChIP-seq data set for H3K4me3 peaks was GSM1003756 (Stamatoyannopoulos et al., 2012). Data were collected as or converted to bigwig format using a combination of BEDtools (Quinlan and Hall, 2010), SAMtools (Li et al., 2009), and analyzed using the bigWigAverageOverBed module from the Kent source tools available from the UCSC genome browser (Kent et al., 2010).

Gene ontology analysis

Gene lists derived from methods above and previous publications (Young et al., 2011) were formatted and uploaded to the AMIGO GO Enrichment tool and analyzed for enrichment in biological processes (Carbon et al., 2009).

Funding was provided by the Howard Hughes Medical Institute and the National Institutes of Health.

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for sending your work entitled “Polycomb and REST-associated histone deacetylases are independent pathways toward a mature neuronal phenotype” for consideration at eLife. Your article has been evaluated by a Senior editor and 3 reviewers, one of whom is a member of our Board of Reviewing Editors, and one of whom, Ramin Shiekhattar, has agreed to reveal his identity.

The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Senior editor has assembled the following comments to help you prepare a new submission.

Although one of the reviewers was positive, two other reviewers raised substantial concerns that preclude acceptance at this time, but we encourage you to read the reviewers’ comments and submit a new manuscript (full comments below). The main issues are related to the biochemistry, where we feel you can easily solve this problem by performing some more experiments as stated by reviewer #1. Reviewer #2 felt that some of the conclusions were not supported by the presented data. Having said this, we believe that a revised manuscript addressing the comments of reviewers 1 & 2 will result in an important addition to the field. The previous data suggesting that Rest and PRC2 physically interact are not conclusive and settling the issue is important, and so we look forward to receive a new manuscript addressing the issues raised.

Reviewer #1:

The manuscript by Oyer et al. reports the observation that different chromatin regulators drive pro-neural and terminal differentiation of neuronal lineages. The authors push the idea that REST and Polycomb are temporally regulated to push the differentiation of ES cells towards a gradually more neuronal fate. In this manuscript the authors describe their attempt to see if there is a link between PRC2 and REST and test the idea of whether these two repressive complexes are working synergistically or in tandem to maintain a more ES cell-like fate and prevent the expression of neuronal genes. The authors used a variety of approaches to identify ESC-specific factors associated with REST, the relationship between H3K27me3 and REST occupancy at target genes and the biological relevance of the loss of REST during differentiation.

The observations presented in this manuscript are interesting, however, there are some points that need clarification and additional experiments to strengthen the claims put forth in the paper. Publication should be pending on whether these points are addressed. The comments of the paper are listed below:

1) During the purification of the REST-associated complexes, the authors do a nice job of validating the candidates by ChIP-qPCR. However, the peptide counts for all of the targets including the bait were relatively low and this may preclude the presence of other interacting protein(s) that may be of interest. More rigorous biochemical purification would benefit the authors in their search for the missing deacetylase, therefore either increasing the number of ESC plates used in the purification should improve the identification of novel factors or the authors should try a different tandem affinity purification strategy to improve the identification of interacting proteins.

2) The use of TSA by the authors as a proxy of histone deacetylation activity should be supported with a figure showing at least the effect of knocking down HDAC1/2, which was identified in from their proteomic analysis.

3) In the Discussion, the authors make a statement regarding the link between REST and PRC2. The statement that “Polycomb family proteins were not present in the REST complexes characterized by mass....” This is not valid reasoning as to why REST and PRC2 are acting independently. These two complexes do not need to be associating together in order to function at same targets.

4) During the purification of the REST complexes, the authors used sonication. This could be a reason why the REST and PRC2 link was not detected by the authors. The association of these two complexes may be nucleic acid dependent and sonication may be disrupting this interaction. The authors need to repeat their purifications using another method to disrupt the cells and cell fractionated to enrich for the nuclear fractions, not whole cell lysates.

Reviewer #2:

In this manuscript, Oyer and collaborators investigated the role of the repressor complex REST in silencing neuronal specific genes in embryonic stem cells (ESCs). Silencing of lineage specific genes in ESCs is often performed by the Polycomb group of proteins. Yet the promoters of those genes are frequently decorated with Polycomb-specific marks (H3K27me3) as well as with active marks (H3K4me3) and are referred to as “bivalent promoters”. Previous works have highlighted the synergistic actions of Polycomb and REST in different cellular model systems (including ESCs; see Science 329, 689, 2010; MCB 31, 2100, 2011; PLOS Gen 8, e1002494, 2011). Oyer and collaborators now report here that the vast majority of REST target genes are not decorated with H3K27me3, and that MS analyses have suggested that Polycomb proteins and REST are not part of the same complex, thus challenging the notion that Polycomb and REST act together in the regulation of neuronal specific genes. They favor a model in which REST acts in combination with HDAC, G9A and KDM1a. Although this topic is of great interest, I find that many of the authors' conclusions are not well supported by the experimental data presented. More stringent and appropriate controls, and more experimental settings, may help to solidify their conclusions.

1) Very surprisingly, the authors do not comment on why there is a dramatic increase (of almost 40%!) of H3K27me3 target genes in Rest-/- cells as compared to wild-type ESCs. Additionally, the bioinformatic analyses of REST ChIP-seq are quite poor overall.

2) It was previously published that REST and Polycomb are co-recruited via the ncRNA HOTAIR. It is thus possible that the PcG and REST do not necessary occupy the same nucleosome. All the analysis performed in Figure 1 is based on a “peak” overlap rather than looking at regions or genes.

3) The authors should also analyze PRC1, since CBX proteins have been demonstrated to interact with REST.

4) The data presented in Table 2 are very confusing. The H3K4me3 ChIP-seq is not mentioned and not properly analyzed in this manuscript, yet it is part of this table. It would be interesting to overlap REST ChIP-seq with H3K4me3 ChIP-seq and include this data to Figure 1.

5) Data presented in Figure 1 should be normalized for nucleosome density (such as for histone H3).

6) The conclusions from Figure 2 are vague and not supported by the data presented. The authors state that “the presence or absence of the 3Me-H3K4 mark is an important part of the chromatin signature orchestrated by REST”. Deletion of Rest affected the H3K4me3 mark of each set of genes in all possible ways.

7) Similarly, for data presented in Figure 3, Rest deletion affected gene expression of ESCs, EBs and neuronal precursors in all possible ways. I couldn't find a common trend in any set of genes. The authors do not describe properly the results obtained or the model system they used. For a reader not familiar with the ESC differentiation protocols, it would be impossible to understand the rationale behind these experiments.

In sum, there is no difference between the 4 groups of genes analysed in Figure 3A. If the authors' only conclusion is that deletion of the Rest repressor leads to an increase H3K4me3, this is in my opinion already well-demonstrated. In any case, the author should comment on the fact that there are no differences in expression of the genes analysed in Figure 3 between wild-type and Rest-/- mature neurons, which is quite surprising.

It is very far fetched to draw any general conclusion when all the analyses performed in Figures 3B-3E are based on 12 genes. And it is unclear why the authors used only 12 of the 14 genes presented in Figure 3A.

8) In my opinion the only interesting (novel) data in this manuscript are those presented in Figure 4B: the authors should start from here and develop this story further!

For example, they should perform this analysis in a genome-wide manner, with overlapping RNA-seq analysis in wild-type and REST -/- cells in the 4 stages (including also mature neurons).

9) Once again, the conclusions of Figure 5 are vague and not supported by the authors' data: “These results suggest that REST repression in ESCs near a TSS is mediated primarily by recruited HDACs that serve as a counterbalance to basal RNA polymerase II activity, and that there is cross talk between HDACs and H3K4 trimethylation”.

The effect of TSA could be completely independent of REST. The correlation with H3K4me3 has nothing to do with REST. This figure only shows correlation between H3K4me3 and histone acetylation.

10) The model presented in Figure 5D is wrong. Sox2 is not “bivalent” in ESCs, it is actually expressed. While is not expressed in neurons.

We submitted previously to eLife a manuscript testing the roles of REST and Polycomb repression in regulating the neuronal lineage in mouse embryonic stem cells. This is an important question because Polycomb has been reported as a general modulator of lineage control in pluripotent cells. As such, many studies focus just on this complex to understand how genes are globally repressed in pluripotency. We provided evidence indicating that the repressor REST is equally efficacious in this role and likely works independently and in parallel for genes expressed in the neuronal lineage. Two of the reviewers indicated that more work was required to substantiate our conclusions, and the editors requested submission of a new manuscript addressing these concerns. We took time to repeat a mass spectrometry study under different conditions, performed new RNA-seq analysis, re-did some of the bioinformatics, and added other new experiments as well. Consequently, this is a much more in-depth and solidified study than the original work and still contains, we feel, the novelty component expected for eLife.

Reviewer #1:

1) During the purification of the REST-associated complexes, the authors do a nice job of validating the candidates by ChIP-qPCR. However, the peptide counts for all of the targets including the bait were relatively low and this may preclude the presence of other interacting protein(s) that may be of interest. More rigorous biochemical purification would benefit the authors in their search for the missing deacetylase, therefore either increasing the number of ESC plates used in the purification should improve the identification of novel factors or the authors should try a different tandem affinity purification strategy to improve the identification of interacting proteins.

Our peptide counts were not dissimilar to other examples in the current literature where novel interacting proteins were identified (Ding, et al, 2012, Cell Res; Costa et al, 2013, Nature), and we identified all known REST interactors using our approach.

However, in response to this concern, we increased the number of plates as suggested and in addition followed the advice in point 4 (below) and performed mass spectrometry analyses using nuclear extracts produced by dounce homogenization, rather than sonicated whole cell extracts as before. Unfortunately, in two biological replicates of the nuclear extract mass spec analysis, we identified primarily nucleolar proteins and no peptides of known REST cofactors, including Rcor1, LSD1/Kdm1a, or G9a, which have been published by several labs as an integral part of a bona fide REST complex and which we identified readily with our previous method. Indeed, to our knowledge no one has identified REST interactors with a REST pull-down using the Dignam method. Therefore, to further test our negative result from the MudPiT analysis we performed a candidate co-immunoprecipitation analysis using nuclear extracts that was not included in the previous submission to confirm these potential interactions. The co-immunoprecipitation analysis confirmed binding of the known interactors of REST but it also identified members of the PRC2 complex Suz12 and Jarid2. However, neither Eed nor Ezh2 were present in the REST pull-downs with this method (new Figure 1–figure supplement 2A). The lack of Eed and Ezh2 is entirely consistent with the lack of REST binding sites marked with H3K27me3, as they are both required for deposition of the H3K27me3 mark. We have included the new results and a discussion of the findings. Finally, we are not sure which deacetylase the reviewer refers to as missing? Perhaps he meant the H3K4me3 demethylase? If the latter, it did not turn up in either of our mass spec analysis methods, indicating that this may be an indirect effect of REST-directed HDAC activity.

2) The use of TSA by the authors as a proxy of histone deacetylation activity should be supported with a figure showing at least the effect of knocking down HDAC1/2, which was identified in from their proteomic analysis.

We have provided a new figure (Figure 4–figure supplement 1) that shows increased levels of H3K9 acetylation at REST binding sites after TSA treatment, which directly inhibits the enzymatic activity of HDACs. We preferred this over the HDAC1/2 knock down experiment because knock down of HDACs is likely to be partial, and we are not sure how we would interpret a negative result. In addition, HDAC1/2 double knockout ESCs shows a profound loss of cell viability (Dovey et al, 2014, PNAS). However, after deletion, these cells show increases in H3K9 acetylation that are similar to those observed in our TSA treated cells. In addition, this report also shows de-repression of several canonical REST targets including neuronal β tubulin, synapsin, and VGF, consistent with our results. We include this new information in the Results section.

With this, we hope the reviewer will give us a pass for the HDAC knock down experiment.

3) In the Discussion, the authors make a statement regarding the link between REST and PRC2. The statement that “Polycomb family proteins were not present in the REST complexes characterized by mass....” This is not valid reasoning as to why REST and PRC2 are acting independently. These two complexes do not need to be associating together in order to function at same targets.

We apologize for our wording. Previous groups had suggested a direct role for REST in recruiting PRC2 to its targets, as well as the possibility that PRC2 directly recruited REST to its targets. Both of these scenarios required biochemical interaction. To better clarify our meaning, we have replaced the sentence in question with the following: “Second, the Polycomb complex member Eed, which is required for H3K27me3 deposition, was absent from the REST complexes characterized by mass spectrometry and co-immunoprecipitation, undermining the likelihood of either repressive complex directly targeting the other.”

4) During the purification of the REST complexes, the authors used sonication. This could be a reason why the REST and PRC2 link was not detected by the authors. The association of these two complexes may be nucleic acid dependent and sonication may be disrupting this interaction. The authors need to repeat their purifications using another method to disrupt the cells and cell fractionated to enrich for the nuclear fractions, not whole cell lysates.

Please see response to point 1, above.

Reviewer #2:

1) Very surprisingly, the authors do not comment on why there is a dramatic increase (of almost 40%!) of H3K27me3 target genes in Rest-/- cells as compared to wild-type ESCs. Additionally, the bioinformatic analyses of REST ChIP-seq are quite poor overall.

Thank you for bringing this to our attention. Addressing this issue further solidified our findings. To address the problem we did the following: First, we went back and completely reanalyzed our results in a manner independent of using Peak Ranger, using MACS only. Secondly, we re-analyzed our H3K27me3 peaks with consideration for how they overlapped with the dataset provided by the ENCODE project’s H3K27me3 ChIP-seq in mESCs, from Bing Ren at LICR. Only those peaks that were called in both our WT dataset and the LICR dataset were deemed ‘true’ H3K27me3 peaks. To ensure that this was an accurate representation of the peaks, we compared the read depth at these sites with four additional published datasets and were satisfied that they were highly correlated and were therefore valid (Figure 1–figure supplement 4). Importantly, with this new analysis, there is no difference in H3K27me3 peaks between WT and REST-/-cells. This result is now entirely consistent with our approach showing lack of correspondence between REST binding sites and PRC2 activity. We have modified the text to state the results and how we did the analysis more clearly.

2) It was previously published that REST and Polycomb are co-recruited via the ncRNA HOTAIR. It is thus possible that the PcG and REST do not necessary occupy the same nucleosome. All the analysis performed in is based on a “peak” overlap rather than looking at regions or genes.

In response to this concern, we extended the REST ‘peak’ 1kb up- and downstream (Broad REST sites) and re-ran the analysis. While the number of overlaps between PRC2 and REST does increase (57 to 270), this quantity is still a very small minority of H3K27me3 sites (2.3%) and REST sites (12.6%). The additional analysis is shown in figure 1A and described in the text.

3) The authors should also analyze PRC1, since CBX proteins have been demonstrated to interact with REST.

In response to reviewer 1 we re-did the proteomics using a different approach and there was still no evidence for PRC1 components in either analysis. We were aware of the CBX result (Ren and Kerppola, 2011, Mol. Cell. Biol.), but it is not interpreted easily because the effects of REST knockdown were opposite depending upon the chromatin context. Further, our current study is focused on PRC2 components and their relationship to REST regulation, a topic of current interest with respect to whether PRC2 alone mediates bivalent chromatin required for pluripotency of all lineages.

4) The data presented in are very confusing. The H3K4me3 ChIP-seq is not mentioned and not properly analyzed in this manuscript, yet it is part of this table. It would be interesting to overlap REST ChIP-seq with H3K4me3 ChIP-seq and include this data to .

Table 2 is no longer included in the manuscript. This is because we took the revision as an opportunity to update the definitions of H3K4me3 and H3K27me3-marked promoters from Mikkelsen et al (2007, Nature) to Young et al. (2011, Nucleic Acids Res.). After including the new definitions, there is no gene ontology difference within REST targets based upon these marks. Regarding the comment about H3K4me3 ChIP-seq, we have overlapped H3K4me3 peaks (Stamatoyannopoulos et al, 2012, Genome Biology) with our REST ChIP seq results and found that 37% of the REST sites located within 20kb of a TSS were also marked by H3K4me3, and 62% of those within 5kb were marked by H3K4me3. We have included this new information and it strengthens our idea that REST targets in ESCs are associated with H3K4me3, as this reviewer already acknowledges in point 7 below.

5) Data presented in should be normalized for nucleosome density (such as for histone H3).

We have conducted histone H3 ChIP and the normalization is now been included for figures 1D, 2A-C, 3E, and 4A, and we note that it has no effect on our conclusions.

6) The conclusions from are vague and not supported by the data presented. The authors state that “the presence or absence of the 3Me-H3K4 mark is an important part of the chromatin signature orchestrated by REST”. Deletion of Rest affected the H3K4me3 mark of each set of genes in all possible ways.

We apologize for the confusion. Our intent was to demonstrate that there is no correlation between the changes observed in H3K27me3 levels (increased, decreased, or unchanged) and the changes in H3K4me3. As the reviewer noticed, the deletion of REST affected the H3K4me3 mark in these H3K27me3-defined classes in all possible ways, indicating that H3K27me3 has no effect on H3K4me3 levels at REST sites. In addition, we have now included more gene promoters in our analysis of H3K4me3 levels after REST deletion (new Figure 4B) and re-analyzed the data. A majority of them gain H3K4me3 (at least 1.5-fold) after loss of REST. We interpret this result to suggest that H3K4me3 is an important part of the chromatin signature of REST.

7) Similarly, for data presented in , Rest deletion affected gene expression of ESCs, EBs and neuronal precursors in all possible ways. I couldn't find a common trend in any set of genes.

The reviewer is exactly right, that there was no trend. We had tried to indicate that the results showed no correlation between changes in H3K27me3 levels and changes in gene expression due to loss of REST. We have now replaced this data with an RNA-seq analysis at the ESC stage showing more clearly this lack of correlation in Figures 3A and 3B.

The authors do not describe properly the results obtained or the model system they used. For a reader not familiar with the ESC differentiation protocols, it would be impossible to understand the rationale behind these experiments. In sum, there is no difference between the 4 groups of genes analysed in .

The reviewer arrived at the correct conclusion, namely that changes in gene expression are not dependent on changes to H3K27me3. To simplify and underscore this result in the resubmission, which will be unexpected to some, we have decided to focus solely on chromatin and gene expression changes at the ESC stage. Indeed, the changes that occur during neuronal differentiation in culture are not likely representative of the in vivo conditions, given the heterogeneity in progenitors and neuronal cell types. For this reason, the differentiation changes might best be studied by single cell transcriptome analysis, which is outside the scope of this study.

If the authors' only conclusion is that deletion of the Rest repressor leads to an increase H3K4me3, this is in my opinion already well-demonstrated. In any case, the author should comment on the fact that there are no differences in expression of the genes analysed in between wild-type and Rest-/- mature neurons, which is quite surprising.

The lack of difference in expression of REST target genes in fully differentiated neurons is not surprising because REST is expressed poorly in these cells; it is down-regulated at terminal differentiation to allow expression of terminal genes. However, as we indicated above, we have now removed this data to make the work more coherent.

It is very far fetched to draw any general conclusion when all the analyses performed in are based on 12 genes. And it is unclear why the authors used only 12 of the 14 genes presented in .

In response to this comment, we did a power analysis to estimate the number of genes we should analyze, given the correlation we observe, to have a falsehood rate < 0.05. Based on this, we extended the analysis to 22 genes and now show that the correlation between H3K4me3 gain and increases in expression are significant (p<0.01, Figure 3E).

8) In my opinion the only interesting (novel) data in this manuscript are those presented in : the authors should start from here and develop this story further!

For example, they should perform this analysis in a genome-wide manner, with overlapping RNA-seq analysis in wild-type and REST -/- cells in the 4 stages (including also mature neurons).

Please see response to point 7 above. We have significantly extended the ESC analysis using RNA-seq, but have eliminated the neuronal differentiation aspect for clarity and in order to focus on PRC2 regulation in stem cells specifically, as the in vitro differentiation may not accurately reflect the in vivo situation.

In regards to the novelty issue, perhaps we obscured some of the novelty for this reviewer in the previous submission. We believe that there is substantial novelty in this work from the point of view of testing the idea, firm in the literature, that Polycomb bivalency is a good proxy for identifying poised genes in all lineages in pluripotent embryonic stem cells, and that PRC2 is required for poising of neuronal genes regulated by REST. In addition, many studies would suggest that simply the presence of polycomb proteins on a site infers repression, and for that reason other repressor mechanisms have been ignored. There are not many repressors that could provide good tests of the bivalent model because the complete set of targets have not been identified, and so it is not easy to discriminate functional significance of the binding. REST is unique: it binds to a unique sequence that allows identity of the binding sites both bioinformatically and by ChIP; the gene targets represent a large set of proteins that are essential for terminal neuronal differentiation, a specific transition during differentiation; and REST is present in ESCs, where these targets are repressed, and minimally expressed at terminal differentiation. Our study shows that Polycomb bivalency is not a general mechanism for poising genes: rather, poising may simply be an active repression mechanism conducted by any repressor (exemplified in this study by REST) balancing RNA Pol II activity, indicated by the H3K4me3 mark. Further, in the neuronal lineage, PRC2 represses proneural genes (Mohn et al, 2008, Mol. Cell, and Burgold et al, 2008, PLoS One), which are not REST targets as identified in our REST ChIP-seq analysis, while REST mediates repression of the later caste of genes required for terminal differentiation, which is a quite considerable number. Our study also demonstrates that in ESCs, the presence of the H3K27me3 mark does not necessarily equate with PRC2-based repression, as we find de-repression after loss of REST even at REST targets that also contain the H3K27me3 mark (e.g. of Calb1 and Glra1 genes). This is an important point and may explain the lack of de-repression observed at some H3K27me3-marked genes in PRC2 mutant cells (Shen et al, 2008, Mol. Cell). We hope the reviewer is more convinced of the novelty component in the new submission.

9) Once again, the conclusions of Figure 5 are vague and not supported by the authors' data: “These results suggest that REST repression in ESCs near a TSS is mediated primarily by recruited HDACs that serve as a counterbalance to basal RNA polymerase II activity, and that there is cross talk between HDACs and H3K4 trimethylation”.

The effect of TSA could be completely independent of REST. The correlation with H3K4me3 has nothing to do with REST. This figure only shows correlation between H3K4me3 and histone acetylation.

We address this concern in several ways. First, we now show a strong correlation between the changes in H3K4me3 due to loss of REST and those due to TSA addition at the specific promoters we have analyzed (new Figure 4B). This correlation suggests that TSA treatment is affecting H3K4me3 levels similarly to loss of REST. Second, we found that a microarray published previously treating ESCs with TSA shows de-repression specifically at REST target genes (new Figure 4C), albeit at lower levels than from loss of REST. Finally, we show that for the genes we analyzed, the change in expression due to TSA treatment was highly correlated with the change in expression due to loss of REST (new Figure 4D). While none of these data prove a direct link between REST, histone deacetylases, H3K4me3, and expression, they all strongly support the model whereby the REST/HDAC complex antagonizes the H3K4me3 mark and subsequent transcription. We have rewritten the Results and Discussion to reflect this idea.

10) The model presented in Figure 5D is wrong. Sox2 is not “bivalent” in ESCs, it is actually expressed. While is not expressed in neurons.

The present focus on ESCs necessitated eliminating this figure.