The N-terminus of Drosophila ESC mediates its phosphorylation and dimerization

Tie, Feng; Siebold, Alex P.; Harte, Peter J.

doi:10.1016/j.bbrc.2005.04.157

Cited by 16 publications

(28 citation statements)

References 22 publications

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“…Expression of FLAG-ESC proteins (full-length, 61 to 425, and 1 to 169) and GST protein in Drosophila S2 cells was driven by the inducible metallothionein promoter of pRMHA3 as previously described (38). pRMHA3 constructs for expressing FLAG-PC and FLAG-ESCL were generated similarly by PCR with NheI-NsiI sites flanking the coding sequence.…”

Section: Methodsmentioning

confidence: 99%

“…We previously showed that the WD region of ESC binds directly to the N terminus of E(Z) (35), while the N terminus of ESC (residues 1 to 60) mediates ESC dimerization and phosphorylation (38). ESC appears to be required for stable association of E(Z) with the 600-kDa complex in vivo or for the stability of E(Z) itself, since we detected no E(Z) in the 600-kDa complex in esc mutant extracts that contain no ESC protein (12).…”

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confidence: 99%

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The N Terminus of Drosophila ESC Binds Directly to Histone H3 and Is Required for E(Z)-Dependent Trimethylation of H3 Lysine 27

Tie

Stratton

Kurzhals

et al. 2007

Molecular and Cellular Biology

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Polycomb group proteins mediate heritable transcriptional silencing and function through multiprotein complexes that methylate and ubiquitinate histones. The 600-kDa E(Z)/ESC complex, also known as Polycomb repressive complex 2 (PRC2), specifically methylates histone H3 lysine 27 (H3 K27) through the intrinsic histone methyltransferase (HMTase) activity of the E(Z) SET domain. By itself, E(Z) exhibits no detectable HMTase activity and requires ESC for methylation of H3 K27. The molecular basis for this requirement is unknown. ESC binds directly, via its C-terminal WD repeats (␤-propeller domain), to E(Z). Here, we show that the N-terminal region of ESC that precedes its ␤-propeller domain interacts directly with histone H3, thereby physically linking E(Z) to its substrate. We show that when expressed in stable S2 cell lines, an N-terminally truncated ESC (FLAG-ESC61-425), like full-length ESC, is incorporated into complexes with E(Z) and binds to a Ubx Polycomb response element in a chromatin immunoprecipitation assay. However, incorporation of this N-terminally truncated ESC into E(Z) complexes prevents trimethylation of histone H3 by E(Z). We also show that a closely related Drosophila melanogaster paralog of ESC, ESC-like (ESCL), and the mammalian homolog of ESC, EED, also interact with histone H3 via their N termini, indicating that the interaction of ESC with histone H3 is evolutionarily conserved, reflecting its functional importance. Our data suggest that one of the roles of ESC (and ESCL and EED) in PRC2 complexes is to enable E(Z) to utilize histone H3 as a substrate by physically linking enzyme and substrate.Polycomb group (PcG) and Trithorax group (TrxG) proteins are required for maintaining stable heritable expression patterns of many developmentally important genes (2, 32). Mutations of PcG genes and TrxG genes cause abnormal development and disease in mammals (3,14,17,40), which has spurred broad interest in the mechanisms underlying PcG protein-mediated gene silencing and TrxG protein-mediated gene expression. Much attention has focused on the covalent modifications of histones by PcG and TrxG protein complexes. Some noteworthy discoveries have included the identification of a histone H3 and H4 methyltransferase activity possessed by the SET domain-containing proteins SU(VAR)3-9, E(Z), TRX, and ASH1 (22).The Drosophila melanogaster 600-kDa ESC/E(Z) complex, also known as Polycomb repressive complex 2 (PRC2), which contains the PcG proteins ESC, E(Z), and SU(Z)12 as well as the histone H4 binding protein p55 (ortholog of mammalian RbAp48 and RbAp46), is recruited to specialized Polycomb response elements (PREs) and methylates histone H3 K27 in the surrounding chromatin of PcG target genes (4,7,16,25). A second PcG complex, PRC1, binds to the methylated H3 K27 and silences the promoter. The precise mechanism of silencing remains poorly understood, but silencing is lost when K27 methylation by E(Z) is perturbed.Mono-, di-, and trimethylated forms of H3 K27 (1me-, 2me-, 3meH3K27) can be detected in vi...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

The N Terminus of Drosophila ESC Binds Directly to Histone H3 and Is Required for E(Z)-Dependent Trimethylation of H3 Lysine 27

Tie

Stratton

Kurzhals

et al. 2007

Molecular and Cellular Biology

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“…Goat anti-GST antibody was from Amersham. Rabbit anti-E(Z), anti-ESC and anti-RPD3 antibodies, and guinea pig anti-SU(Z)12 and anti-ESCL antibodies were described previously (Tie et al, 2001;Tie et al, 2003;Tie et al, 2005;Tie et al, 2007). Rabbit anti-GCN5 antibody was kindly provided by Jerry Workman (Kusch et al, 2003).…”

Section: Antibodiesmentioning

confidence: 99%

“…Drosophila S2 Cells were transiently transfected with plasmid DNA (pMT-CBP and pAct-GST-H3) using SuperFect Transfection Reagent (Qiagen) as described (Tie et al, 2005). After a 24-hour transfection, cells were treated with a final concentration of 0.5 mM CuSO 4 and 5 mM sodium butyrate for 2 days, then whole-cell extracts were prepared for western analysis.…”

Section: Transient Transfection Assaymentioning

confidence: 99%

CBP-mediated acetylation of histone H3 lysine 27 antagonizesDrosophilaPolycomb silencing

et al. 2009

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Trimethylation of histone H3 lysine 27 (H3K27me3) by Polycomb repressive complex 2 (PRC2) is essential for transcriptional silencing of Polycomb target genes, whereas acetylation of H3K27 (H3K27ac) has recently been shown to be associated with many active mammalian genes. The Trithorax protein (TRX),which associates with the histone acetyltransferase CBP, is required for maintenance of transcriptionally active states and antagonizes Polycomb silencing, although the mechanism underlying this antagonism is unknown. Here we show that H3K27 is specifically acetylated by Drosophila CBP and its deacetylation involves RPD3. H3K27ac is present at high levels in early embryos and declines after 4 hours as H3K27me3 increases. Knockdown of E(Z)decreases H3K27me3 and increases H3K27ac in bulk histones and at the promoter of the repressed Polycomb target gene abd-A, suggesting that these indeed constitute alternative modifications at some H3K27 sites. Moderate overexpression of CBP in vivo causes a global increase in H3K27ac and a decrease in H3K27me3, and strongly enhances Polycomb mutant phenotypes. We also show that TRX is required for H3K27 acetylation. TRX overexpression also causes an increase in H3K27ac and a concomitant decrease in H3K27me3 and leads to defects in Polycomb silencing. Chromatin immunoprecipitation coupled with DNA microarray (ChIP-chip) analysis reveals that H3K27ac and H3K27me3 are mutually exclusive and that H3K27ac and H3K4me3 signals coincide at most sites. We propose that TRX-dependent acetylation of H3K27 by CBP prevents H3K27me3 at Polycomb target genes and constitutes a key part of the molecular mechanism by which TRX antagonizes or prevents Polycomb silencing.

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“…One interpretation of this result is that the larger complexes include additional subunits for which recruitment is Pcl2 dependent. Alternatively, high molecular weight species could arise through multimerisation of core PRC2 complexes, an idea favoured by recent studies revealing that the N-terminus of the core PRC2 protein Eed/Esc interacts with histone H3 and dimerises in a phosphorylationdependent manner (Tie et al, 2005;Margueron et al, 2009). Although we cannot differentiate between these possibilities, the absence of additional stoichiometric components in purified Pcl2-PRC2 and the fact that Pcl2 depletion affects the biochemical properties of total PRC2, only a small proportion of which is associated with Pcl2 at any given time, lead us to favour the idea that Pcl2 mediates the formation of higher-order PRC2 complexes.…”

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Polycomblike 2 facilitates the recruitment of PRC2 Polycomb group complexes to the inactive X chromosome and to target loci in embryonic stem cells

et al. 2011

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SUMMARYPolycomb group (PcG) proteins play an important role in the control of developmental gene expression in higher organisms. In mammalian systems, PcG proteins participate in the control of pluripotency, cell fate, cell cycle regulation, X chromosome inactivation and parental imprinting. In this study we have analysed the function of the mouse PcG protein polycomblike 2 (Pcl2), one of three homologues of the Drosophila Polycomblike (Pcl) protein. We show that Pcl2 is expressed at high levels during early embryogenesis and in embryonic stem (ES) cells. At the biochemical level, Pcl2 interacts with core components of the histone H3K27 methyltransferase complex Polycomb repressive complex 2 (PRC2), to form a distinct substoichiometric biochemical complex, Pcl2-PRC2. Functional analysis using RNAi knockdown demonstrates that Pcl2-PRC2 facilitates both PRC2 recruitment to the inactive X chromosome in differentiating XX ES cells and PRC2 recruitment to target genes in undifferentiated ES cells. The role of Pcl2 in PRC2 targeting in ES cells is critically dependent on a conserved PHD finger domain, suggesting that Pcl2 might function through the recognition of a specific chromatin configuration. KEY WORDS: ES cell, Polycomb, X inactivation, MousePolycomblike 2 facilitates the recruitment of PRC2 Polycomb group complexes to the inactive X chromosome and to target loci in embryonic stem cells Li et al., 2010; Landeira et al., 2010) but its function is as yet undetermined. Finally, the Polycomblike (Pcl) protein associates with PRC2 in Drosophila (O'Connell et al., 2001;Tie et al., 2003;Papp and Muller, 2006) and in mammalian cells (Cao et al., 2008;Sarma et al., 2008) and has been proposed to have a role in stimulating H3K27me3 activity and/or targeting of the complex (Nekrasov et al., 2007;Cao et al., 2008;Sarma et al., 2008). In this study we have analysed the function of the mouse Pcl2 (Mtf2 -Mouse Genome Informatics) protein, one of three homologues of Pcl found in mammalian cells. We find that Pcl2 is expressed at high levels during early embryogenesis and in ES cells. Pcl2 interacts with the core PRC2 complex to form a stable and distinct biochemical complex, Pcl2-PRC2. Functional analysis using RNAi knockdown demonstrates that Pcl2-PRC2 is important in PRC2 recruitment to the Xi in differentiating XX ES cells and also for PRC2 recruitment to target genes in undifferentiated ES cells. A conserved PHD finger domain in Pcl2 is required for PRC2 targeting in ES cells. MATERIALS AND METHODS Cell lines and embryosGrowth and maintenance of trophoblast stem (TS) and ES cell lines were as described previously (Penny et al., 1996;Mak et al., 2002). ES cell differentiation was achieved by embryoid body formation via removal of LIF from the medium. The MP fibroblast cell line was derived in house from a male PGK mouse.Preimplantation and postimplantation mouse embryos were obtained from timed mating of C57B16ϫCBA F1 animals as described previously (Sheardown et al., 1997). Immunofluorescence (IF) and RNA fluorescent in ...

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The N-terminus of Drosophila ESC mediates its phosphorylation and dimerization

Cited by 16 publications

References 22 publications

The N Terminus of Drosophila ESC Binds Directly to Histone H3 and Is Required for E(Z)-Dependent Trimethylation of H3 Lysine 27

The N Terminus of Drosophila ESC Binds Directly to Histone H3 and Is Required for E(Z)-Dependent Trimethylation of H3 Lysine 27

CBP-mediated acetylation of histone H3 lysine 27 antagonizesDrosophilaPolycomb silencing

Polycomblike 2 facilitates the recruitment of PRC2 Polycomb group complexes to the inactive X chromosome and to target loci in embryonic stem cells

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