Allele-specific enhancer interaction at the Peg3 imprinted domain.

The parental allele specificity of mammalian imprinted genes has been evolutionarily well conserved, although its functional constraints and associated mechanisms are not fully understood. In the current study, we generated a mouse mutant with switched active alleles driving the switch from paternal-to-maternal expression for Peg3 and the maternal-to-paternal expression for Zim1. The expression levels of Peg3 and Zim1, but not the spatial expression patterns, within the brain showed clear differences between wild type and mutant animals. We identified putative enhancers localized upstream of Peg3 that displayed allele-biased DNA methylation, and that also participate in allele-biased chromosomal conformations with regional promoters. Most importantly, these data suggest for the first time that long-distance enhancers may contribute to allelic expression within imprinted domains through allele-biased interactions with regional promoters.

Introduction

In eutherians, a subset of genes is expressed mainly from one parental allele due to an epigenetic mechanism termed genomic imprinting; there are about 100–200 imprinted genes in mammalian genomes [1,2]. The mono-allelic expression and parental-allele specificity have been well preserved for most imprinted genes during eutherian evolution, although the biological impetus for this dosage and allele specificity is not fully understood. The majority of imprinted genes are expressed in embryos, placentas and brain, and genetic studies have further demonstrated roles in controlling fetal growth rates and maternal-caring behaviors [1,2]. The majority of imprinted genes are found only in the eutherian mammals, and it has been conjectured that genomic imprinting may have emerged in the eutherian lineage to cope with viviparity and placentation [3-6]. Imprinted genes tend to be clustered in specific regions of chromosomes, forming imprinted domains, which are regulated through small genomic regions termed ICRs (Imprinting Control Regions). Genetic studies have also demonstrated that mutations within these ICRs usually disrupt the imprinting and transcription of the individual genes [1,2]. It is, however, currently unknown what other types of DNA elements may be involved in the allelic expression of imprinted genes besides the known ICRs.

The Peg3 imprinted domain is localized in a 500-kb genomic interval in human chromosome 19q13.4/proximal mouse chromosome 7 that is evolutionarily well conserved among mammals [7-10]. This domain contains paternally expressed Peg3, Usp29, Zfp264, APeg3 and maternally expressed Zim1, Zim2, Zim3 (Fig 1) [10]. As seen in other domains, the imprinting of this domain is controlled through an ICR, the Peg3-DMR (Differentially Methylated Region), which encompasses a 4-kb genomic interval containing the bidirectional promoter for Peg3 and Usp29 [11]. According to the results from a mutant allele termed KO2, deletion of this ICR results in complete abrogation of the transcription of paternally expressed Peg3 and Usp29, and the concurrent biallelic expression of Zim1 through reactivation of its paternal allele [12]. The maternal-specific DNA methylation of this ICR is established during oogenesis through unknown transcription-mediated mechanisms involving an oocyte-specific alternative promoter called U1, which is localized 20-kb upstream of the Peg3-DMR [13]. Maternal deletion of this upstream promoter causes complete removal of oocyte-specific DNA methylation on the ICR, resulting in biallelic expression of Peg3/Usp29 [14]. Overall, this series of studies demonstrated that the imprinting of the Peg3 domain is mediated through the Peg3-DMR and the U1 alternative promoter.

Fig 1

Generation of the Peg3/Zim1 mutant with switched active alleles.

(A) The genomic structure of the mouse Peg3 imprinted domain. The paternally and maternally expressed genes are indicated with blue and red, respectively. The direction of each gene is indicated with an arrow. The 4-kb Peg3-DMR is indicated with a grey box, while the oocyte-specific U1 alternative promoter is indicated with a small arrow. (B) Breeding scheme. Female U1 heterozygotes were crossed with male KO2 heterozygotes, deriving four possible genotypes. Among these four genotypes, shown are the two genotypes: WT and U1/KO2 with the maternal and paternal expression of Peg3 and Zim1, respectively. The schematic representations for the second and third groups have been omitted for simplicity.

Besides the two regulatory regions, the Peg3 domain also contains a large number of evolutionarily conserved regions (ECRs) that are localized in the middle 200-kb transcribed region of Usp29 [15,16]. These ECRs are usually marked with two histone modifications, H3K4me1 (mono-methylation on lysine 4 of histone 3) and H3K27ac (acetylation on lysine 27 of histone 3), suggesting potential enhancer roles for the transcription of Peg3 domain. One ECR, ECR18, has been shown to physically interact with the promoter of Peg3/Usp29, and also recently demonstrated to be a shared enhancer between the paternally expressed Peg3/Usp29 and the maternally expressed Zim1 [17]. However, the functional contribution of the majority of these ECRs to the imprinting of the Peg3 domain has not been examined. Recently, we have been able to derive a mouse mutant line with the switching of active alleles, which is driving the maternal expression of Peg3/Usp29 and the paternal expression of Zim1 [18]. Interestingly, initial investigation of this mutant revealed differences in the expression levels and patterns of genes in the Peg3 domain. In the current study, we further characterized these expression differences with various experimental approaches. The results suggest that the putative enhancers localized upstream of Peg3 may be responsible for the observed differences. Further, these enhancers are allele-biased, suggesting that they contribute to allele-specific expression within the Peg3 domain.

Results

Generation of the Peg3/Zim1 mutant with switched active alleles

To generate the mutant with switched active alleles, we used the following mouse breeding scheme involving two mutant alleles, U1 and KO2, targeting the U1 alternative promoter and the Peg3-DMR, respectively (Fig 1). Maternal and paternal transmission of U1 and KO2 mutant alleles is predicted to derive the following 4 genotypes. The first group, referred herein as ‘WT’, inherits the WT allele from both parents, thus maintaining the normal paternal and maternal expression of Peg3/Usp29 and Zim1, respectively (Fig 1B). The second group inherits maternal WT and paternal KO2, subsequently no expression of Peg3/Usp29 but biallelic expression of Zim1. The third group inherits maternal U1 and paternal WT, thus biallelic expression of Peg3/Usp29 but no expression of Zim1. Finally, the fourth group, referred as ‘U1/KO2’, inherits maternal U1 and paternal KO2, deriving the maternal and paternal expression of Peg3/Usp29 and Zim1, respectively, which are opposite to the parental-specific expression of the imprinted genes in the WT group (Fig 1B). With this breeding scheme, 10 female heterozygotes for U1 were crossed with 5 male heterozygotes for KO2, producing a total of 158 pups for 18 litters with the average litter size being 8.72. According to the initial results, these four genotypes were all equally represented among the surviving pups, suggesting no major embryonic lethality associated with any of these four genotypes [18]. Among these four groups, the pups belonging to the two groups, WT and U1/KO2, were further analyzed in the current study: 32 pups (15 females and 17 males) for WT and 36 pups (21 females and 15 males) for U1/KO2 (Fig 1B). More detailed results regarding the breeding experiments are available from the previous study [18].

Expression level comparison between WT and U1/KO2

The two groups of pups were analyzed in the following manner. We performed a series of qRT-PCR analyses to compare the expression levels of Peg3 and Zim1 between WT and U1/KO2 mice (Fig 2). This series of analyses did not include the remaining 5 imprinted genes, Usp29, APeg3, Zim2, Zim3 and Zfp264, mainly due to their very low expression levels in the adult tissues that had been selected for the current study. A set of 15 tissues was harvested from each of the 8 adult 2-month-old mice, representing two WT and two U1/KO2 with both sexes (WT-F and WT-M, UK-F and UK-M with UK used as an abbreviation for the U1/KO2 genotype in Fig 2). We isolated total RNA and prepared cDNA from the following tissues: hypothalamus, olfactory bulb, cortex, cerebellum, kidney, and gonadal fat (Fig 2 and S1–S3 Files). The results derived from this series of expression analyses are summarized as follows.

Fig 2

Expression level comparison of Peg3 and Zim1 between WT and U1/KO2.

Expression levels of Peg3 and Zim1 were compared between WT and U1/KO2, in short referred to as ‘UK.’ For this series of analyses, four individual sets of WT and UK were used for isolating total RNA and subsequent cDNA synthesis, which were then used for qRT-PCR analyses. Expression levels of each gene was first normalized with the levels of β-actin, and later compared between WT and UK. The results sets derived from the hypothalamus (A) and olfactory bulb (B) were presented with the average values and standard deviations. The statistical significance is indicated in the following manner: *, p < 0.05; **, p < 0.01; and ***, p < 0.001.

First, the expression levels of Peg3 were 12% higher in the hypothalamus of U1/KO2 than WT (Mann-Whitney U test, p-value < 0.00001; Fig 2A). This was also the case for Zim1, exhibiting 27% higher expression levels in U1/KO2 than in WT (Mann-Whitney U test, p-value < 0.00001). Second, the expression levels of Peg3 and Zim1 were lower in two other regions of the brain, including the olfactory bulbs and cerebellum. In the case of olfactory bulbs, the expression levels of Peg3 and Zim1 were 12% and 15% lower in U1/KO2 than in WT (Mann-Whitney U test, p-value = 0.03 for Peg3 and p-value = 0.0012 for Zim1; Fig 2B). This was also the case for cerebellum with Peg3 and Zim1 showing 22% and 18% lower levels in U1/KO2 than in WT (Mann-Whitney U test, p-value < 0.00001 for Peg3 and p-value = 0.0455 for Zim1; S1 File). On the other hand, the expression levels of Peg3 and Zim1 in cortex were indistinguishable mainly due to large variation among individuals with the same genotype and sex (S1 File).

Third, compared to the different brain regions, expression level differences of Peg3 and Zim1 between WT and U1/KO2 were neither obvious nor uniform in the other adult tissues, including kidney and gonadal fat (S2 File). In the case of these two tissues, the male set seemed to show some difference between WT and U1/KO2, whereas the female set did not yield any major difference. In the male kidney, the expression levels of Peg3 and Zim1 were significantly higher in U1/KO2 than in WT (26% for Peg3 and 61% for Zim1). In the male gonadal fat, on the other hand, the expression levels of Peg3 and Zim1 were significantly lower in U1/KO2 than in WT (37% for Peg3 and 43% for Zim1; S2 File). Although the observed differences in these two tissues were more dramatic than those observed from the brain regions, the expression levels of Peg3 and Zim1 were 10 to 100-fold lower in these non-neuronal tissues than in the brain regions based on the relative Ct (threshold cycle) values detected through qRT-PCR analyses. Thus, the differences observed in the brain regions might be still more functionally significant than those from the non-neuronal tissues. Fourth, we also analyzed the expression levels of several genes that are expressed and closely linked to the functions of Peg3, including Avp, Oxt, Oxtr and Ghrh in the hypothalamus set (S3 File). The expression levels of these genes were all higher in U1/KO2 than in WT, although this pattern was detected mainly in the female set. This sex-biased pattern agrees with those observed from the previous study, showing more obvious up-regulation of these genes in the female set [18]. Overall, the expression levels of Peg3 and Zim1 displayed clear differences between WT and U1/KO2 in several adult tissues, particularly in the three regions of adult brain, including the hypothalamus, olfactory bulbs and cerebellum.

Spatial expression pattern of Zim1 between WT and U1/KO2

The known functions of Peg3 are closely associated with the physiological roles played by the hypothalamus, including milk provision, neonatal growth, and nurturing behaviors [19-22]. The expression levels of both Peg3 and Zim1 in the hypothalamus are also the highest among the different regions of the adult brains. Thus, we previously performed an initial series of immunostaining experiments to monitor the spatial expression patterns of Peg3 with the sectioned samples prepared from the four genotypes. The data revealed the overall ubiquitous expression of Peg3 within the adult brains, with no major difference in the spatial expression patterns among the 4 genotypes [18]. To expand on these previous results, we performed a similar series of immunostaining experiments to monitor the spatial expression patterns of Zim1, which are known to be more restricted than those of Peg3 in terms of developmental stage and tissue specificity [23]. This series of immunostaining experiments used a set of 8 adult 2-month-old mice representing each of the four genotypes with both sexes. The harvested brains were sectioned and subsequently immunostained with an anti-ZIM1 polyclonal antibody (Fig 3 and S4 File). The results from this series of analyses are as follows. First, the main expression sites of Zim1 within the hypothalamus include the paraventricular nucleus (PVN), supraoptic nucleus (SON), and the ependymal cells of the 3rd ventricles. The detection in the ependymal cells appeared to be unique to Zim1, since similar expression patterns have not been observed from the immunostaining with anti-PEG3 antibody [18,24]. In contrast, the expression patterns within the PVN and SON were very similar to those observed from Peg3. This similar pattern between Peg3 and Zim1 also supports the prediction that these two genes may share long-distance enhancers. According to the surveys of the ENCODE dataset and also our own results [16], the middle 200-kb region has the majority of potential enhancers within this imprinted domain. Thus, we believe that the shared enhancers are likely localized within the middle 200-kb region. (Fig 4) [15,16]. Second, the spatial expression patterns of Zim1 were also similar with no major difference between WT and U1/KO2: both groups displayed the highest expression within PVN, SON and the ependymal cells (Fig 3). Thus, the spatial expression patterns of Zim1 within the hypothalamus were not affected by switching of the active allele, from maternal to paternal. This further suggests that allele switching does not disturb the transcriptional programs that direct the spatial expression pattern of Zim1, at least within the adult hypothalamus.

Fig 3

Spatial expression patterns of Zim1 within the hypothalamus of WT and U1/KO2.

Immunostaining of ZIM1 (green) and OXT (red) were performed using the hypothalamus prepared from a set of 2-month-old mice of WT and U1/KO2 with two sexes (A-D). OXT-immunoreactive neuronal cells are detected mainly within the PVN (Paraventicular nucleus) and SON (Supraoptic nucleus) areas of the hypothalamus. ZIM1-immunoreactive neuron cells are also detected within the similar areas of the hypothalamus with higher levels being detected in PVN, SON and the ependymal cells of the 3rd ventricles. However, there was no major difference between WT and U1/KO2.

Fig 4

DNA methylation levels of several ECRs localized upstream of Peg3.

The schematic diagram shows the relative positions of 19 ECRs (Evolutionarily Conserved Regions) within the mouse Peg3 domain (upper panel). The neonatal brain of F1 mice derived from the crossing of C57BL/6J (B6) females and PWD/PhJ (PWD) males were used for isolating genomic DNA, which were then used for the analyses of DNA methylation levels. The DNA methylation levels of six individual ECRs were summarized and presented with open and closed circles indicating unmethylated and methylated CpG sites (lower panel). The methylation levels of each allele for a given ECR were calculated and presented. In the case of ECR19, a presumable SNP was not found from the F1 hybrid that had been used for the current study, thus the results were presented without allele sorting.

DNA methylation levels of ECRs

To further characterize the expression difference of Peg3 and Zim1 between the two genotypes, we tested potential involvement of the 19 ECRs that are localized in the 200-kb upstream region of Peg3 (Fig 4). As an initial step, we measured the allele-specific DNA methylation levels of the ECRs with the following strategy. In brief, F1 hybrid mice derived from the crossing of male PWD/PhJ and female C57BL/6J were used to differentiate the maternal and paternal allele using single nucleotide polymorphisms (SNPs) detected between the two strains. Genomic DNA isolated from neonatal brains was first treated with bisulfite conversion protocol [25,26], and subsequently amplified with the 6 individual sets of primers targeting the following ECRs: ECR2, ECR3, ECR9, ECR11, ECR17, and ECR19. The remaining ECRs were not included in the current survey, since they do not have more than 3 CpG sites or SNPs between C57BL/6J and PWD/PhJ. The amplified PCR products were individually cloned and sequenced. The results are summarized as follows. First, ECR2, ECR3, ECR9 and ECR19 displayed relatively low levels of DNA methylation in neonatal brain, ranging from 8 to 37%. In contrast, ECR11 and ECR17 showed high levels of DNA methylation, ranging from 64 to 98%. Second, ECR2 and ECR17 exhibited different levels of DNA methylation between two alleles. Both showed higher levels of DNA methylation in the maternal than paternal alleles: 37% vs 27% for ECR2 (Mann-Whitney U test, p-value = 0.07078) and 83% vs 64% for ECR17 (Mann-Whitney U test, p-value = 0.0476). These methylation level differences for ECR2 and ECR17 appeared to be significant, although the functional relevance is currently unknown. Overall, some of the ECRs maintain different levels of DNA methylation between the two alleles, supporting the possibility that this subset of ECRs may contribute to the allele-specific expression of the imprinted genes in the Peg3 domain.

Chromosomal conformation comparison between WT and U1/KO2

We further tested potential involvement of ECRs in the allele-specific expression of the Peg3 domain with the Chromosomal Conformation Capture protocol (3C), in which potential interactions between promoters and enhancers can be determined for a given genomic interval [27,28]. For this series of experiments, we used the following strategy. We designed a set of oligonucleotides that are derived from the regions 100 bp-downstream of NcoI sites, which were then named after the adjacent ECRs (vertical blue lines in Fig 5). We also included the two primers that are derived from the promoter regions of Peg3 and Zim1, which were subsequently used as anchor or base primers for PCR amplification (Zim1p and Peg3p primers). The amplification efficiency of these ECR primers with each of two base primers were tested using the template DNA that had been prepared through digestion and re-ligation of the genomic DNA isolated from the two BAC clones covering this 200-kb interval with minimal overlap (upper gel panel in Fig 5) [15]. For the actual 3C experiments, we used three sets of individual tissues—neonatal brains, adult hypothalamus and placenta—harvested from WT and U1/KO2 mice. The 3C libraries from each tissue were first analyzed with a fixed number of PCR cycles (middle and bottom gel panels in Fig 5), which were then analyzed with qPCR. To monitor the quality of each library, we also used an independent set of primers as an internal control, which is designed to detect potential interactions between the promoter and 130-kb upstream enhancer of the Snrpn locus (S7 and S8 Files).

Fig 5

3C analyses on ECRs.

The upper panel details the genomic structure of Peg3 domain, the relative positions of 19 ECRs and associated primers (light blue), and the two base primers (green). The position of U1 is indicated with *. The three gel images on the bottom panels represent the results from the two sets of control experiments with a BAC library, and also from the neonatal brains of wild type (WT) and the mutant with switched active alleles (U1/KO2).

The results derived from this series of 3C experiments are summarized as follows. In WT-neonatal brain samples, the overall enrichment levels of PCR products detecting potential ECR’s interactions with the promoter of Peg3 were generally greater than those with the promoter of Zim1 (middle panels in Fig 5; blue versus red bars in Fig 6A). This may reflect the fact that the promoter activity of Peg3 is stronger than that of Zim1 in vivo. The observed ECR’s interaction with Peg3 and Zim1 are also likely allele-specific: Peg3 on the paternal and Zim1 on the maternal allele. This is based on the results from a set of independent 3C with the neonatal brains of KO2 pups lacking the active paternal allele of Peg3 (Fig 1), which showed no detectable enrichment with the promoter of Peg3, but much greater levels of enrichment with the promoter of Zim1 (S9 File). Also, the enrichment levels with the promoter of Peg3 were quite variable among individual ECRs: three ECRs, ECR17, ECR18, and ECR19, showed much greater levels of enrichment than the remaining ECRs (Fig 6A). By contrast, the enrichment levels with the promoter of Zim1 were overall similar between individual ECRs. Finally, the overall enrichment patterns detected from the WT samples were quite different from those observed from the U1/KO2 samples (Fig 6B). It is notable that the enrichment levels at ECR17, ECR18, and ECR19 become somewhat similar between the promoters of Peg3 and Zim1. On the other hand, the enrichment levels at ECR10 and ECR16 showed much greater levels of interaction with both Peg3 and Zim1 promoters in the U1/KO2 than in WT samples.

Fig 6

qPCR analyses of 3C results.

The results from the neonatal brains of WT and U1/KO2 were further analyzed with qPCR. Relative enrichment levels of each amplicon detecting potential interaction of a given ECR with the promoters of Peg3 (blue bars) and Zim1 (red bars) in each library were first normalized with those derived from the BAC library. Also, successful construction of 3C libraries was monitored through measuring the enrichment level of an independent amplicon measuring the interaction between the promoter and the 130-kb upstream enhancer of Snrpn. The subsequent Ct values were shown upper right.

For each ECR, the enrichment levels of a given promoter were further compared between two alleles (Fig 7). This series of comparisons was feasible, since the two 3C libraries from the WT and U1/KO2 samples displayed a similar range of Ct values, 32.7 and 32.1, respectively, at the internal control Snrpn locus. According to the results, the following four types of enrichment or interaction patterns were observed among individual ECRs. The first type showed greater levels of the enrichment on the paternal allele with the promoters of both genes (A). This type includes ECR7, ECR11 and ECR17. The second type also displayed greater levels of enrichment on the paternal allele but only with the promoter of Peg3 (B). The third type showed similar levels of the enrichment between the two alleles, thus biallelic interaction (C). Finally, the fourth type tended to show higher enrichment levels on the switched alleles with both promoters: the maternal allele for Peg3 and the paternal allele for Zim1. This type includes ECR3, ECR10, and ECR16 (D). In this case, some properties associated with these ECRs might have been affected or changed by the switching of active alleles of two promoters. Thus, these ECRs tend to show greater levels of enrichment with the newly activated promoters: Peg3 on the maternal allele and Zim1 on the paternal allele. On the other hand, the first two types (A, B) tend to maintain greater levels of enrichment on the paternal allele, and thus paternal-biased. These four types of the enrichment patterns were also observed from the samples prepared from the hypothalamus of 2-month-old adult mice, but not from the samples from the placentas (S5–S7 Files). In the case of 14.5-dpc (days post coitum) placenta, the interaction seemed to be mostly biallelic except the paternal-biased enrichment at three ECRs, ECR11, ECR17 and ECR18 (S6 and S7 Files). Thus, the observed four types of enrichment patterns may be tissue-specific, mostly detected in the neuronal tissues. This series of 3C analyses were repeated with two additional sets of neonatal brains, including one female set, which were shown to be reproducible. Overall, the ECRs indeed interact with the promoters of Peg3 and Zim1, and also a subset of ECRs, including ECR7, ECR11, ECR17 and ECR19, tend to interact better or more frequent on the paternal than maternal alleles.

Fig 7

Four different types of the enrichment patterns.

For a given ECR, the enrichment levels representing its potential interaction with the promoters of Peg3 or Zim1 were compared between two alleles, which were summarized with the average values with standard deviations. For a given promoter, the enrichment levels measured from the paternal and maternal alleles were indicated with blue and red bars. The values for the paternal and maternal alleles of Peg3 were derived from WT and U1/KO2, respectively. On the other hand, the values for the maternal and paternal alleles of Zim1 were derived from WT and U1/KO2, respectively. The statistical significance is indicated in the following manner: *, p < 0.05; **, p < 0.01; and ***, p < 0.001.

Discussion

In the current study, we compared the expression levels and patterns of Peg3 and Zim1 in WT mice and U1/KO2 animals with switched active alleles. We found that the expression levels of Peg3 and Zim1 were altered in the U1/KO2 mice, suggesting functional non-equivalence between the two alleles. Follow-up studies further identified ECRs localized upstream of Peg3 that are allele-biased, in terms of both DNA methylation levels and interactions with the promoters of Peg3 and Zim1 (Fig 8). Thus, this suggests that some long-distance enhancers also contribute to the allelic expression of genes within the Peg3 domain, in addition to known DMRs and the promoters.

Fig 8

Schematic representation of allele-biased features of ECRs.

The 500-kb genomic interval of the Peg3 domain harbors paternally expressed Peg3, Usp29, Zfp264 (blue) and maternally expressed Zim1, Zim2, Zim3 (red). The middle 200-kb region is filled with 19 ECRs that are putative enhancers for the transcription of the Peg3 domain. This schematic diagram summarizes the results from 3C results, revealing the presence of several allele-biased ECRs, including ECR7, ECR11, ECR17, and ECR19: the interaction of these ECRs with the promoters of Peg3 and Zim1 is stronger or more frequent on the paternal than maternal allele, thus paternal-biased. This may provide an explanation for the differences observed between WT and U1/KO2 with switched active alleles. Switching the active alleles of Peg3 and Zim1 may disrupt the allele-specific interactions that are already programmed between the promoters and these ECRs, thus causing some effects on the expression levels of the Peg3 domain.

The results from expression profiling demonstrated that two imprinted genes, Peg3 and Zim1, showed clear differences between two alleles (Fig 2). The differences were more obvious within the three regions of adult brain, including the hypothalamus, olfactory bulbs and cerebellum. Interestingly, the expression level changes were not unidirectional among these brain regions: the expression levels of Peg3 and Zim1 were slightly greater in the hypothalamus, but lower in the olfactory bulbs and cerebellum of the U1/KO2 animals (Fig 2 and S1 File). On the other hand, two independent series of immunostaining experiments demonstrated that the spatial expression patterns of Peg3 and Zim1 within the hypothalamus were overall similar between WT and U1/KO2 (Fig 3), suggesting that the switching of active alleles does not interfere with transcriptional programs directing the spatial expression patterns of Peg3 and Zim1. This finding further suggests that the expression level differences occur within the same set of cells, but not from ectopic expression of Peg3 and Zim1 in U1/KO2 mice. Nonetheless, we cannot rule out the possibility that there may be some subtle differences in the spatial expression patterns of Peg3 and Zim1 in U1/KO2 mice, but in a very cell-type-specific manner within the hypothalamus. This is further supported by the fact that the expression levels of the hormonal genes, Oxt, Avp, and Ghrh, of the hypothalamus exhibited differences between WT and U1/KO2 (S3 File). The latter gene in particular is consistent with the previous observation that the switching resulted in the increased and decreased body weights among the females and males, respectively, at weaning age [18], indicating effects of the switching on growth hormone pathways. Although unlikely, however, we cannot rule out another possibility that some of the observed differences might be caused by some unknown effects of the two mutant alleles, U1 and KO2, that have been used for the switching. Overall, the switching of active alleles appeared to cause clear changes in the expression levels of Peg3 and Zim1, and suggested that some regions other than the promoters of Peg3 and Zim1 might differ in activity and function between two alleles.

In particular, our results show that several ECRs localized upstream of Peg3 do indeed differ in terms of DNA methylation and promoter interaction between the two alleles, and that their altered activity may be responsible for the observed differences between WT and U1/KO2 (Figs 4–7). DNA methylation analyses indicated that two ECRs, ECR2 and ECR17, exhibited slightly lower levels of DNA methylation on the paternal than maternal allele (Fig 4). Furthermore, 3C experiments demonstrated that the interaction of four ECRs—ECR7, ECR11, ECR17 and ECR19—with the promoters of Peg3 and Zim1 tend to be stronger or more frequent on the paternal than maternal allele (Fig 7). This suggests that some unknown properties associated with these ECRs may be biased toward the paternal allele (Fig 8). The molecular nature of these paternal-biased properties is currently unknown; however, the bias is likely to be due to epigenetic modifications, since the two alleles of these ECRs are identical in terms of DNA sequence. Consistent with this prediction, these ECRs are indeed associated with two histone marks, H3K4me1 and H3K27ac [15,16]. According to recent studies involving ATAC-seq, the open chromatin structure of some of these ECRs turns out to be also allele-biased in the neural precursor cells: maternal-biased open structure for ECR2 and ECR17 and paternal-biased open structure for ECR19 [29]. Interestingly, the pioneer factor MyoD, a well-known E-box-binding protein, has been shown to bind to a subset of ECRs, yet the list of the target sites identified within the Peg3 domain overlaps quite well with the three ECRs, including ECR7, ECR11, ECR17 [16]. This overlap may not be a simple coincidence, but rather a strong hint suggesting some shared features among these ECRs. In this case, the paternal biased properties described in this study may be one of these shared features. Overall, the identification of the paternal-biased ECRs from the Peg3 domain is a novel and significant finding, opening door to new insights regarding potential mechanisms for the monoallelic or allele-biased expression patterns that are frequently associated with mammalian genomes [30,31].

Materials and methods

Ethics statement

All the experiments related to mice were performed in accordance with National Institutes of Health guidelines for care and use of animals, and also approved by the Louisiana State University Institutional Animal Care and Use Committee (IACUC), protocol #16–060.

Mouse breeding

In the current study, we used two mutant strains that have been previously characterized, Peg3 and Peg3 strains [12,14]. Female heterozygotes for Peg3 were crossed with male heterozygotes for Peg3. The subsequent pups were analyzed in terms of sex and genotype. For genotyping, genomic DNA was isolated from either clipped ears or tail snips by incubating the tissues overnight at 55°C in the lysis buffer (0.1 M Tris-Cl, pH 8.8, 5 mM EDTA, pH 8.0, 0.2% SDS, 0.2 M NaCl, 20 μg/ml Proteinase K). The isolated DNA was subsequently genotyped using the following set of primers: for the KO2 allele, Primer A (5’-TGACAAGTGGGCTTGCTGCAG-3’), B (5’-GGATGTAAGATGGAGGCACTGT-3’), and D (5’-AGGGGAGAACAGACTACAGA-3’); for the U1 allele, P1 (5’-TAGCAAGGGAGAGGGCCTAG-3’), P2 (5’-GGAAGCCTCCATCCGTTTGT-3’), and P3 (5’-AGCACAGCTAGAAATACACAGA-3’). The sex of the pups was determined through PCR using the following primer set: mSry-F (5’-GTCCCGTGGTGAGAGGCACAAG-3’) and mSry-R (5’-GCAGCTCTACTCCAGTCTTGCC-3’).

RNA isolation, cDNA synthesis, and qRT-PCR analyses

Total RNA was isolated from the various tissues of adult mice using a commercial kit (Trizol, Invitrogen) according to manufacturer’s instructions. The total RNA was reverse-transcribed using the M-MuLV kit (Invitrogen), and the subsequent cDNA was used as a template for quantitative real-time PCR. This analysis was performed with the iQ SYBR green supermix (Bio-Rad) using the ViiA 7 Real-Time PCR System (Life Technologies). All qRT-PCR reactions were carried out for 40 cycles under standard PCR conditions. The analyses of the results from qRT-PCR were described previously [32]. Statistical significance of potential difference in expression levels of a given gene between two samples was tested with Mann-Whitney U test (https://www.socscistatistics.com/tests/mannwhitney/default2.aspx). The information regarding individual primer sequences is available as S7 File.

Immunohistochemistry

Each mouse was anesthetized with an intraperitoneal injection of a ketamine/xylazine cocktail (87.5 mg/kg ketamine; 12.5 mg/kg xylazine) at a dosage of 0.1 ml per 20-gram body weight. The animals were then transcardially perfused with 0.1 M sodium phosphate-buffered saline (PBS: pH 7.2–7.4) and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB: pH 7.2–7.4). Mice were decapitated, and heads were post-fixed overnight in the same fixative. Coronal sections were transected from the hypothalamus at 40 μm thickness using a vibratome (Leica VT1200 S, Leica, Mannheim, Germany), and placed in PBS containing 0.5% Triton X‐100 (PBST). The free‐floating brain sections were incubated with an in-house primary antibody against ZIM1 and the primary PS38 antibody against oxytocin-neurophysin (provided by H. Gainer, NIH) at dilutions of 1:1000 and 1:500, respectively. This incubation was carried out in PBST for 48–72 hours at 4°C with continuous gentle agitation. Sections were washed three times with fresh PBST, followed by incubation with goat anti-rabbit antibody conjugated with DyLight 488 (Jackson ImmunoResearch, West Grove, PA) and goat antibody conjugated with DyLight 649 (Jackson ImmunoResearch, West Grove, PA). Both incubations were performed at 1:400 dilution in PBST overnight. The sections were washed three times with PBST, mounted on slides, and cover-slipped with an anti-fading agent that consists of 4.8g PVA, 12g glycerol, 12 mL dH2O, 24 mL 0.2M Tris-HCl, and 1.25g DABCO (1,4-diazabicyclo[2.2.2]octane). Fluorescence images were acquired digitally (Eclipse 80i equipped with a digital camera, DS‐QiMc, Nikon, Tokyo, Japan). ImageJ software was used to process the images in dynamic range with minimal alterations.

DNA methylation analysis

DNA was first isolated from the brains of one-day-old neonates of the F1 hybrid that had been derived from the crossing of female C57BL/6J and male PWD/PhJ. The isolated DNA was treated with the bisulfite conversion protocol [25,26]. The converted DNA was subsequently used as a template for PCR reactions targeting each ECR. The amplified products were individually cloned into a commercial vector, and on average 16 clones for each PCR product were sequenced for its final DNA methylation level. The information regarding the sequences of oligonucleotides for each ECR is available as S7 File.

3C (Chromatin Conformation Capture)

The 3C method was performed as detailed in the Current Protocols in Molecular Biology Handbook unit 21.11 in Supplement 74 [27,28]. In short, the two mouse BAC (bacterial artificial chromosome) clones, RP23-178C5 (Invitrogen) and RP23-117K9 (CHORI), were used for generating the control template libraries. These BACs cover the majority of the Peg3 domain (nucleotide positions 6,610,343–6,929,458 in mouse chromosome 7) with an approximately 9,000 bp overlap (6,793,948–6,802,969). The purified DNA from these two BACs (total 10 μg with an equal ratio) was digested with NcoI, religated, and finally prepared for the control template libraries. For the actual 3C experiments, we harvested the following three tissues of WT and U1/KO2 mice: the one-day-old neonatal brains, the hypothalamus of 2-month-old adult mice, and the 14.5-d.p.c. placentas. Each tissue was homogenized in PBS, crosslinked with 1% formaldehyde for 10 minutes, and finally divided into several fractions at the concentration of 10 mg per aliquot. Each aliquot was used for NcoI digestion, religation, and DNA purification.

For PCR analysis, each primer was designed from a region 100-bp downstream of a given NcoI site. The efficiency and compatibility of a given primer set were tested using serial dilutions of the control template libraries that had been prepared from the two BAC clones. For an initial survey, a fixed number of PCR cycles was performed using each base primer along with a panel of ECR primers. For qPCR analysis, 1 μl of either the control or the 3C libraries from mouse tissues was used as a template with SYBR Green Premix reagents (BioRad). The parameters for PCR are as follows: 95°C for 4 mins, 40 repetitions of the following cycle of 95°C for 15 sec, 65°C for 30 sec, 72°C for 30 sec. A camera capture setting was included after the 65°C step to monitor the formation of PCR product. A melt curve step ranging from 55–95°C with a hold of 10 sec and a temperature increment of 0.5°C was included at the end of the PCR to monitor the quality of PCR products.

This file contains the summary of the expression level comparison of Peg3 and Zim1 in the cortex (A) and cerebellum (B) between WT and U1/KO2.

(TIF)

Click here for additional data file.

This file contains the summary of the expression level comparison of Peg3 and Zim1 in the kidney (A) and gonadal fat (B) between WT and U1/KO2.

(TIF)

Click here for additional data file.

This file contains the summary of the expression level comparison of Avp (A), Oxt (B), Oxtr (C), and Ghrh (D) in the hypothalamus between WT and U1/KO2.

(TIF)

Click here for additional data file.

This file contains the magnified view of the PVN areas of the hypothalamus that have been immunostained with anti-ZIM1 antibody.

(TIF)

Click here for additional data file.

This file contains the summary of the 3C results that have been derived from the hypothalamus of 2-month-old adult mice of WT (A) and U1/KO2 (B).

(TIF)

Click here for additional data file.

This file contains the summary of the 3C results that have been derived from the 14.5-dpc placentas of WT (A) and U1/KO2 (B).

(TIF)

Click here for additional data file.

This file contains the compiled raw data sets that have been derived from qRT-PCR-based expression surveys and also from qPCR-based 3C analyses.

This file also contains the information regarding all the oligonucleotides used for expression, DNA methylation and 3C analyses.

(XLSX)

Click here for additional data file.

This file contains the 3C results demonstrating the interaction of the promoters of Snrpn and Ube3a with several ECRs that are localized upstream of Snrpn.

(TIF)

Click here for additional data file.

This file contains the summary of the 3C results that have been derived from the mutant animals with the paternal deletion of KO2, demonstrating the allele-specific interaction of the promoter of Peg3 with ECRs.

(TIF)

Click here for additional data file.

6 Aug 2019

PONE-D-19-17945

Allele-specific enhancer interaction at the Peg3 imprinted domain

PLOS ONE

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Reviewer #1: This is a novel study of gene regulation at the imprinted Peg3 locus using mouse KO lines that have been described in several previous papers from the same group. The mice harbour two different mutations that together switch allelic expression of imprinted genes on the maternal and paternal alleles. Here, by comparing expression, methylation and enhancer/promoter interactions they infer quantitative differences may exist in the interactions between the promoters and distal enhancer regions that are allele-specific.

The data generally look sound and my main advice is that the authors exercise greater caution in the way they choose to interpret their findings. Although they show some quantitative differences to be statistically significant they are relatively subtle. While these differences might be due to allele-specific in promoter/enhancer interactions involving epigenetic changes this remains to be proven. The observed differences could also be a direct consequence of the two different gross changes that have been introduced to the mutant loci. Thus, I would like to see a clear caveat to this effect included in the discussion

Some specific points:

Introduction (p3, para 1). Is it true to say “The majority of imprinted genes are found only in placental mammals”?

Introduction (p3, para 1). The sentence beginning “The mechanisms through imprinted…” does not make grammatical sense.

Introduction (p3, para 2). In what context is the Peg3 domain “well conserved”, do you mean specifically between mouse and human?

Introduction (p3, para 2). Reference to Figure 1 would be helpful.

Results (p5, para 1). The sentence beginning “The schematic representations…” belongs in the appropriate figure legend.

Results (p6, para 2). The last line, beginning “on the other hand..” should perhaps refer to expression ‘differences’ being ‘indistiguishable’ rather than ‘levels’ being ‘inconclusive’.

Results (p6, para 3). Make clear you are talking specifically about male kidney and male gonadal fat.

Results (p6, para 3). In the sentence beginning “Although the observed differences..” it is not clear whether the comment “at least ranging from 10 to 100 fold…” refers to brain or non-neural tissues. The whole sentence needs a rethink.

Results (p7, para 1). “Fourth we analysed the exp[ression levels…” in which tissues etc?

Results (p7, para 2). “The results from this series…” Remove this unnecessary statement and others that are similar elsewhere.

Results (p7, para 2). You state that Peg3 expression is ubiquitous in adult brain then later that Zim1 is uniquely expressed in ependymal cells; one of these statements must be wrong.

Results (p8, para 1). You note that Zim1 expression is “very similar” to Peg3 in PVN and SON. If Peg3 expression is ubiquitous and Zim1 expression more restricted, ZIm1 expression must form a sub-set of Peg3 expression and I don’t see how this forms good evidence they “share long-distance enhancers”. In addition, you specifically indicate enhancers “localized in the middle 200-kb region” of the domain but this cannot be inferred from expression patterns alone.

Results (p8, para 2). Why give a DNA methylation range (“8-37%” for enhancers with low levels but not for those with high methylation levels? You also state that maternal versus paternal differences “appeared to be significant”, but on what basis (statistical test)?

Results (p9, para 2). Not clear that BAC DNA provides a good control for amplification efficiency for 3C experiments involving native cellular DNA.

Results (p9, para 1). I can’t find any primer details for the 3C experiments. Also, why was the Snrpn control chosen, has it been validated previously, perhaps in a prior publication?

Results (p10, para 1 and 2). Some potentially important data on Zim1 is ‘not shown’ and there is a lack of quantification until Figure 7. It is not clear to me how this comparison has been done, including the stats, and I am struggling to evaluate its validity.

Discussion (p12, par 2). Four ECRs are indicated (ECR7, 11, 17 and 19), which is different to the 3 highlighted on page 10 of the Results (ECR17, 18 and 19).

Discussion (p13, par 1). The section beginning “Interestingly, the pioneer factor MyoD…” and ending with the sentence “Further, this suggests…” comes across as a rather clumsy piece of speculation. I would consider a careful rewrite or omitting this entirely.

Reviewer #2: In the manuscript entitled “Allele-specific enhancer interaction at the Peg3 imprinted domain” by Kim et al. identified putative enhancers localized upstream of Peg3 that displayed allele-biased DNA methylation, and they suggested that some of them participate in allele-biased chromosomal conformations with regional Peg3 and Zim1 promoters and that such long-distance enhancers may contribute to allelic expression of Peg3 and Zim1. In the previous report, the authors reported their interesting double KO mice with switched active alleles of Peg3 and Zim1, that is maternally expressed Peg3 and paternally expressed Zim1 by deletion of both Peg3-DMR (ICR) and oocyte-specific alternative promoter (U1) of Peg3. I agree that their switched mouse is a very good tool for elucidating imprinting mechanism and identification of the paternal-biased ECRs from the Peg3 domain is also a novel and very important finding, however, I don’t think that their results actually support their main conclusion (suggestion) that the long-distance enhancers may contribute to allelic expression of Peg3 and Zim1.

Major comments

1. Imprinted domains are very unique because expression profiles of both of the paternal and maternal alleles are different. Therefore, the authors need to carry out 3C experiment on paternal and maternal alleles separately. The authors insisted that the observed ECR’s interaction with Peg3 and Zim1 are likely allele-specific: Peg3 on the paternal and Zim1 on the maternal allele. However, it is not guaranteed.

In the U1/KO2 mice with maternally expressed Peg3 and paternally expressed Zim1, the interaction of ECR’s and Peg3/Zim1 promoters (Fig. 6B) is far from the reversed version of wild type (Fig. 6A), suggesting that the pattern of interaction between the ECR’s and Peg3/Zim1 promoters is more complex and represents mixed pattern of both of the paternal and maternal interaction. Therefore, they should present the interaction profiles of paternal and maternal alleles in the U1/KO2 case, separately.

2. The authors suggested that the long-distance enhancers may contribute to allelic expression of Peg3 and Zim1, however, which ones contribute to the change of Peg3/Zim1 allelic expression although I agree that the expression levels of Peg3 and Zim1 are affected by some of the ECR’s by modifying chromosome conformation around Peg3 and Zim1 promoters?

3. DNA methylation analysis was carried out only using wild type mice but not using U1/KO2 mice. However, it is very important to know the DNA methylation status of the ECR2 and ECR17 in U1/KO2 mice because their methylation difference may cause change of interaction between these ECRs and Peg3/Zim1 promoters.

4. I don’t think that interaction between ECRs and Peg3/Zim1 promoter in Fig. 8 reflects the authors experimental data and their explanation in the text. For one example, why do the fourth type (ECR3, ECR10 and ECR16) tended to show higher enrichment levels on the switched alleles with both promoter (the maternal allele for Peg3 and the paternal allele for Zim1) is classified by biallelic interaction because no enrichment was observed in the wild type (Fig. 6).

Minor comments

1. Although it is necessary to explain the generation of a mouse mutant (U1/KO2), Figure 1 may be redundant because it’s the same as the previous report (ref. 18). The authors should include the information of the position of U1 promoter in the ECR region.

2. In Fig. 4 (on DNA methylation levels of ECRs), authors described that ECR2 and ECR17 exhibited different levels of DNA methylation between paternal and maternal alleles. However, it is difficult to say that the difference of methylation levels are significant between two alleles because it was provided the data of one sample.

3. In Figure 3, images of immunostaining are low resolution.

Reviewer #3: Allele-specific enhancer interaction at the Peg3 imprinted domain

Kim et al.

The authors look at the role of ECRs in the control of imprinted gene expression in the Peg3 domain.

They also investigate the consequences of a double switch so the paternal chromosome behaves maternally

and the maternal chromosome behaves paternally.

Major comments.

For the gene expression data shown in Figure 1 only 8 animals appear to have been studied, this is far too few to draw

conclusions from and perform meaningful statistical analyses. The error bars shown are for technical replicates - I expect on biological replicates they would be much greater.

Are the wildtype samples littermates of the mutants? The expression levels of mutants should be normalised to wildtype litter mates to ensure they had the same in utero environment and they are exactly the same age.

Also, it is not clear if the genetic background of the chromosome that each deletion is on is the same. Many knockouts are derived on non-C57B/6 strains and the results seen could purely be down to genetic background differences.

For the methylation analyses the authors used C57BL/6J females crossed with PWD/PhJ males. However,

for robust methylation analysis reciprocal crosses should be used to eliminate any genetic background effects.

In the 3C analysis the authors do not explain how they are able to distinguish the maternal and paternal alleles. Is this through SNPs or are the base primers located within the deletion so they are only interrogating interactions

on one chromosome? As this provides a major part of their discussion their methods should be described more

fully.

In Figure 5, the middle panel for the Peg3 promoter wild-type - the band for ECR17 is much larger than expected - why is this?

They mention a further set of 3Cs in KO2 pups lacking the paternal Peg3 allele - these data should be in the supplementary material.

The authors mention a that the 3C analyses were repeated twice. The data from all three biological replicates should be combined for the main figures rather that showing them separately. This would allow the statistical analyses to be performed on biological replicates rather than technical replicates (as I assume are being shown in Figure 6).

Minor comments

What do the authors mean by "placental mammals". To make it clearer authors should use the accepted

terminology:

Eutherians - mammals that are not marsupials or monotremes

Therians - eutherian and marsupials

Line 5 should read eutherian evolution - as only 5 genes are known to be imprintied in marsupials

Line 9 should read the majority of imprinted genes are found only in eutherian mammals.

Line 10 should read imprinting may have evolved (or emerged) in the therian lineage. - A biological process can not be invented>

Line 11 the sentence beginning "The mechanisms through imprinting " needs re-wording as it does not make sense.

In paragraph 3 of the introduction the authors should say that the ECRs are located within the Usp29 transcript.

The orientation of the region in figures switches from figure to figure which make it difficult for a non-expert in this region

to follow what is going on.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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17 Sep 2019

Response to Review Comments

Reviewer #1: This is a novel study of gene regulation at the imprinted Peg3 locus using mouse KO lines that have been described in several previous papers from the same group. The mice harbour two different mutations that together switch allelic expression of imprinted genes on the maternal and paternal alleles. Here, by comparing expression, methylation and enhancer/promoter interactions they infer quantitative differences may exist in the interactions between the promoters and distal enhancer regions that are allele-specific.

The data generally look sound and my main advice is that the authors exercise greater caution in the way they choose to interpret their findings. Although they show some quantitative differences to be statistically significant they are relatively subtle. While these differences might be due to allele-specific in promoter/enhancer interactions involving epigenetic changes this remains to be proven. The observed differences could also be a direct consequence of the two different gross changes that have been introduced to the mutant loci. Thus, I would like to see a clear caveat to this effect included in the discussion

------- Response: We have included a sentence describing this potential caveat of the current study in the second paragraph of the Discussion section.

Some specific points:

Introduction (p3, para 1). Is it true to say “The majority of imprinted genes are found only in placental mammals”?

-----Response: Yes, that is correct.

Introduction (p3, para 1). The sentence beginning “The mechanisms through imprinted…” does not make grammatical sense.

------- Response: We have removed that sentence.

Introduction (p3, para 2). In what context is the Peg3 domain “well conserved”, do you mean specifically between mouse and human?

------- Response: The Peg3 domain is well conserved among several mammals, including human, mouse, sheep, dog, and cow.

Introduction (p3, para 2). Reference to Figure 1 would be helpful.

------- Response: We have added a reference pointer to Fig 1 in that paragraph.

Results (p5, para 1). The sentence beginning “The schematic representations…” belongs in the appropriate figure legend.

------- Response: We have moved this sentence to the legend of Fig 1.

Results (p6, para 2). The last line, beginning “on the other hand..” should perhaps refer to expression ‘differences’ being ‘indistinguishable’ rather than ‘levels’ being ‘inconclusive’.

------- Response: We have corrected this as suggested.

Results (p6, para 3). Make clear you are talking specifically about male kidney and male gonadal fat.

------- Response: We have added the term 'male' in the two sentences.

Results (p6, para 3). In the sentence beginning “Although the observed differences..” it is not clear whether the comment “at least ranging from 10 to 100 fold…” refers to brain or non-neural tissues. The whole sentence needs a rethink.

------- Response: We have further clarified this by modifying the sentence.

Results (p7, para 1). “Fourth we analysed the expression levels…” in which tissues etc?

------- Response: We have added the phrase 'in the hypothalamus set.'

Results (p7, para 2). “The results from this series…” Remove this unnecessary statement and others that are similar elsewhere.

------- Response: We put a summary statement at the end of the corresponding paragraph to emphasize the main point of each experiments. We have modified several summary sentences not to be redundant.

Results (p7, para 2). You state that Peg3 expression is ubiquitous in adult brain then later that Zim1 is uniquely expressed in ependymal cells; one of these statements must be wrong.

------- Response: Peg3 is expressed in the majority of neuron cells in the hypothalamus, whereas the expression of Zim1 was more limited to the small areas, such as PVN and SON. Also, we detected high expression levels of Zim1 in the ependymal cells from the current study. Overall, we do not feel these statements are contracting to each other.

Results (p8, para 1). You note that Zim1 expression is “very similar” to Peg3 in PVN and SON. If Peg3 expression is ubiquitous and Zim1 expression more restricted, ZIm1 expression must form a sub-set of Peg3 expression and I don’t see how this forms good evidence they “share long-distance enhancers”. In addition, you specifically indicate enhancers “localized in the middle 200-kb region” of the domain but this cannot be inferred from expression patterns alone.

------- Response: We agree with the reviewer on this point that we need to have additional evidence to support the statements. However, we also strongly believe that the following scenario is most likely. Although Peg3 expression is more ubiquitous than Zim1 expression in the hypothalamus, both genes share a similar spatial expression pattern, the expression within the PVN and SON areas. This unique spatial expression pattern between these two genes is a strong indication that they share a set of cis-regulatory elements, or enhancers. Further, according to the surveys of the ENCODE dataset and also our own results (Kim and Ye 2016), the middle 200-kb region has the majority of potential enhancers within this imprinted domain. Thus, we believe that the shared enhancers are likely localized within the middle 200-kb region.

Results (p8, para 2). Why give a DNA methylation range (“8-37%” for enhancers with low levels but not for those with high methylation levels? You also state that maternal versus paternal differences “appeared to be significant”, but on what basis (statistical test)?

------- Response: We have included the methylation level range of another set of ECRs with high methylation levels. We have also included the p values derived from statistical analyses in the text.

Results (p9, para 2). Not clear that BAC DNA provides a good control for amplification efficiency for 3C experiments involving native cellular DNA.

------- Response: Purified BAC DNA goes through a series of similar digestion and ligation, which is subsequently used for PCR amplification to test the feasibility of a given 3C scheme as well as the amplification efficiency of each amplicon. We believe that this is a standard practice for 3C experiments. However, we also agree with the reviewer that this may not be an ideal control for native cellular DNA.

Results (p9, para 1). I can’t find any primer details for the 3C experiments. Also, why was the Snrpn control chosen, has it been validated previously, perhaps in a prior publication?

------- Response: We are the first group reporting this particular 3C on the Snrpn locus. The information regarding the primers has been included in Supporting information 7. We are also providing an additional set of the detailed information regarding this series of 3C as Supporting information 8.

Results (p10, para 1 and 2). Some potentially important data on Zim1 is ‘not shown’ and there is a lack of quantification until Figure 7. It is not clear to me how this comparison has been done, including the stats, and I am struggling to evaluate its validity.

------- Response: This set of results has been included as Supporting information 9. The results in Fig 7 have been derived by comparing the relative enrichment values of each amplicon or interaction between WT and U1/KO2 animals. The detailed information regarding the raw vales and stats have been included as Supporting information 7.

Discussion (p12, par 2). Four ECRs are indicated (ECR7, 11, 17 and 19), which is different to the 3 highlighted on page 10 of the Results (ECR17, 18 and 19).

------- Response: The ECR18 was not allele-biased but is known to be a target site for MyoD, thus has been included in this statement. We have corrected this mistake.

Discussion (p13, par 1). The section beginning “Interestingly, the pioneer factor MyoD…” and ending with the sentence “Further, this suggests…” comes across as a rather clumsy piece of speculation. I would consider a careful rewrite or omitting this entirely.

------- Response: We have removed this speculative sentence along with two references.

Reviewer #2: In the manuscript entitled “Allele-specific enhancer interaction at the Peg3 imprinted domain” by Kim et al. identified putative enhancers localized upstream of Peg3 that displayed allele-biased DNA methylation, and they suggested that some of them participate in allele-biased chromosomal conformations with regional Peg3 and Zim1 promoters and that such long-distance enhancers may contribute to allelic expression of Peg3 and Zim1. In the previous report, the authors reported their interesting double KO mice with switched active alleles of Peg3 and Zim1, that is maternally expressed Peg3 and paternally expressed Zim1 by deletion of both Peg3-DMR (ICR) and oocyte-specific alternative promoter (U1) of Peg3. I agree that their switched mouse is a very good tool for elucidating imprinting mechanism and identification of the paternal-biased ECRs from the Peg3 domain is also a novel and very important finding, however, I don’t think that their results actually support their main conclusion (suggestion) that the long-distance enhancers may contribute to allelic expression of Peg3 and Zim1.

Major comments

1. Imprinted domains are very unique because expression profiles of both of the paternal and maternal alleles are different. Therefore, the authors need to carry out 3C experiment on paternal and maternal alleles separately. The authors insisted that the observed ECR’s interaction with Peg3 and Zim1 are likely allele-specific: Peg3 on the paternal and Zim1 on the maternal allele. However, it is not guaranteed.

In the U1/KO2 mice with maternally expressed Peg3 and paternally expressed Zim1, the interaction of ECR’s and Peg3/Zim1 promoters (Fig. 6B) is far from the reversed version of wild type (Fig. 6A), suggesting that the pattern of interaction between the ECR’s and Peg3/Zim1 promoters is more complex and represents mixed pattern of both of the paternal and maternal interaction. Therefore, they should present the interaction profiles of paternal and maternal alleles in the U1/KO2 case, separately.

------- Response: We are very certain that the interaction of Peg3 with ECRs is paternal-specific and the interaction of Zim1 with ECRs is maternal-specific. In particular, the U1/KO2 sample has only one allele for the Peg3 promoter, which is the maternal allele in this case. Thus, the results from the Peg3 promoter represent the interaction of the maternal allele of Peg3 promoter with ECRs. If the interaction between Peg3 or Zim1 and ECRs is purely based on the promoter strength and associated properties of each gene, but not influenced by ECRs, we should have seen the reversed patterns of 3C results between WT and U1/KO2. However, the results turned out to be far from the reversed patterns, which were also noted by the reviewer. In fact, this has been one of the main evidence supporting our conclusion that ECRs are not biallelic but rather allele-biased.

We also confirmed the allele-specific interaction between Peg3 promoter and ECRs by performing 3C with the samples from the mutant with the paternal deletion of the Peg3 promoter so that the base primer for Peg3 (Peg3p) binds to only one allele, which is the maternal allele. This control experiment did not show any enrichment or interaction, indicating that the repressed maternal allele of Peg3 is not normally available for the interaction with ECRs. This is also the case for Zim1 promoter: the repressed paternal allele of Zim1 promoter is not available for the interaction with ECRs. This set of control experiments has been included as Supporting information 9.

2. The authors suggested that the long-distance enhancers may contribute to allelic expression of Peg3 and Zim1, however, which ones contribute to the change of Peg3/Zim1 allelic expression although I agree that the expression levels of Peg3 and Zim1 are affected by some of the ECR’s by modifying chromosome conformation around Peg3 and Zim1 promoters?

------- Response: We believe that the ones showing allele-biased properties in 3C and DNA methylation should be responsible for the observed differences. So, ECR7, ECR11, ECR17, ECR19 should be the potential candidates.

3. DNA methylation analysis was carried out only using wild type mice but not using U1/KO2 mice. However, it is very important to know the DNA methylation status of the ECR2 and ECR17 in U1/KO2 mice because their methylation difference may cause change of interaction between these ECRs and Peg3/Zim1 promoters.

------- Response: We have tested the majority of ECRs in U1/KO2 and also in U1 and KO2 animals, and the results indicated no major difference in DNA methylation levels of the ECRs in various mutant alleles. Thus, we believe that the DNA methylation levels of ECRs are independent of these mutations.

4. I don’t think that interaction between ECRs and Peg3/Zim1 promoter in Fig. 8 reflects the authors experimental data and their explanation in the text. For one example, why do the fourth type (ECR3, ECR10 and ECR16) tended to show higher enrichment levels on the switched alleles with both promoter (the maternal allele for Peg3 and the paternal allele for Zim1) is classified by biallelic interaction because no enrichment was observed in the wild type (Fig. 6).

------- Response: I agree with the reviewer on this point. We have modified the figure accordingly.

Minor comments

1. Although it is necessary to explain the generation of a mouse mutant (U1/KO2), Figure 1 may be redundant because it’s the same as the previous report (ref. 18). The authors should include the information of the position of U1 promoter in the ECR region.

------- Response: Although redundant, we feel that the current manuscript should stand by itself as a separate unit for the readers, so we have included this particular figure. We have included the position information of U1 relative to the positions of ECRs in the legend of Fig 5.

2. In Fig. 4 (on DNA methylation levels of ECRs), authors described that ECR2 and ECR17 exhibited different levels of DNA methylation between paternal and maternal alleles. However, it is difficult to say that the difference of methylation levels are significant between two alleles because it was provided the data of one sample.

------- Response: We agree with the reviewer on this point. However, given our previous results, the DNA methylation methylation levels seem to be quite stable among biological replicates, since this analysis usually captures the average methylation levels of million cells. Thus, we are very confident that the results presented in this study is likely reproducible.

3. In Figure 3, images of immunostaining are low resolution.

------- Response: The images with higher resolution have been included as Supporting information 4.

Reviewer #3: Allele-specific enhancer interaction at the Peg3 imprinted domain

The authors look at the role of ECRs in the control of imprinted gene expression in the Peg3 domain.

They also investigate the consequences of a double switch so the paternal chromosome behaves maternally

and the maternal chromosome behaves paternally.

Major comments.

For the gene expression data shown in Figure 1 only 8 animals appear to have been studied, this is far too few to draw conclusions from and perform meaningful statistical analyses. The error bars shown are for technical replicates - I expect on biological replicates they would be much greater.

------- Response: Yes, the error bars are for technical replicates, but we have repeated each set of 3C experiments with 2 to 3 sets of biological replicates, which have been included as Supporting information 5-7. We are reporting only the results that are consistent and reproducible among these biological replicates.

Are the wildtype samples littermates of the mutants? The expression levels of mutants should be normalised to wildtype littermates to ensure they had the same in utero environment and they are exactly the same age.

------- Response: Yes, the WT animals were all littermates of the U1/KO2 animals.

Also, it is not clear if the genetic background of the chromosome that each deletion is on is the same. Many knockouts are derived on non-C57B/6 strains and the results seen could purely be down to genetic background differences.

------- Response: The U1 allele has been derived from the C57BL/6J background, whereas the KO2 allele from 129/SvJ. The animals with the KO2 allele have been backcrossed more than 10 generations with C57BL/6J. Now, both alleles are on the same genetic background, thus we do not anticipate any artifacts that might be caused by genetic differences.

For the methylation analyses the authors used C57BL/6J females crossed with PWD/PhJ males. However,

for robust methylation analysis reciprocal crosses should be used to eliminate any genetic background effects.

------- Response: Yes, we agree with the reviewer on this point. At the same time, we do not believe that the allelic methylation level differences observed from the two ECRs were caused by genetic background differences based on all the previous results that have been derived from the same cross (Kim et al, 2012; He et al, 2016 and 2017)

In the 3C analysis the authors do not explain how they are able to distinguish the maternal and paternal alleles. Is this through SNPs or are the base primers located within the deletion so they are only interrogating interactions on one chromosome? As this provides a major part of their discussion their methods should be described more fully.

------- Response: The base primer for Peg3 is localized within the deleted region of the KO2 allele, thus the interaction of Peg3 in U1/KO2 was derived from the maternal allele. In the case of WT animals, we are very confident that the interactions of the Peg3 promoter with ECRs were derived from the paternal allele based on the following control experiment. We performed a set of control experiments with the animals with the paternal deletion of KO2, lacking the paternal allele of Peg3 promoter. In this series of control experiments, the interaction of Peg3 promoter with ECRs on the maternal allele was very minimal or marginal, indicating that the repressed maternal allele of Peg3 promoter is not available for the interaction with ECRs. Thus, the interactions of Peg3 with ECRs in the WT animals were mostly derived from the paternal allele. This is also the case for the paternal allele of Zim1 promoter. Thus, we are confident that the 3C experiments with two base primers measure allele-specific interaction with ECRs. This has been included as Supporting information 9.

In Figure 5, the middle panel for the Peg3 promoter wild-type - the band for ECR17 is much larger than expected - why is this?

------- Response: We confirmed later that this band represents the genuine interaction between Peg3 and ECR17, but not some artifactual fragment amplified from an unrelated locus. The different-size product was found to have been amplified due to another NcoI site nearby ECR17.

They mention a further set of 3Cs in KO2 pups lacking the paternal Peg3 allele - these data should be in the supplementary material.

------- Response: We are providing this result as Supporting information 9.

The authors mention that the 3C analyses were repeated twice. The data from all three biological replicates should be combined for the main figures rather that showing them separately. This would allow the statistical analyses to be performed on biological replicates rather than technical replicates (as I assume are being shown in Figure 6).

------- Response: It was not feasible to perform statistical analyses on the combined results of biological replicates, mainly due to the fact that the quality of 3C libraries varied quite a bit among individual biological replicates.

Minor comments

What do the authors mean by "placental mammals". To make it clearer authors should use the accepted

terminology:

Eutherians - mammals that are not marsupials or monotremes

Therians - eutherian and marsupials

------- Response: Yes, we have replaced placental mammals with eutherians.

Line 5 should read eutherian evolution - as only 5 genes are known to be imprinted in marsupials

------- Response: Yes, we have corrected this.

Line 9 should read the majority of imprinted genes are found only in eutherian mammals.

------- Response: Yes, we have corrected this.

Line 10 should read imprinting may have evolved (or emerged) in the therian lineage. - A biological process can not be invented>

------- Response: Yes, we have corrected this.

Line 11 the sentence beginning "The mechanisms through imprinting " needs re-wording as it does not make sense.

------- Response: We have removed this sentence.

In paragraph 3 of the introduction the authors should say that the ECRs are located within the Usp29 transcript.

------- Response: Yes, we have included this information.

The orientation of the region in figures switches from figure to figure which make it difficult for a non-expert in this region to follow what is going on.

------- Response: We have modified Fig 1 to display the consistent orientation of the Peg3 domain as shown in the other figures.

Submitted filename: Response_to_reviewer_comments.docx

Click here for additional data file.

2 Oct 2019

PONE-D-19-17945R1

Allele-specific enhancer interaction at the Peg3 imprinted domain

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #3: (No Response)

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Reviewer #3: Partly

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Reviewer #3: No

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Reviewer #1: The authors have made efforts to address most of my earlier comments. The few remaning points are as follows:

1. Introduction (p3, para 1). Is it true to say “The majority of imprinted genes are found only in placental mammals”?

-----Response: Yes, that is correct.

Reviewer reply: I think this needs clarification still. Are you indicating that imprinting is restricted to mammals among animals (which is not contentious) or do you mean to say that most imprinted genes do not have orthologues outside of mammals (which I would contend)?

2. Introduction (p3, para 2). In what context is the Peg3 domain “well conserved”, do you mean specifically between mouse and human?

------- Response: The Peg3 domain is well conserved among several mammals, including human, mouse, sheep, dog, and cow.

Reviewer response: Please make this clear, e.g. “The Peg3 imprinted domain is localized in 500-kb genomic interval in human chromosome 19q13.4/proximal mouse chromosome 7 that is evolutionarily well conserved among mammals”

3. Results (p7, para 2). You state that Peg3 expression is ubiquitous in adult brain then later that Zim1 is uniquely expressed in ependymal cells; one of these statements must be wrong.

------- Response: Peg3 is expressed in the majority of neuron cells in the hypothalamus, whereas the expression of Zim1 was more limited to the small areas, such as PVN and SON. Also, we detected high expression levels of Zim1 in the ependymal cells from the current study. Overall, we do not feel these statements are contracting to each other.

Reviewer response: Then this is still unclear.

4. Results (p8, para 1). You note that Zim1 expression is “very similar” to Peg3 in PVN and SON. If Peg3 expression is ubiquitous and Zim1 expression more restricted, ZIm1 expression must form a sub-set of Peg3 expression and I don’t see how this forms good evidence they “share long-distance enhancers”. In addition, you specifically indicate enhancers “localized in the middle 200-kb region” of the domain but this cannot be inferred from expression patterns alone.

------- Response: We agree with the reviewer on this point that we need to have additional evidence to support the statements. However, we also strongly believe that the following scenario is most likely. Although Peg3 expression is more ubiquitous than Zim1 expression in the hypothalamus, both genes share a similar spatial expression pattern, the expression within the PVN and SON areas. This unique spatial expression pattern between these two genes is a strong indication that they share a set of cis-regulatory elements, or enhancers. Further, according to the surveys of the ENCODE dataset and also our own results (Kim and Ye 2016), the middle 200-kb region has the majority of potential enhancers within this imprinted domain. Thus, we believe that the shared enhancers are likely localized within the middle 200-kb region.

Reviewer response: Your ‘beliefs’ need to be supported by briefly setting out the logic you have presented here.

Reviewer #3: The authors do not address the point raised about the expression data. In the response they mention 3C experiments rather than expression.

"but we have repeated each set of 3C experiments with 2 to 3 sets of biological replicates, which have been

included as Supporting information 5-7. We are reporting only the results that are consistent and reproducible among these biological replicates."

The point being raised was that the expression data from 4 KOs and 4 WTs is not enough and they have not addressed this. Have they repeat the expression data on more individuals? At least 6 animals from each genotype across a number of litters are required. The error bars should then reflect the differences in expression across multiple individuals NOT technical replicates. These data should all be incorporated into 1 graph and shown in Figure 1.

I previously asked what was the genetic background the mutations were generated in . This question is not about the background of the genome as a whole but rather the region in close proximity to the deletions. The authors should note that even after backcrossing for 10 generations the DNA in close proximity to the deletion (which is selected for) is unlikely to have had a crossover event and thus will still be 129/SvJ in the case of the KO2 mice. As the KO2 region is close to genes it is highly likely that the genes located on the KO2 chromosome are 129/SvJ. Therefore the U1/KO2 mice Zim1 will be on a SvJ background but in Wildtypes on BL6. Differences in the sequences of genes and cis regulatory elements could explain the subtle differences in expression levels in Zim1.

On page 9

"The observed ECR's interaction with Peg3 and Zim1 are also likely allele-specific: Peg3 on the paternal and Zim1 on the maternal allele"

This statement is naive: enhancers have been shown to interact with promoters even in tissues where the gene is not expressed (data from Eileen Furlong's lab). The authors show in Supp Fig.9 that upon paternal transmission of KO2 there is reduced interaction of Peg3 promoter with ECRs. But the Zim1 data in this cross is biallelic. To fully characterise the interactions the authors should also perform 3Cseq on maternal KO2 heterozygotes and to interrogate the paternal chromosome separately.

All the data presented in Figure 7 relies on the assumption of allele specific interactions in the WT and U1/KO2 mice but this has not been established in the 3Cs presented. Supp_Figure 9A clearly shows that some interaction occurs between the maternal Peg3 promoter and the ECRs on a gel even though it was not detected in the qPCRs.

In the light of this Figure 7 should be re-labelled to show the mouse genotype, wildtype or U1/KO2, NOT Mat or Pat ECRs to make it clear that these were not allele specific 3Cs.

All the minor comments have been addressed except on page 3 they should say imprinting emerged in the therian lineage as imprinting has been observed in marsupials too.

**********

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6 Oct 2019

Responses to Reviews' Comments

Reviewer #1: The authors have made efforts to address most of my earlier comments. The few remaining points are as follows:

1. Introduction (p3, para 1). Is it true to say “The majority of imprinted genes are found only in placental mammals”?

Response: Yes, that is correct.

Reviewer reply: I think this needs clarification still. Are you indicating that imprinting is restricted to mammals among animals (which is not contentious) or do you mean to say that most imprinted genes do not have orthologues outside of mammals (which I would contend)?

-----Response: We mean to say that the majority of imprinted genes do not have orthologues outside of mammals.

2. Introduction (p3, para 2). In what context is the Peg3 domain “well conserved”, do you mean specifically between mouse and human?

Response: The Peg3 domain is well conserved among several mammals, including human, mouse, sheep, dog, and cow.

Reviewer response: Please make this clear, e.g. “The Peg3 imprinted domain is localized in 500-kb genomic interval in human chromosome 19q13.4/proximal mouse chromosome 7 that is evolutionarily well conserved among mammals”

-----Response: We mean to say that the Peg3 domain is well conserved among several mammals, including human, mouse, sheep, dog, and cow. We have modified this sentence to further clarify this point.

3. Results (p7, para 2). You state that Peg3 expression is ubiquitous in adult brain then later that Zim1 is uniquely expressed in ependymal cells; one of these statements must be wrong.

Response: Peg3 is expressed in the majority of neuron cells in the hypothalamus, whereas the expression of Zim1 was more limited to the small areas, such as PVN and SON. Also, we detected high expression levels of Zim1 in the ependymal cells from the current study. Overall, we do not feel these statements are contracting to each other.

Reviewer response: Then this is still unclear.

-----Response: Our understanding is that two genes, Peg3 and Zim1, share several enhancers, but each of these two genes can have its own unique expression pattern in specific cell types, as shown in the ependymal cells of the hypothalamus.

4. Results (p8, para 1). You note that Zim1 expression is “very similar” to Peg3 in PVN and SON. If Peg3 expression is ubiquitous and Zim1 expression more restricted, ZIm1 expression must form a sub-set of Peg3 expression and I don’t see how this forms good evidence they “share long-distance enhancers”. In addition, you specifically indicate enhancers “localized in the middle 200-kb region” of the domain but this cannot be inferred from expression patterns alone.

Response: We agree with the reviewer on this point that we need to have additional evidence to support the statements. However, we also strongly believe that the following scenario is most likely. Although Peg3 expression is more ubiquitous than Zim1 expression in the hypothalamus, both genes share a similar spatial expression pattern, the expression within the PVN and SON areas. This unique spatial expression pattern between these two genes is a strong indication that they share a set of cis-regulatory elements, or enhancers. Further, according to the surveys of the ENCODE dataset and also our own results (Kim and Ye 2016), the middle 200-kb region has the majority of potential enhancers within this imprinted domain. Thus, we believe that the shared enhancers are likely localized within the middle 200-kb region.

Reviewer response: Your ‘beliefs’ need to be supported by briefly setting out the logic you have presented here.

-----Response: We have modified this paragraph to make more logical as suggested.

Reviewer #3: The authors do not address the point raised about the expression data. In the response they mention 3C experiments rather than expression.

"but we have repeated each set of 3C experiments with 2 to 3 sets of biological replicates, which have been included as Supporting information 5-7. We are reporting only the results that are consistent and reproducible among these biological replicates."

The point being raised was that the expression data from 4 KOs and 4 WTs is not enough and they have not addressed this. Have they repeat the expression data on more individuals? At least 6 animals from each genotype across a number of litters are required. The error bars should then reflect the differences in expression across multiple individuals NOT technical replicates. These data should all be incorporated into 1 graph and shown in Figure 1.

-----Response: We have used these 4 sets of KO and WT derived from the previous breeding experiments, which took place 2 years ago throughout a span of 6 months. This was the maximum number of WT and KO littermate pairs with matching age at that time. Although we understand the reviewer's point, it is not feasible to perform another round of breeding experiments, which may take another 6 month to 1 year. At the same time, we are very confident with the conclusion derived from this set of the results given the associated statistical significances.

I previously asked what was the genetic background the mutations were generated in . This question is not about the background of the genome as a whole but rather the region in close proximity to the deletions. The authors should note that even after backcrossing for 10 generations the DNA in close proximity to the deletion (which is selected for) is unlikely to have had a crossover event and thus will still be 129/SvJ in the case of the KO2 mice. As the KO2 region is close to genes it is highly likely that the genes located on the KO2 chromosome are 129/SvJ. Therefore the U1/KO2 mice Zim1 will be on a SvJ background but in Wildtypes on BL6. Differences in the sequences of genes and cis regulatory elements could explain the subtle differences in expression levels in Zim1.

-----Response: When we first started working on this domain 20 years ago, we have assembled and sequenced two BAC contigs from the 129 and B6 strains. Some of these have been published (Kim et al, 2000 Genome Research 10:1138-1147; Kim et al, 2001 Genomics 74:129-141). According to this survey, we have not found any SNPs between these two strains in the ECRs, promoter and exon regions of the imprinted genes, including Peg3/Usp29 and Zim1. Thus, we believe that the expression difference reported in the current manuscript cannot be caused by the sequence variations between B6 and 129.

On page 9

"The observed ECR's interaction with Peg3 and Zim1 are also likely allele-specific: Peg3 on the paternal and Zim1 on the maternal allele"

This statement is naive: enhancers have been shown to interact with promoters even in tissues where the gene is not expressed (data from Eileen Furlong's lab). The authors show in Supp Fig.9 that upon paternal transmission of KO2 there is reduced interaction of Peg3 promoter with ECRs. But the Zim1 data in this cross is biallelic. To fully characterise the interactions the authors should also perform 3Cseq on maternal KO2 heterozygotes and to interrogate the paternal chromosome separately.

All the data presented in Figure 7 relies on the assumption of allele specific interactions in the WT and U1/KO2 mice but this has not been established in the 3Cs presented. Supp_Figure 9A clearly shows that some interaction occurs between the maternal Peg3 promoter and the ECRs on a gel even though it was not detected in the qPCRs.

In the light of this Figure 7 should be re-labelled to show the mouse genotype, wildtype or U1/KO2, NOT Mat or Pat ECRs to make it clear that these were not allele specific 3Cs.

-----Response: We actually performed a series of 3C using the sample with the maternal transmission of KO2. However, the results cannot be used as a control demonstrating the allele-specific interaction mainly due to the fact that the maternal deletion of KO2 has a very unusual trans-allelic effects, up-regulating the remaining paternal allele (Bretz and Kim, 2018 PLoS ONE 13:e0206112).

Regarding re-labeling Fig 7, both WT and U1/KO2 have their own parental alleles (MAT and PAT). Thus, it will make very busy figures with many labelings. Thus, we included the following sentences in the figure legend. The values for the paternal and maternal alleles of Peg3 were derived from WT and U1/KO2, respectively. On the other hand, the values for the maternal and paternal alleles of Zim1 were derived from WT and U1/KO2, respectively.

All the minor comments have been addressed except on page 3 they should say imprinting emerged in the therian lineage as imprinting has been observed in marsupials too.

Submitted filename: Responses_to_revieres_comments_2.docx

Click here for additional data file.

10 Oct 2019

Allele-specific enhancer interaction at the Peg3 imprinted domain

PONE-D-19-17945R2

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Reviewers' comments:

14 Oct 2019

PONE-D-19-17945R2

Allele-specific enhancer interaction at the Peg3 imprinted domain

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