Mediator coordinates PIC assembly with recruitment of CHD1

Department of Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, California 90095, USA.
Genes & development (Impact Factor: 10.8). 10/2011; 25(20):2198-209. DOI: 10.1101/gad.17554711
Source: PubMed


Murine Chd1 (chromodomain helicase DNA-binding protein 1), a chromodomain-containing chromatin remodeling protein, is necessary for embryonic stem (ES) cell pluripotency. Chd1 binds to nucleosomes trimethylated at histone 3 Lys 4 (H3K4me3) near the beginning of active genes but not to bivalent domains also containing H3K27me3. To address the mechanism of this specificity, we reproduced H3K4me3- and CHD1-stimulated gene activation in HeLa extracts. Multidimensional protein identification technology (MuDPIT) and immunoblot analyses of purified preinitiation complexes (PICs) revealed the recruitment of CHD1 to naive chromatin but enhancement on H3K4me3 chromatin. Studies in depleted extracts showed that the Mediator coactivator complex, which controls PIC assembly, is also necessary for CHD1 recruitment. MuDPIT analyses of CHD1-associated proteins support the recruitment data and reveal numerous components of the PIC, including Mediator. In vivo, CHD1 and Mediator are recruited to an inducible gene, and genome-wide binding of the two proteins correlates well with active gene transcription in mouse ES cells. Finally, coimmunoprecipitation of CHD1 and Mediator from cell extracts can be ablated by shRNA knockdown of a specific Mediator subunit. Our data support a model in which the Mediator coordinates PIC assembly along with the recruitment of CHD1. The combined action of the PIC and H3K4me3 provides specificity in targeting CHD1 to active genes.

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    • "Immobilized template assays were performed as described ( Kitada et al . 2012 ) using G5E4T assembled into chromatin ( Lin et al . 2011 ; Kuryan et al . 2012 ) and incubated with either Ino80C or a mix of Sir3 and Sir2 / 4 in 25 mL of binding buffer ( 100 mM KOAc , 20 mM HEPES at pH 7 . 6 , 1 mM EDTA , 10% glycerol ) for 1 h at 30°C with a subsequent 45 - min incubation of the second protein ."
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    • "Chromodomain helicase DNA-binding protein 1 (Chd1) is a conserved protein associated with decondensed chromatin and transcription in several species (Sims et al., 2005; Stokes et al., 1996; Woodage et al., 1997). Chd1 binds with high specificity to H3K4me2/3 (Flanagan et al., 2005; Sims et al., 2007) and enhances the transcriptional activity of RNAP II on a synthetic chromatin template (Lin et al., 2011). Chd1 has recently been shown to remove the barrier of promoter-proximal nucleosomes and facilitate release of RNAP II into productive elongation (Skene et al., 2014). "
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    • "The first of these identified is human CHD1, which binds H3K4me3 through its tandem chromodomains [28] [29]. Utilizing both in vitro reconstituted transcriptional assays as well as in vivo binding assays, CHD1 was shown to be recruited to chromatin via interaction with the mediator complex, but that H3K4me3 stabilized its chromatin interaction [30]. Aside from an ability to enhance chromatin binding and ultimately transcription, the downstream mechanisms facilitated by the CHD1–H3K4me3 interaction are not entirely clear; however, studies show that CHD1 plays a major role in the deposition of H3.3 implying that H3K4me3 may be linked to transcription-coupled histone variant deposition at gene promoters [31]. "
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