Epigenetic cellular memory mechanisms that involve polycomb and trithorax group of proteins are well conserved across metazoans. The cis-acting elements interacting with these proteins, however, are poorly understood in mammals. In a directed search we identified a potential polycomb responsive element with 25 repeats of YY1 binding motifthatwe designate PRE-PIK3C2B as it occurs in the first intron of human PIK3C2B gene. It down regulates reporter gene expression in HEK cells and the repression is dependent on polycomb group of proteins (PcG). We demonstrate that PRE-PIK3C2B interacts directly with YY1 in vitro and recruits PRC2 complex in vivo. The localization of PcG proteins including YY1 to PRE-PIK3C2B in HEK cells is decreased on knock-down of either YY1 or SUZ12. Endogenous PRE-PIK3C2B shows bivalent marking having H3K27me3 and H3K4me3 for repressed and active state respectively. In transgenic Drosophila, PRE-PIK3C2B down regulates mini-white expression, exhibits variegation and pairing sensitive silencing (PSS), which has not been previously demonstrated for mammalian PRE. Taken together, our results strongly suggest that PRE-PIK3C2B functions as a site of interaction for polycomb proteins.
Ino80 is well known as a chromatin remodeling protein with the catalytic function of DNA dependent ATPase and is highly conserved across phyla. Ino80 in human and Drosophila is known to form the Ino80 complex in association with the DNA binding protein Ying-Yang 1 (YY1)/Pleiohomeotic (Pho) the Drosophila homologue. We have earlier reported that Ino80 sub-family of proteins has two functional domains, namely, the DNA dependent ATPase and the DNA binding domain. In the background of the essential role of dIno80 in development, we provide evidence of Pho independent function of dIno80 in development and analyze the dual role of dIno80 in activation as well as repression in the context of the homeotic gene Scr (sex combs reduced) in imaginal discs. This differential effect of dIno80 in different imaginal discs suggests the contextual function of dIno80 as an Enhancer of Trithorax and Polycomb (ETP). We speculate on the role of dIno80 as a chromatin remodeler on one hand and a potential recruiter of epigenetic regulatory complexes on the other.
The chromatin remodeling protein, dIno80 (Drosophila Ino80) regulates homeotic genes. We show that Ino80, along with Trx and ETP (Enhancer of Trithorax and Polycomb) proteins, interacts with two Polycomb/Trithorax Responsive Elements (PRE/TRE), iab-7 and bxd PRE in flies and the larval imaginal discs. In S2 cells, dIno80 localizes to the endogenous iab-7and bxd-PREs. The localization of Ino80 and Pleiohomeotic (Pho) at the PRE is sensitive to the cellular abundance of each other; when levels of Ino80 are limiting, there is increased Pho enrichment, and Pho knock-down leads to increased enrichment of Ino80. We demonstrate that over-expression of dIno80 rescues the pupal lethality in pleiohomeotic (pho) deficient flies, which suggests that dIno80 has a role in cellular memory. The apparent competition between Pho and Ino80 for binding at the PRE indicates that Ino80 may act as a potential recruiter of the regulatory complex in addition to being a chromatin remodeler.
Author SummaryThe null mutants of Pho and dIno80 show lethality at different stages of development in the fly, implying that they may function independent of each other. The observation that Pho-lethality can be rescued by overexpression of dIno80 with significant penetrance and that Ino80 has its own DNA binding domain, led us to predict that Ino80 may have Pho-independent functions, perhaps through non-canonical complexes. In the current study, we show that dIno80 interacts with bxd and iab-7 PRE in cooperation with Polycomb and Trithorax proteins and regulate the homeotic genes. The effect of knock-down or mutation of dIno80 results in altered phenotype in adult flies and rescue of Lac-Z expression in imaginal discs, in parallel with similar effect of Pho mutation or knock-down. We provide evidence of direct interaction of dIno80 with iab7-and bxd-PRE using chromatin immunoprecipitation. The dIno80 localization in and around the PRE sequence was enhanced in the absence of Pho, indicating competition between Pho and dIno80 for binding at the PRE.
The epigenetic mechanisms regulating developmental gene expression are examples of a strategy to generate unique expression profiles with global regulators controlling several genes. In a simplified view, a common set of tools, that include DNA motif recognizing proteins (recruiters), binding/interacting surfaces (ARPs- actin related proteins), epigenetic writers (histone methyltransferases, acetylases), readers (chromatin remodeling proteins, PRC1 members) and erasers (demethylases, deacetylases) form complexes which not only regulate transcription, but also retain the transcriptional memory through mitosis. There are two arms of epigenetic regulation: covalent modification of DNA and the post-translational modification of histones. In this review, we discuss both of these aspects briefly to illustrate functional diversity. We discuss our efforts at utilization of the genome sequence data for de novo identification of new players and their functional validation in this remarkable process.
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