dREAM co-operates with insulator-binding proteins and regulates expression at divergently paired genes

Abstract

dREAM complexes represent the predominant form of E2F/RBF repressor complexes in Drosophila. dREAM associates with thousands of sites in the fly genome but its mechanism of action is unknown. To understand the genomic context in which dREAM acts we examined the distribution and localization of Drosophila E2F and dREAM proteins. Here we report a striking and unexpected overlap between dE2F2/dREAM sites and binding sites for the insulator-binding proteins CP190 and Beaf-32. Genetic assays show that these components functionally co-operate and chromatin immunoprecipitation experiments on mutant animals demonstrate that dE2F2 is important for association of CP190 with chromatin. dE2F2/dREAM binding sites are enriched at divergently transcribed genes, and the majority of genes upregulated by dE2F2 depletion represent the repressed half of a differentially expressed, divergently transcribed pair of genes. Analysis of mutant animals confirms that dREAM and CP190 are similarly required for transcriptional integrity at these gene pairs and suggest that dREAM functions in concert with CP190 to establish boundaries between repressed/activated genes. Consistent with the idea that dREAM co-operates with insulator-binding proteins, genomic regions bound by dREAM possess enhancer-blocking activity that depends on multiple dREAM components. These findings suggest that dREAM functions in the organization of transcriptional domains.

Cohesin-dependent and independent mechanisms support chromosomal contacts between promoters and enhancers

“…Other factors associated with cohesinindependent interactions include chromatin regulatory proteins such as NuRD-related factor PWWP2A, DREAM complex subunit LIN-9 and histone demethylase PHF8, which are known to be involved in the transcriptional control of the cell cycle (Liu et al, 2010;Pünzeler et al, 2017;Sadasivam and DeCaprio, 2013). Both PWWP2A and LIN-9 associate with H2A.Z histone variant (Latorre et al, 2015;Pünzeler et al, 2017), while interestingly, the DREAM complex was found to possess insulator activity in Drosophila (Bohla et al, 2014;Korenjak et al, 2014). Finally, an enrichment for H3K27me3 histone mark associated with Polycomb Repressive Complexes is consistent with the independent findings of a very recent study in ES cells (Rhodes et al, 2020).…”

The Dm-Myb Oncoprotein Contributes to Insulator Function and Stabilizes Repressive H3K27me3 PcG Domains

“…To better explore the relationship between Myb and insulator proteins, we focused on divergently paired genes (DPGs) because insulator proteins as well as some dREAM complex members have been shown to be present at these sites (Korenjak et al, 2014;Nè gre et al, 2010;Yang et al, 2012). We found that Myb bound to 84% of all Drosophila DPGs (p = 2.32 3 10 À275 compared to shuffled control), similar to the percentage of DPGs bound by CP190 and BEAF-32 (each approximately 86%), but much higher than the binding observed for other insulator-associated factors (Figure 2A).…”

“…Recent studies have shown that several components of dREAM can bind insulator sites (Bohla et al, 2014;Korenjak et al, 2014), and RNAi depletion of Mip40, Mip130, and E2f2 resulted in impairment of enhancer-blocking function at specific sites (Bohla et al, 2014). Furthermore, components of dREAM (Myb, Mip120, Mip130, Rbf1, E2f2, and DP) were shown to directly interact with dCTCF and/or CP190 (Bohla et al, 2014;Korenjak et al, 2014). Interestingly, double knockdown of dCTCF and CP190 resulted in loss of dREAM (Mip40, Mip120, Mip130, and E2f2) at some shared sites (Bohla et al, 2014).…”

“…All components of the complex are present at the majority of target promoters (Georlette et al, 2007), and the absence of both Myb and E2f2 causes variegated expression of a variety of genes (Wen et al, 2008). Several studies have implicated Myb as an epigenetic regulator of gene transcription (Bohla et al, 2014;Korenjak et al, 2014;Sim et al, 2012;Wen et al, 2008); however, no clear evidence exists regarding specific mechanisms by which this epigenetic regulation is achieved.…”

L(3)mbt and the LINT complex safeguard tissue identity in the Drosophila ovary

“…However, the phenotype of lint1 loss of function is weaker than the l(3)mbt phenotype suggesting that other protein complexes contribute to L(3)mbt boundary function. L(3)mbt also associates with the dREAM complex, some components of which have been shown to cooperate with insulator proteins to maintain transcriptional integrity at a subset of divergently-paired genes in Kc167 cells (Bohla et al 2014;Korenjak et al 2014). Further, E2f2 binding sites that possess enhancer-blocking properties are all co-occupied by L(3)mbt in Kc cells ).…”