Regulation of gene expression via histone post-translational modification has been the focus of many studies, and understanding how various epigenetic constellations impact gene transcription has become very important, especially as more histone marks are being identified and implicated in both transcriptional activation as well as repression. Studies by various groups have shown that there are different molecular effectors capable of recognizing and binding specific histones marks; however, their mode of action remains obscure. For instance, recent work by Xhemalce and Kouzaridez showed that acetylation of H3K4, a mark generally associated with gene activation, plays a major role in the formation of repressive heterochromatin in Schizosaccharomyces pombe
by promoting a switch in binding of HP1-like proteins to methylated H3K9 (38
). This finding redefines the notion that there is a strict code of histone modifications that specifies a particular transcriptional outcome, and suggests that both activation and repression-specific marks could play dual roles during gene transcription. Another example of the complexity of mechanisms used to regulate chromatin structure comes from studies where WDR5, a component of MLL complexes that binds methylated H3K4, can be found in association with the H3(Me3
)K27-specific demethylase KDM6A/UTX (39
), indicating that histone-binding proteins can interact with both histone methyltransferases as well as histone demethylases to promote either gene activation or repression.
In this study, we have identified BRD7 as a component of the PRMT5–hSWI–SNF complex, and using stable cell lines that express epitope-tagged BRD7 complexes we have shown that PRMT5 and hSWI–SNF subunits co-purify with His-BRD7. We have also determined that BRD7 interacts with specific hSWI–SNF subunits including BRG1, BRM and BAF60, as well as PRMT5 and MEP50. Moreover, we have found that BRD7 can also interact with components of the PRC2 repressor complex in vitro
. Our previous work showed that hSWI–SNF-associated PRMT5 and its epigenetic marks are enriched at the promoter region of ST7 and RBL2
tumor suppressor genes (23
). Therefore, we investigated the involvement of BRD7 and PRC2 in PRMT5 target gene regulation. Both ChIP and ChIP-re-ChIP analyses indicated that BRD7 and PRC2 co-localize with PRMT5 on ST7 and RBL2
target promoters in vivo
. To gain a better understanding of the role played by BRD7 in ST7 and RBL2
transcriptional regulation, we knocked down its expression and measured the ST7 and RBL2
mRNA levels. Our findings clearly show that reducing expression of BRD7 triggers transcriptional derepression of ST7 as evidenced by the increase in ST7 mRNA levels in BRD7 knockdown cells; however, transcription of RBL2
was not affected even though recruitment of both PRMT5 and PRC2 was compromised in BRD7 knockdown cells.
To determine if lack of recruitment of PRMT5 and PRC2 could result in a decrease in methylation of histones H3 and H4, we used ChIP assays to measure the levels of PRMT5 and PRC2-induced epigenetic marks at the ST7 and RBL2
promoters in both control as well as BRD7 knockdown cells. In accordance with the real-time RT–PCR results (C), we discovered that H3(Me2
)R3 and H3(Me3
)K27 epigenetic marks were completely removed from the ST7 promoter region. When we checked the promoter region of RBL2
, we found that while methylation of H3R8 and H3K27 was reduced in the absence of BRD7, symmetric methylation of H4R3 was unaffected, raising the possibility that another type II PRMT might be involved in RBL2
transcriptional regulation. To date the only other type II PRMT known for its ability to symmetrically methylate H4R3 is PRMT7 (34
). Therefore, we tested its association with the RBL2
promoter in WaC3CD5 cells. Our results indicated that even though PRMT7 was recruited to two other tumor suppressor genes, GAS1 and GAS2, its association with the RBL2
promoter was not increased.
Since it has previously been shown that lysine methylation marks are removed by specific KDMs, and recent studies have indicated that histone arginine methylation can be erased by Jumonji C domain-containing RDMs, we checked if there was altered recruitment of lysine and arginine demethylases to the ST7 and RBL2 promoters.
ChIP analysis of H3(Me3)K27-specific demethylases showed that while both KDM6A/UTX and KDM6B/JMJD3 were efficiently recruited to the ST7 promoter, only KDM6A/UTX was associated with RBL2. In addition, recruitment studies of the H4(Me2)R3-specific demethylase JMJD6 revealed that its association with the ST7 promoter is highly enriched in BRD7 knockdown cells, while its binding to the RBL2 promoter is lacking in these cells. These data are in complete agreement with the ChIP results obtained using antibodies specific to PRMT5- and PRC2-induced methylation marks (). Although it is not clear if binding of BRD7 to PRMT5 and PRC2 target genes precludes recruitment of JMJD6, KDM6A/UTX and KDM6B/JMJD3, our results provide important clues about the function of BRD7 in recruiting PRMT5 and PRC2 repressors, and suggest that transcriptional activation of repressed genes does not only require release of enzymes involved in inducing specific epigenetic marks, but also efficient recruitment of different combinations of lysine and arginine-specific demethylases.