RNF40 regulates gene expression in an epigenetic context-dependent manner
© The Author(s). 2017
Received: 2 December 2016
Accepted: 23 January 2017
Published: 16 February 2017
Monoubiquitination of H2B (H2Bub1) is a largely enigmatic histone modification that has been linked to transcriptional elongation. Because of this association, it has been commonly assumed that H2Bub1 is an exclusively positively acting histone modification and that increased H2Bub1 occupancy correlates with increased gene expression. In contrast, depletion of the H2B ubiquitin ligases RNF20 or RNF40 alters the expression of only a subset of genes.
Using conditional Rnf40 knockout mouse embryo fibroblasts, we show that genes occupied by low to moderate amounts of H2Bub1 are selectively regulated in response to Rnf40 deletion, whereas genes marked by high levels of H2Bub1 are mostly unaffected by Rnf40 loss. Furthermore, we find that decreased expression of RNF40-dependent genes is highly associated with widespread narrowing of H3K4me3 peaks. H2Bub1 promotes the broadening of H3K4me3 to increase transcriptional elongation, which together lead to increased tissue-specific gene transcription. Notably, genes upregulated following Rnf40 deletion, including Foxl2, are enriched for H3K27me3, which is decreased following Rnf40 deletion due to decreased expression of the Ezh2 gene. As a consequence, increased expression of some RNF40-“suppressed” genes is associated with enhancer activation via FOXL2.
Together these findings reveal the complexity and context-dependency whereby one histone modification can have divergent effects on gene transcription. Furthermore, we show that these effects are dependent upon the activity of other epigenetic regulatory proteins and histone modifications.
KeywordsRNF40 H2Bub1 Broad H3K4me3 Ezh2 Epigenetics Enhancer FOXL2
Over the past decade, significant advances have been made in the understanding of the functional role of post-translational modifications of the four core histones. The monoubiquitination of histone H2B on lysine 123 in yeast or lysine 120 in mammals is catalyzed by Bre1 in yeast and the obligate RNF20/RNF40 heterodimeric complex in mammals [1–4]. While its precise mechanisms of action remain largely unknown, H2Bub1 has been suggested to play multiple roles in chromatin-associated molecular processes including gene transcription [5, 6], DNA damage response , DNA replication , and messenger RNA (mRNA) processing [9, 10].
A significant amount of accumulating data suggests that high H2Bub1 levels are coupled with gene activation and the opening of the chromatin structure [11, 12]. However, while H2Bub1 occupancy is generally correlated with gene expression levels, small interfering RNA (siRNA)-mediated knockdown of either RNF20 or RNF40 affected only a subset of H2Bub1-occupied genes in human cells [6, 11]. While H2Bub1 occupancy is tightly coupled with transcriptional elongation rates , knockdown of RNF20 generally did not affect RNA Polymerase II elongation rate in HCT116 cells .
Stimulus-induced genes, which presumably require rapid changes in chromatin structure to become active, appear to particularly require H2Bub1 to facilitate recruitment of the FACT histone chaperone complex and induce dynamic changes in chromatin structure [15–17]. One study proposed that the depletion of H2Bub1 had a stronger impact on rapidly transcribed genes, while having fewer effects on highly transcribed genes . In contrast to its apparent positive role in transcription, H2Bub1 was also reported to repress transcription of a subset of genes by blocking the recruitment of the transcriptional elongation factor TFIIS to chromatin . Notably, the vast majority of genes whose expression increased in RNF20-depleted human cells did not display significant levels of H2Bub1, thereby suggesting that “repressive” functions of H2Bub1 likely occur via indirect mechanisms .
H2Bub1 was shown to promote the activity of the SET1/COMPASS methyltransferase complex to directly stimulate H3K4 trimethylation [19, 20]. Thus, the trans-histone crosstalk between H2Bub1 and H3K4me3 may be important for the regulation of Trithorax- and Polycomb-regulated genes such as the Hox genes . However, until now genome-wide data investigating this trans-histone crosstalk and its functions on gene transcription are lacking, therefore leaving the importance of this crosstalk unclear. Interestingly, recent studies showed that a loss of H2Bub1 impaired stem-cell differentiation with decreased induction of lineage-specific genes [21–23]. In other recent studies, a class of cell identity-related genes which display broad H3K4me3 domains near their transcriptional start sites (TSSs) was identified [24–27]. However, the mechanisms governing broad H3K4me3 domains and its transcriptional functions as well as any potential connection to H2Bub1 remain unknown. In addition, while increased breadth of H3K4me3 peaks is highly correlated with positive transcription elongation factors , the regulatory relationship between H3K4me3 domain width and the elongation machinery is unknown.
In this study, we performed genome-wide studies for H2Bub1, H3K4me3, H3K27me3, and H3K27ac occupancy in inducible Rnf40 knockout mouse embryo fibroblasts (MEF) and observed that low and moderate levels of H2Bub1 are particularly associated with RNF40-dependent gene expression changes. Interestingly, RNF40-mediated H2B monoubiquitination is required for the formation and maintenance of broad H3K4me3 domains. Consistently, RNF40-dependent genes show broader H3K4me3 peaks near the TSS, which are associated with an increased elongation rate. In addition, the CDK9-RNF20/RNF40 axis-driven H2B monoubiquitination promotes the broadening of H3K4me3 peaks to facilitate tissue-specific gene transcription. While downregulation of gene expression in response to Rnf40 deletion appears to be largely mediated by H3K4me3-dependent histone crosstalk, the upregulation of many genes, including Foxl2, was dependent upon a loss of Ezh2 transcription and decreased H3K27me3 near TSSs. Many other upregulated genes not displaying significant H3K27me3 prior to Rnf40 loss were found to be associated with the activation of FOXL2-bound enhancers and dependent upon Foxl2 expression. Together these findings uncover a previously unknown function of RNF40-mediated H2B monoubiquitination in promoting the broadening of H3K4me3 peaks to increase the transcriptional elongation rates of tissue-specific genes, as well as in the indirect repression of gene transcription via the maintenance/activation of PRC2 and indirect repression of Foxl2 transcription and provide further insight into the context-dependent intricacies of epigenetic regulation.
RNF40-regulated genes display low and moderate H2Bub1 occupancy
To investigate the role of RNF40-directed H2B monoubiquitination in gene expression, we developed a conditional knockout mouse in which exons 3 and 4 of the mouse Rnf40 gene were flanked by LoxP sites (Additional file 1: Figure S1C). This mouse was subsequently crossed to a transgenic line expressing a ubiquitously expressed tamoxifen-inducible Cre recombinase (Rosa26-CreERT2) and MEF were then obtained from homozygous Rnf40 loxP/loxP embryos containing the CreERT2 transgene . Deletion of Rnf40 and the resulting loss of H2Bub1 were effectively achieved by treating MEFs with 250nM 4-hydroxytamoxifen (4-OHT) (Fig. 1d). After categorizing genes globally into four clusters based on their degree of H2Bub1 occupancy from high to non-enriched (Fig. 1c), we further observed that genes displaying either undetectable or abundant levels of H2Bub1 were largely unaffected in their expression levels. In contrast, genes displaying low (L) or moderate (M) H2Bub1 occupancy were highly regulated in Rnf40-deficient MEFs (Fig. 1c and e). In agreement with earlier work , loss of H2Bub1 resulted in changes in expression of a select subset of genes (Fig. 1f).
We next examined the effects of loss of H2Bub1 on the occupancy of H3K4me3, H3K27ac, and H3K27me3 near the TSS. SmoothScatter analysis showed that active marks (H3K4me3 and H3K27ac) were most strongly decreased on genes displaying high levels of H2Bub1 and slightly increased on non-/low-H2Bub1 marked genes (Additional file 1: Figure S1D). Consistent with the dynamic pattern of gene regulation (Fig. 1e), the active and repressive histone marks near the TSS of “L” and “M” gene clusters were significantly altered in Rnf40-deficient MEFs (Additional file 1: Figure S1E). Notably, genes in the highly regulated clusters (L and M) displayed a high degree of occupancy of both active and repressive marks (Fig. 1c). We hypothesized that the significant differential expression of the “L” and “M” genes may be associated with changes in the active and repressive histone modifications in Rnf40-deficient MEFs.
Transcriptional dependency on H2Bub1 is linked to widespread narrowing of H3K4me3 peaks
We next sought to characterize the relationship between decreased H3K4me3 and gene regulation following Rnf40 deletion. The change in overall H3K4me3 abundance near the TSS displayed little correlation with the regulation of gene expression in response to Rnf40 deletion (R = 0.208) (Additional file 1: Figure S2D). Recent studies showed that H3K4me3 peak width may play a potentially important function in gene transcription [24, 25]. Consistently, we observed that the width of H3K4me3 peaks is correlated to gene expression levels in MEF (Additional file 1: Figure S2E). Interestingly, H2Bub1 loss affected the width of H3K4me3 peaks much more than the height, thereby resulting in a significant narrowing of H3K4me3 peaks (Fig. 2c; Additional file 1: Figure S2F). In general, the summits of H3K4me3 peaks surrounding the TSS were shifted by ca. 50 bp towards the 5’ end of the gene after Rnf40 deletion (Fig. 2c). Importantly, the downregulation of RNF40-dependent genes was associated with the narrowing of H3K4me3 peak width (R = 0.38) (Additional file 1: Figure S2G). In addition, RNF40-dependent genes (downregulated following Rnf40 deletion) displayed significantly wider H3K4me3 peaks than those found on genes upregulated or unchanged (Fig. 2d). In comparison, H3K4me3 abundance on RNF40-dependent genes was significantly lower than unchanged genes (Fig. 2e). Consistent with the observed changes in gene regulation, the width of H3K4me3 peaks on downregulated genes was significantly shortened in Rnf40-deleted MEFs (Fig. 2f). In contrast to the specificity observed in H3K4me3 peak breadth, overall H3K4me3 abundance was significantly decreased irrespective of whether the associated gene was upregulated, downregulated, or unregulated in response to Rnf40 deletion (Fig. 2g). Together these data indicate that transcriptional dependency on RNF40 is tightly coupled to the formation of broader H3K4me3 domains and less to overall H3K4me3 abundance.
Given the proposed importance of broad H3K4me3 domains for cell identity and function [24–27] and the correlation between changes in H3K4me3 peak breadth and H2Bub1, we next sought to examine whether the presence of broad H3K4me3 domains also correlated with changes in gene expression and was predictive for the requirement of RNF40 for gene expression. We therefore used the previously described approach [24, 25] to identify genes with top 5% broadest H3K4me3 domains, as well as similar numbers of genes displaying sharp H3K4me3 domains and random control genes in MEFs (Fig. 2h; Additional file 1: Figure S2H). In agreement with a recent report , broad H3K4me3 domains were correlated to gene expression (Fig. 2i). Importantly, loss of H2Bub1 resulted in a more significant narrowing of broad H3K4me3 peaks compared to sharp or random control H3K4me3 peaks (Fig. 2j; Additional file 1: Figure S2I–K). Moreover, the expression levels of genes displaying broad H3K4me3 peaks were significantly decreased in response to Rnf40 deletion, whereas genes displaying sharp H3K4me3 peaks were moderately, but significantly, increased in their gene expression and randomly chosen control genes were unchanged in their expression (Fig. 2k). Taken together, we conclude that transcriptional dependency on H2Bub1 is closely coupled to the narrowing of H3K4me3 peak width.
H2Bub1 selectively regulates transcription elongation rate and facilitates the spreading of H3K4me3 into the gene body
Release of RNA Polymerase II (Pol II) from transcriptional pausing is a crucial step in regulating the transcription of many genes. Recent findings documented that the spreading of H3K4me3 into the transcribed regions of active genes is inversely correlated to the Pol II pausing index . Consistently, Pol II showed a clear enrichment on the gene body and 3’ end of genes with broad H3K4me3 domains in MEFs compared to genes with sharp H3K4me3 peaks and random control genes, while displaying no apparent differences in occupancy near the TSS (Additional file 1: Figure S3A). To further examine the association of H3K4me3 and transcriptional elongation, we examined the correlation between H3K4me3 and elongation rates using data available from HeLa cells . Indeed, H3K4me3 peaks were significantly broader on genes displaying a high elongation rate (Additional file 1: Figure S3B), while there was no significant difference in the height of H3K4me3 peaks between genes displaying high and low elongation rates (Additional file 1: Figure S3C).
Next, we sought to examine whether H2Bub1 occupancy and transcription elongation rates are correlated on genes classified based on the width of the TSS-associated H3K4me3 domains. In contrast to a previous study which reported that H2Bub1 abundance is tightly coupled to transcription elongation rates , we found that genes displaying sharp H3K4me3 domains and low elongation rates (Additional file 1: Figure S3H) actually displayed the highest levels of H2Bub1 near the 5’ end while genes with broad H3K4me3 domains displaying high elongation rates showed significantly lower H2Bub1 occupancy near the 5’ end. Consistently, RNF40-dependent genes (which also display broader H3K4me3 domains, Fig. 2d) showed significantly higher Pol II enrichment (Fig. 3d) but lower H2Bub1 occupancy (Fig. 3e) across the gene body and 3’ end regions compared to unchanged genes. Therefore, these data indicate that the association between H2Bub1 occupancy and transcriptional elongation is intimately connected to H3K4me3 spreading into the gene body.
In order to further determine the role of H2Bub1 on transcriptional elongation, we analyzed individual RNF40-dependent genes to confirm the effects of RNF40 loss on gene expression and H3K4me3 occupancy. The Myl9 and Psrc1 genes displayed a clear enrichment of H2Bub1 across the gene body as well as broad H3K4me3 peaks near the TSS (Fig. 3f). Importantly, Rnf40 deletion resulted in the narrowing of the TSS-associated H3K4me3 domain, decreased H3K27ac occupancy and Myl9 and Psrc1 mRNA levels. These effects were further confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and ChIP-qPCR for H2Bub1 and H3K4me3 (Additional file 1: Figure S3I–K). Consistent with a role of H2Bub1 in promoting transcriptional elongation, Pol II occupancy on the gene body, but not near the TSS, of the Myl9 and Psrc1 genes was significantly decreased in response to Rnf40 deletion (Fig. 3g and h).
In order to confirm the specificity of the observed effects and the importance of RNF40-mediated ubiquitin ligase activity, we re-expressed wild-type RNF40 or a RING finger-deleted RNF40 in Rosa26-CreERT2, Rnf40 loxP/loxP MEFs. Rnf40 mRNA levels were restored to nearly normal levels in HA-ΔRING and HA-RNF40 MEFs after deleting the endogenous Rnf40 gene (Fig. 3i). Importantly, re-expression of wild-type, but not ΔRING RNF40 was able to rescue RNF40-dependent gene expression (Myl9 and Psrc1) (Fig. 3j and k), thereby confirming the specificity of the observed effects and reinforcing the importance of RNF40-mediated ubiquitin ligase activity in controlling the expression of RNF40-dependent genes.
CDK9 is required for the establishment of broad H3K4me3 peaks and increased tissue-specific gene transcription via CDK9-RNF20/RNF40-H2Bub1 axis
A previous study indicated that the spreading of H3K4me3 into the gene body is highly associated with the recruitment of the positive elongation machinery (CDK9, AFF4, ELL2, and TCEA1) . Since H2B monoubiquitination depends directly upon CDK9 activity  and CDK9, the WAC adaptor protein, and RNF20/40 are similarly required for the induction of adipocyte-specific genes , we examined whether CDK9 is also required for the establishment of broad H3K4me3 domains on differentiation-specific genes using the newly reported CDK9-specific inhibitor LDC000067 . Consistent with the effects elicited by siRNA-mediated CDK9 depletion , CDK9 inhibition led to a significant decrease in the induction of PPARG and RASD1 during adipocyte differentiation (ADI) (Fig. 4c and d). In agreement with the reported importance of the CDK9-WAC-RNF20/RNF40 axis , H2Bub1 occupancy on PPARG and RASD1 were significantly decreased in adipocytes following CDK9 inhibition (Fig. 4e and f). Importantly, H3K4me3 occupancy across the gene body, but not proximal to the TSS, was significantly decreased in response to CDK9 inhibition (Fig. 4e and f). Thus, we propose that the H2Bub1-H3K4me3 trans-histone pathway driven by the CDK9-RNF20/RNF40 axis is particularly important for cell lineage specification-associated broadening of H3K4me3 domains during cell fate specification (Fig. 4g).
Increased gene expression in Rnf40-deficient MEFs is related to the loss of the Ezh2 expression
We next sought to characterize the relationship between H3K27me3 occupancy near the TSS and the induction of gene expression following Rnf40 deletion. Thus, we identified a gene set enriched for H3K27me3, which displayed a greater than two-fold decrease in H3K27me3 levels surrounding the TSS (Additional file 2: Table S1), and could observe that a large fraction of these genes was upregulated in Rnf40-deficient MEFs (Additional file 1: Figure S6D). Moreover, GSEA analysis using genes displaying decreased H3K27me3 occupancy in Rnf40-deficient MEFs further confirmed an enrichment of genes upregulated following Rnf40 deletion (Fig. 6b). In addition, GSEA analysis of mRNA-seq data confirmed a significant enrichment of EZH2 target genes that were upregulated in Rnf40-null MEFs (Additional file 1: Figure S6E). Together these findings suggest that the deficiency of Rnf40 and H2Bub1 results in a global decrease in H3K27me3 levels near the TSS of PRC2 target genes via decreased expression of Ezh2, thereby leading to a derepression of PRC2 target gene transcription.
In order to confirm that the observed effects were, indeed, due to decreased PRC2 activity, we compared the effects of treating Rnf40 +/+ MEFs with the EZH2 inhibitor EPZ6438  to those observed following Rnf40 deletion. The profiles for several PRC2 target genes (Chd5, Nat8l, Kcnc3, Foxl2, Foxl2os, and Tgfa; Fig. 6d, Additional file 1: Figure S6I) demonstrate that loss of RNF40 led to significant decreases in H3K27me3 occupancy at promoters, while H3K27ac occupancy and mRNA levels increased and H3K4me3 levels were largely unaffected. Consistent with the importance of this mechanism in vivo, Rnf40 deletion in vivo also led to a significant increase of Nat8l expression (Additional file 1: Figure S6H). Consistent with the global decrease in H3K27me3 levels and a central role for decreased Ezh2 expression in mediating increases in gene expression following Rnf40 deletion (Fig. 6c), EZH2 inhibition significantly increased the expression of several PRC2 target genes (Chd5, Nat8l, Kcnc3, Foxl2, Foxl2os, and Tgfa) identified to be upregulated following the loss of Rnf40 (Fig. 6e, additional file 1: Figure S6F), while non-PRC2-target genes (e.g. Prsc1) were unaffected by EZH2 inhibition (Additional file 1: Figure S6G). Importantly, increased expression of Nat8l and Foxl2 resulting from Rnf40 deletion could be rescued by re-expressing wild-type but not a methyltransferase-deficient mutant (H689A) of EZH2 (Fig. 6f and g) . Moreover, ChIP-qPCR for H3K27me3 confirmed that overexpressing wild-type but not mutant EZH2 was sufficient to restore H3K27me3 occupancy on the Nat8l and Foxl2 genes in the absence of RNF40 (Fig. 6h and i). Together these data suggest that the upregulation of a subset of genes following a loss of H2Bub1 elicited by Rnf40 deletion can largely be attributed to a loss of Ezh2 expression, rather than a direct repressive function of H2Bub1 (Fig. 6j).
H2Bub1 coordinates homeobox gene activation and repression
Given the important functions of Hox genes in stem-cell self-renewal and differentiation [37, 38], we also investigated two individual Hox genes (HOXB2 and HOXC10) with different epigenetic contexts which are induced during adipocyte differentiation. As seen in ChIP-seq profiles for each gene (Additional file 1: Figure S7C), the TSS of both HOXB2 and HOXC10 were significantly enriched for H3K4me3 near the TSS, while HOXC10 also displayed significant H3K27me3 occupancy upstream of the TSS. Consistently, the expression of both HOXB2 and HOXC10 was significantly increased during adipocyte differentiation. This effect coincided with increased occupancy of H3K4me3 on the HOXB2 gene and decreased occupancy of H3K27me3, but no significant change of H3K4me3 on the HOXC10 gene during adipocyte differentiation (Additional file 1: Figure S7C). Consistent with the epigenetic context of both genes and the differential effects of RNF40 loss on Polycomb-repressed and H3K4me3-dependent genes, RNF40 depletion led to downregulation of the H3K4me3 occupied HOXB2 gene and upregulation of the H3K27me3 and H3K4me3 co-occupied HOXC10 gene (Fig. 7f and g). Together, these data suggest that H2Bub1 differentially regulates Hox genes in a context-dependent manner by coordinating the equilibrium between active (H3K4me3 and H3K27ac) and repressive (H3K27me3) histone modifications not only in MEFs, but also in a biologically relevant differentiation system.
Transcriptional activation of a subset of RNF40-suppressed genes is associated with enhancer activation
To uncover potential transcription factors which may contribute to enhancer activation and upregulation of genes in Rnf40 –/– MEFs, we performed a sequence-based motif analysis of the upregulated gene-associated enhancers and identified a significant enrichment of Forkhead box protein binding motifs (Fig. 8b). Consistent with our observation that the expression of Foxl2 was significantly increased in Rnf40 –/– MEFs (Fig. 6e), we identified 3223 enhancers in our study which were occupied by FOXL2 in a published ChIP-seq dataset  (Additional file 1: Figure S8G). In addition, GREAT analysis of those regions identified the FOXL2-enriched enhancer-associated genes, which contained more than 25% (166/672) of the upregulated genes (Fig. 8c) and 100 genes (more than 60%) which were upregulated and displayed enhancer activation following Rnf40 deletion (Additional file 1: Figure S8H). Consistent with increased enhancer activation, the H3K27ac occupancy surrounding these FOXL2-enriched distal regions was significantly increased in Rnf40 –/– MEFs (Fig. 8d and e).
In order to confirm the role of FOXL2 in the upregulation of this subset of genes in Rnf40 –/– MEFs, we examined the effects of siRNA-mediated FOXL2 depletion in MEFs following Rnf40 deletion. Consistent with a previous study demonstrating the importance of FOXL2 for their expression , we observed that both the Esr2 and Efna5 genes were significantly upregulated following Rnf40 deletion as well as following EZH2 inhibitor treatment (Fig. 8f). Furthermore, FOXL2-targeted genes (Esr2 and Efna5) were increased in the ovary of a Rnf40-deleted mouse (Additional file 1: Figure S8I). Importantly and consistent with an indirect effect mediated by FOXL2, these effects were blocked by FOXL2 depletion (Fig. 8f). Moreover, ChIP-seq profiles confirmed that H3K27ac occupancy on each of those genes was increased at FOXL2-bound enhancers following Rnf40 deletion (Fig. 8g; Additional file 1: Figure S8J). Together these data support a central role for FOXL2 in mediating enhancer activation and increased gene expression of a subset of genes whose expression increases following Rnf40 deletion (Fig. 8h).
The complex regulatory network of post-translational histone modifications has long been hypothesized to play a significant role in controlling the timely activation or repression of gene transcription . Here we investigated the genome-wide occupancy of H2Bub1 and examined the effects of its loss following Rnf40 deletion on other post-translational histone modifications at proximal and enhancer regions, and investigated the relation of these alterations to changes in gene transcription. In addition to providing a genome-wide confirmation of the previously reported H2Bub1-H3K4me3 trans-histone tail crosstalk [42, 43], our work describes a previously unknown role for RNF40-mediated H2B monoubiquitination in the establishment and maintenance of broad H3K4me3 domains, which appear to selectively promote the transcriptional elongation of tissue-specific genes. In addition, we provide the first mechanistic explanation by which the loss of RNF40 can lead to increased gene expression. Specifically, a subset of PRC2-repressed genes is upregulated following Rnf40 deletion via decreased Ezh2 expression and a resulting decrease in H3K27me3 and a concordant increase in H3K27ac occupancy near their TSSs (Fig. 6j). We also identified an additional group of “RNF40-suppressed genes,” which is associated with increased enhancer activity via upregulation of Foxl2 gene expression (as a consequence of decreased Ezh2 expression and PRC2 activity) in Rnf40-null MEFs (Fig. 8h).
RNF40-regulated genes display low and moderate H2Bub1 occupancy
In order to obtain efficient activation of gene transcription, the signals enabling transcriptional activity, including active histone modifications, appear to require a certain threshold in order to facilitate gene expression . According to our data, genes which display the highest occupancy of H2Bub1 and other active histone modifications appear to be more robustly expressed and less sensitive to changes in the presence of individual histone modifications. We hypothesize that, even in the absence of H2Bub1, these genes retain sufficient additional active signals to maintain high levels of transcription. In contrast, inactive or lowly active genes, such as poised genes marked by the PRC2 complex, may require higher levels of additional activation signals to switch from a repressed to an active state and therefore be more dependent upon individual histone modifying enzymes. Furthermore, there seems to be a complex regulatory mechanism acting on genes marked by varying degrees of both active and repressive histone modifications as we observed for low to moderate H2Bub1-occupied genes, whose transcription highly depends upon changes in histone modifications facilitated by the recruitment of tissue-specific transcription factors . Thus, this class of genes appears to be particularly vulnerable to changes in expression elicited by the loss of either active or repressive marks.
RNF40-mediated H2Bub1 governs H3K4me3 peak width to increase transcription elongation rate
Consistent with the H2Bub1-H3K4me3 trans-histone crosstalk model in which H2Bub1 facilitates the trimethylation of H3K4 by the SET/COMPASS complex , our study shows that the absence of H2Bub1 results in a decrease, but not a total loss, of H3K4me3 levels genome-wide. Notably, the decrease in H3K4me3 occupancy was most apparent at regions downstream of the TSS, which were also occupied by H2Bub1. Following loss of H2Bub1, the width of H3K4me3 peaks was affected much more than their height, thereby resulting in a significant narrowing of the peaks toward the TSS. We speculate that the bulk of H3K4me3 near the TSS may be catalyzed by SET/COMPASS or other H3K4 methyltransferases in an RNF40/H2Bub1-independent manner, but that transcriptional elongation-associated spreading of H3K4me3 into the gene body is highly dependent upon RNF20/40-mediated H2B monoubiquitination. This effect can also be observed on the Hoxc gene cluster where H3K4me3 on each of the Hoxc genes decreases, but some degree of H3K4me3 remains and becomes more focused around the TSS. These effects closely resemble those observed in Mll1- deficient MEFs , suggesting that H2Bub1 may be capable of directing MLL-dependent H3K4 methylation downstream of the TSS.
Although H2Bub1 had broad effects on H3K4me3 occupancy genome-wide, the H2Bub1-H3K4me3 crosstalk selectively regulated a subset of genes in response to Rnf40 deletion. We further determined that transcriptional dependency on H2Bub1 is highly linked to the narrowing of H3K4me3 peak width. Consistent with the finding that increasing H3K4me3 width is associated with increased transcription elongation rates , RNF40-dependent genes show broader H3K4me3 peaks and higher transcription elongation rates compared to RNF40-independent and RNF40-suppressed genes. Moreover, given that the width of H3K4me3 peaks is highly dependent on H2Bub1, the transcription of genes with the broad H3K4me3 domains depended on RNF40 more than genes with sharp H3K4me3 peaks near the TSS and random control genes.
Previous work from the Oren lab reported that high H2Bub1 occupancy is associated with high transcription elongation rates , but that RNF20 depletion did not affect elongation rates of RNF20-dependent genes . However, in this work we examined the context-dependency of the effects of RNF40/H2Bub1 loss on transcription and observed that RNF40-dependent genes with broader H3K4me3 and higher elongation rates showed significantly lower occupancy of H2Bub1 across the gene body than RNF40-independent genes with narrower H3K4me3 peaks and lower elongation rates. Given the correlation between the positive elongation machinery and H3K4me3 width , we suggest that the function of H2Bub1 in facilitating transcriptional elongation is closely linked to the RNF40-dependent broadening of H3K4me3. Although our work has revealed that H2Bub1 is essential for broadening H3K4me3 peaks, it remains to be determined how H2Bub1 selectively facilitates the broadening of a subset of genes. We hypothesize that some regulators may function to prevent the spreading of H3K4me3 domain. While changes in the activity or recruitment of specific methyltransferases such as UpSET in Drosophila melanogaster or MLL5 in humans may explain these effects , another possibility would be the specific recruitment or exclusion of the RACK7 histone demethylase complex, which has been shown to suppress the broadening of H3K4me3 at promoters and enhancers .
Recent studies uncovered a previously unrecognized association of broad TSS-associated H3K4me3 domains with the expression of tumor suppressor and cell identity genes [24, 25]. Consistent with potential tumor suppressor functions of RNF20/40 and H2Bub1 and their requirement for stem cell differentiation [17, 23], we observed a widening of H3K4me3 peaks on RNF40-dependent lineage-specific genes during adipocyte differentiation. Consistently, we previously demonstrated that CDK9-WAC-RNF20/RNF40-directed H2B monoubiquitination is required for tissue-specific gene transcription . Moreover, we demonstrate that the activity of CDK9, the catalytic component of the Positive Transcription Elongation Factor-b complex, is required for the spreading of H3K4me3 peaks into the gene body of RNF20/40-dependent differentiation-induced genes. Additionally, and consistent with a direct role of broad H3K4me3 domains in facilitating transcriptional elongation, Wdr82 deletion in BMDM cells resulted in a shortening of H3K4me3 peaks  and decreased the transcription elongation rate specifically at genes with broad H3K4me3 domains. Therefore, we suggest that CDK9, RNF20/RNF40, H2Bub1, and broad H3K4me3 domains cooperatively facilitate transcriptional elongation of tissue-specific genes. Given that broad H3K4me3 domains are a particular epigenetic hallmark of cell identity and tumor suppressor genes [24, 25], further studies on the CDK9-RNF20/RNF40-H2Bub1-broad H3K4me3 axis to determine the functional interconnectivity between WDR82, H2Bub1, and transcriptional elongation will likely reveal important insight into the epigenetic regulatory mechanisms controlling important processes such as embryogenesis and tumorigenesis. Moreover, the extent and molecular mechanisms by which broad H3K4me3 domains and H2Bub1 promote transcriptional elongation as well as the specific mechanisms leading to spreading of H3K4me3 at only a subset of genes remain to be determined.
Loss of H2Bub1 causes de-repression of a subset of genes
Consistent with findings following RNF20 knockdown , we find that the vast majority of “RNF40-suppressed” genes do not display significant levels of H2Bub1, thereby suggesting that their regulation may occur through more indirect mechanisms. Consistently, we find that the Ezh2 gene, encoding the catalytic component of the PRC2 complex, which mediates H3K27 methylation, displays a significant level of H2Bub1 occupancy and requires RNF40 for its full expression. The regulation of RNF40-mediated H2Bub1 on Ezh2 expression is not isolated to cultured MEF cells, but was confirmed in human cell lines and various tissues from a global conditional in vivo Rnf40 knockout mouse model.
Furthermore, consistent with a central role for EZH2 as a central mediator of H2Bub1-dependent “gene repression,” small molecule inhibition of EZH2 activity resulted in a similar derepression of H3K27me3-targeted genes which were upregulated in Rnf40-deficient MEFs. The expression of this subset of PRC2-regulated genes is likely related to a dynamic antagonism between H3K27me3 and H3K27ac at p300/CBP and PRC2 co-targeted sites [49–51]. Thus, the upregulation of “RNF40-suppressed” genes appears to be related to a shift in the balance between H3K27me3 and H3K27ac, whereby decreased H3K27 methylation enables the acetylation of the same residue at these loci.
In addition to direct PRC2-regulated genes, we also observed the upregulation of genes which demonstrated increased activity of nearby enhancers. This increased enhancer activity was associated with the derepression of Foxl2 as a consequence of Ezh2 downregulation in Rnf40 –/– MEFs. Interestingly, the promoter of the FOXL2 gene was found to be hypermethylated in ovary granulosa cell tumors concomitant with increased EZH2 expression . However, whether these effects coincide with changes in RNF40 activity or H2Bub1 occupancy in ovary granulosa cell tumorigenesis remains unknown.
We further determined that genes upregulated following Rnf40 deletion were enriched for developmental regulators, further supporting a critical function of RNF40 in directing cell fate determination. Consistent with a context-dependent function of H2Bub1 in regulating different groups of genes, while we previously demonstrated a central role for RNF20/40-dependent H2B monoubiquitination in differentiation to the osteoblast and adipocyte lineages , another group reported that H2Bub1 levels decrease during myoblast differentiation . Interestingly, while the regulatory role of RNF40 in ovarian development remains to be determined, the FOXL2-regulated genes Esr2 and Enf5a, which are required for ovary development , were increased in the ovary following global conditional Rnf40 deletion. Thus, it is possible that the RNF20/40-H2Bub1 pathway may promote cell differentiation to one lineage and suppress that of another lineage in a given epigenetic context while promoting differentiation to other lineages in a different context.
In conclusion, we provide evidence and insight into the apparent discrepancy between the association of H2Bub1 with active gene transcription and the unexpected finding that a nearly equal fraction of genes are up- or downregulated following its loss. Our results support a model in which the direct function of RNF40-mediated H2B monoubiquitination increases transcriptional elongation by promoting the spreading of H3K4me3 into the 5’ transcribed region of a select subgroup of genes. However, given the finding that the Ezh2 gene is a major target of RNF40 and H2Bub1, and the demonstration that the effects of Rnf40 deletion on these “H2Bub1-suppressed” genes can be mimicked by inhibition of EZH2 catalytic activity, our data support a model in which “suppression” of gene transcription by H2Bub1 is mediated via indirect effects through PRC2. These findings, together with our results supporting a role for H2Bub1 in controlling H3K4me3 on RNF40-dependent genes, provide important insight into the enigmatic role of H2Bub1 in transcription. Further studies examining the effects of Rnf40 deletion in additional cell types and tissues, in conjunction with in vivo disease models, will shed further light into the biological and mechanistic functions of H2Bub1 and further elucidate its context-dependent function.
Conditional Rnf40 knockout mouse model and isolation of inducible knockout MEFs
All animal work was performed in agreement with the Institutional Animal Care and Use Committee and the Institutional Guidelines for Humane Use of Animals in Research. The construct for generating conditional Rnf40 knockout mice was generated using a construct containing two loxP sites surrounding exons 3 and 4 of the Rnf40 gene and a neomycin selection cassette was surrounded by two short flippase recognition target (FRT) sites. The targeting construct was electroporated in MPI II ES cells and targeted clones were identified by quantitative and long-range PCR. Following the generation of chimeras and verification of germline transmission, the neomycin cassette was removed to generate Rnf40 loxP mice by crossing to a transgenic mouse line expressing the FLP recombinase in all tissues . The Rnf40 loxP mice were next crossed to a transgenic line expressing a tamoxifen-inducible Cre recombinase (CreERT2) inserted into the ubiquitously expressed Rosa26 locus . The inducible Rnf40 knockout MEFs were obtained by intercrossing Rosa26-CreERT2, Rnf40 loxP/wt mice. Finally, MEFs were isolated from 13.5 postcoitum mouse embryos as previously described  and homozygous Rosa26-CreERT2, Rnf40 loxP/loxP embryos were utilized to generate MEFs.
In order to examine the effects of in vivo Rnf40 deletion, two Rosa26-CreERT2, Rnf40 loxP/loxP and Rnf40 wt/wt mice were treated with tamoxifen every other day. 5% Tamoxifen (w/v; Sigma-Aldrich) was dissolved in ethanol and subsequently mixed 1:10 with sunflower oil and injected intraperitoneally every other day for one week with a total dose of 1.5 mg tamoxifen per day. Weight and general health status were monitored daily and two weeks after commencing injections various tissues were harvested.
Primary MEFs were cultured in high-glucose GlutaMAX™-DMEM (Invitrogen) supplemented with 10% FBS Superior (Biochrom), 1% penicillin–streptomycin (Sigma-Aldrich), and 1% non-essential amino acids (Invitrogen) at 37 °C, 5% CO2. For deletion of the conditional Rnf40 allele, MEFs were passaged in growth medium supplemented with 250 nM of 4-hydroxytamoxifen (4-OHT). After five days, cells were grown for another three days in the absence of 4-OHT. Where indicated, Rnf40 +/+ MEFs were treated with 1 μM EPZ6438 (Selleck Chemicals) for three days and forward and reverse transfection of siRNA were performed in Rnf40 +/+ and Rnf40 –/– MEFs using RNAiMAX (Thermo Scientific) according to the manufacturer’s instructions. Non-targeting siRNA (D-001210-05-50, Dharmacon) was used as a negative control. Targeted mouse Foxl2 SMARTpool siRNAs (L-043309-01-0005, Dharmacon) contained the sequences 5’-GCGCAGUCAAAGAGGCCGA-3’, 5’-ACUCGUACGUGGCGCUCAU-3’, 5’-UAGCCAAGUUCCCGUUCUA-3’, and 5’-CGGGACAACACCGGAGAAA-3’.
The constructs for overexpressing wild-type Rnf40, RING finger-deleted Rnf40 (ΔRING-RNF40), wild-type Ezh2, and SET domain-mutated Ezh2 (H689A) were generated by cloning Rnf40, ΔRING-RNF40, Ezh2, and H689A PCR products into a pSG5-HA-hygromycin vector in which the expressed HA-tagged fusion protein and hygromycin resistance gene were expressed from a single open reading frame and separated by a P2A sequence resulting in the production of two separate polypeptides. The primers for cloning are listed in Additional file 4: Table S3. Expression constructs were transfected into Rosa26-CreERT2, Rnf40 loxP/loxP MEFs using Lipofectamine 2000 (Thermo Fisher Scientific). Four days after transfection, hygromycin-resistant MEFs were selected by using 300 μg/mL of hygromycin for approximately two months.
hMSC-Tert cells  were passaged in phenol red-free low-glucose MEM (Invitrogen) supplemented with 10% FBS and 1% penicillin–streptomycin (Sigma-Aldrich) at 37 °C, 5% CO2. For adipocyte differentiation , hMSC were cultured in MEM supplemented with 15% FBS, 2 × 10−6 M insulin, 0.45 mM isobutylmethyl-xanthine, 10−5 M troglitazone, and 10−4 M dexamethasone. For knockdown of RNF40, hMSC were transfected with RNF40  or non-targeting siRNA (D-001210-05-50, Dharmacon) using RNAiMAX (Thermo Scientific) 16 h prior to the induction of adipocyte differentiation according to the manufacturer’s instructions. For inhibiting CDK9, hMSC were treated with 5 μM of LDC000067 2 h prior to the induction of adipocyte differentiation.
Western blotting and qRT-PCR
Protein extraction, western blot analysis, RNA isolation, reverse transcription, and qPCR were performed as previously described . The primary antibodies for western blot and primers for qRT-PCR are listed in Additional file 4: Table S3 and Additional file 5: Table S4, respectively.
ChIP, ChIP-qPCR, ChIP-seq, and RNA-seq library preparation
ChIP for histone modifications was performed as described using 10 min crosslinking  or 15 min for RNA Polymerase II. Antibodies for ChIP and their dilutions are listed in Additional file 5: Table S4. ChIP qRT-PCR analysis of the occupancy of Pol II, H2Bub1, H3K4me3, and H3K27me3 was performed as described previously . Immunoprecipitated DNA of each sample was quantitated using a Qubit 3.0 (Life technologies). A total of 5 ng of DNA was sonicated to obtain 200 bp fragments using the Bioruptor® Pico (Diagenode) and sequencing libraries were prepared using the NEBNext Ultra DNA library preparation kit according to the manufacturer’s protocol (New England Biolabs).
Total RNA was isolated from wild-type and Rnf40-null MEF cells at passage 3. Total RNA quality was verified using a Bioanalyzer 2100 (Agilent) and libraries were prepared from 1 μg of total RNA using the NEXTflex™ Rapid Directional RNA-Seq Kit according to the manufacturer’s protocol (Bio Scientific).
Each RNA and ChIP DNA library was quantified using a Qubit 3.0 (Life Technologies) and fragment sizes was checked using the Bioanalyzer 2100 (Agilent). Finally, 75 bp single-end sequencing for H3K4me3 and 51 bp single-end sequencing for other histone modifications were performed with single indexing using the NextSeq or HiSeq 2500 (Illumina) platforms, respectively, as described before . ChIP-seq and RNA-seq experiments in each condition were performed in duplicate and triplicate, respectively (Additional file 6: Table S5).
Gene expression data
Whole-genome gene expression analysis from Rnf40 +/+ and Rnf40 –/– MEFs were generated as previously described . Sequences were mapped to the mouse reference transcriptome (UCSC mm9) and differential gene expression of each sample was normalized using DESeq. Significant differentially expressed genes were classified as follows: downregulated genes (down), baseMean > 15, p value < 0.05, log2 fold change < –1; upregulated genes (up), baseMean > 15, p value < 0.05, log2 fold change > 1; unchanged genes (unch), baseMean > 15, p value > 0.8, –0.2 < log2 fold change < 0.2.
The enrichment scores were calculated using GSEA as described before  and gene expression data were sorted by fold changes under Rnf40 –/– versus Rnf40 +/+ conditions. Gene Ontology (GO) enrichment analysis for significantly downregulated and upregulated gene clusters were performed using DAVID 6.7 . The significant enriched GO terms (FDR < 0.05) were shown as a bubble plot generated from REViGO .
The ChIP-seq raw data were mapped to the mouse reference genome (UCSC mm9) using Bowtie (version 1.0.0) . To identify significant peaks, we used Model-based Analysis of ChIP-seq (MACS) (version 1.0.0) for peak calling with the input of each condition as control and p value < 0.00001 cutoff for peak detection . Coverage was determined by normalizing the filtered reads per hundred million. The bigwig data were visualized in Integrative Genomics Viewer (version 2.3.14) . The tables containing mouse genome elements (TSS, gene bodies, etc.) and CpG island were obtained from UCSC Table Browser . The average signal of H3K4me3, H3K27me3, and H3K27ac near TSS (±1 kb) and H2Bub1 in gene bodies were computed using ComputeMatrix in deepTools . The heatmapper in deepTools was used to create heatmaps of each ChIP. CEAS (version 1.0.0) and aggregate profile analyses were performed in Galaxy/Cistrome . The H3K27me3 targeted distal regions were obtained by considering only the regions further than 5 kb upstream or downstream of gene bodies. Active enhancers were defined as enriched for (+) H3K4me1 and H3K27ac but negative (–) for H3K4me3 enrichment. Differential binding (DiffBind) analysis of H3K27me3 near TSS (±1 kb) and distal regions or H3K27ac on enhancers under Rnf40 +/+ versus Rnf40 –/– conditions was performed as described before . Enhancer associated coding genes were identified using the Genomic Regions Enrichment of Annotations Tool (GREAT version 3.0.0) . Sequence-based motif analysis for upregulated genes associated with enhancers in Rnf40 –/– MEFs was performed using oPOSSUM (version 3.0) . The input file contained the regions (±150 bp) surrounding H3K27ac peak centers on enhancers associated with genes upregulated in Rnf40 –/– MEFs. The remaining enhancers not increased following Rnf40 deletion were utilized as background. Broad H3K4me3 peak regions and peak height were determined using MACS2 . Genes with the top 5% broadest H3K4me3 domains were defined as broad H3K4me3 genes, while genes with the top 5% H3K4me3 occupancy, not overlapping with the broach domain peaks were defined as “sharp” H3K4me3 peaks (see Fig. 2h).
We acknowledge S. Laufer, M. Miehe, and A. Domke-Shibamiya for their help performing MEF isolation, J. Spötter for MEF genotyping and mouse colony maintenance, G. Salinas-Riester for RNA sequencing, and the members of the Johnsen group for discussion.
This work was funded by The China Scholarship Council (CSC) (201206170048 to WX); the German Academic Exchange Service (DAAD) (to SN); the German Ministry for Science and Education (BMBF)-funded iBONE consortium (01KU1401A), the German Research Foundation (DFG; JO 815/3-1), and the Deutsche Krebshilfe (111600) (to SAJ).
Availability of data and materials
The data in our study have been deposited in the Gene Expression Omnibus (GEO) database under the accession number GSE72239. The published data accession numbers are in the Additional file 6: Table S5.
SAJ and WX conceived and designed the experiments; WX performed bioinformatic analyses; WX and VK performed the experiments; WX, FW, and RLK performed the mice experiments; MH and SB performed the H3K4me3 ChIP-seq data in hMSC; DI and AG helped to sequence RNA and ChIP libraries; SAJ and AM generated the conditional Rnf40 knockout mice; SAJ and WX wrote the paper. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
All animal studies were performed in compliance with the requirements of the German Animal Welfare Act. Preparation of mouse embryo fibroblasts was performed under approval of the University Hospital Hamburg-Eppendorf ethics committee (approval number ORG 673). In vivo Rnf40 deletion was performed under approval of the state of Lower Saxony (approval number 33.9-42502-04-15/2039).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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