17-10034 Sigma-AldrichChIPAb+ EED - ChIP Validated Antibody and Primer Set

During B lymphopoiesis, recombination of the locus encoding the immunoglobulin κ-chain complex (Igk) requires expression of the precursor to the B cell antigen receptor (pre-BCR) and escape from signaling via the interleukin 7 receptor (IL-7R). By activating the transcription factor STAT5, IL-7R signaling maintains proliferation and represses Igk germline transcription by unknown mechanisms. We demonstrate that a STAT5 tetramer bound the Igk intronic enhancer (E(κi)), which led to recruitment of the histone methyltransferase Ezh2. Ezh2 marked trimethylation of histone H3 at Lys27 (H3K27me3) throughout the κ-chain joining region (J(κ)) to the κ-chain constant region (C(κ)). In the absence of Ezh2, IL-7 failed to repress Igk germline transcription. H3K27me3 modifications were lost after termination of IL-7R-STAT5 signaling, and the transcription factor E2A bound E(κi), which resulted in acquisition of H3K4me1 and acetylated histone H4 (H4Ac). Genome-wide analyses showed a STAT5 tetrameric binding motif associated with transcriptional repression. Our data demonstrate how IL-7R signaling represses Igk germline transcription and provide a general model for STAT5-mediated epigenetic transcriptional repression.

X-chromosome inactivation (XCI) depends on the noncoding Xist gene. Xist transcription is negatively regulated by its antisense partner Tsix, whose disruption results in nonrandom XCI in females. However, males can maintain Xist in a repressed state without Tsix, indicating participation of additional factor(s) in the protection of the single male X from inactivation. Here, we provide evidence that the histone methyltransferase Eed is also involved in the process. Male embryonic stem cells with Eed-null and Tsix mutations (X(Delta)Y Eed-/-) showed Xist hyperactivation upon differentiation, whereas cells with either mutation alone did not. Impaired X-linked gene expression was observed in the X(Delta)Y Eed-/- ES cells at the onset of differentiation. The Xist promoter in the X(Delta)Y Eed-/- cells showed elevated histone H3-dimethyl lysine 4 modifications and lowered CpG methylation, which are characteristics of open chromatin. Hence, we identified Eed as an additional major player in the regulation of Xist expression. The synergy of Polycomb group proteins and antisense Tsix transcription in Xist gene regulation explains why males can repress Xist without Tsix.

PcG proteins mediate heritable transcriptional silencing by generating and recognizing covalent histone modifications. One conserved PcG complex, PRC2, is composed of several proteins including the histone methyltransferase (HMTase) Ezh2, the WD-repeat protein Eed, and the Zn-finger protein Suz12. Ezh2 methylates histone H3 on lysine 27 (H3K27), which serves as an epigenetic mark mediating silencing. H3K27 can be mono-, di-, or trimethylated (1mH3K27, 2mH3K27, and 3mH3K27, respectively). Hence, either PRC2 must be regulated so as to add one methyl group to certain nucleosomes but two or three to others, or distinct complexes must be responsible for 1m-, 2m-, and 3mH3K27. Consistent with the latter possibility, 2mH3K27 and 3mH3K27, but not 1mH3K27, are absent in Suz12-/- embryos, which lack both Suz12 and Ezh2 protein. Mammalian proteins required for 1mH3K27 have not been identified. Here, we demonstrate that unlike Suz12 and Ezh2, Eed is required not only for 2m- and 3mH3K27 but also global 1mH3K27. These results provide a functionally important distinction between PRC2 complex components and implicate Eed in PRC2-independent histone methylation.

Human Enhancer of Zeste homolog (Ezh2) is a histone lysine methyltransferase (HKMT) associated with transcriptional repression. Ezh2 is present in several distinct complexes, one of which, PRC2, we characterized previously. Here we report an additional Ezh2 complex, PRC3. We show that the Ezh2 complexes exhibit differential targeting of specific histones for lysine methylation dependent upon the context of the histone substrates. This differential targeting is a function of the associated Eed protein within each complex. We found that Eed protein is present in four isoforms, which represent alternate translation start site usage from the same mRNA. These Eed isoforms selectively associate with distinct Ezh2-containing complexes with resultant differential targeting of their associated HKMT activity toward histone H3-K27 or histone H1-K26. Our data provide evidence for a novel mechanism regulating the substrate specificity of a chromatin-modifying enzyme through disparate translational products of a regulatory subunit.

Polycomb-group (PcG) proteins form multimeric protein complexes, which are involved in maintaining the transcriptional repressive state of genes over successive cell generations. Components of PcG complexes and their mutual interactions have been identified and analysed through extensive genetic and biochemical analyses. Molecular mechanisms underlying PcG-mediated repression of gene activity, however, have remained largely unknown. Previously we reported the existence of two distinct human PcG protein complexes. The EED/EZH protein complex contains the embryonic ectoderm development (EED) and enhancer of zeste 2 (EZH2; refs 9,10) PcG proteins. The HPC/HPH PcG complex contains the human polycomb 2 (HPC2; ref. 11), human polyhomeotic (HPH), BMI1 (ref. 13 ) and RING1 (refs 14, 15) proteins. Here we show that EED (refs 4, 5, 6, 7, 8) interacts, both in vitro and in vivo, with histone deacetylase (HDAC) proteins. This interaction is highly specific because the HDAC proteins do not interact with other vertebrate PcG proteins. We further find that histone deacetylation activity co-immunoprecipitates with the EED protein. Finally, the histone deacetylase inhibitor trichostatin A (ref. 17) relieves transcriptional repression mediated by EED, but not by HPC2, a human homologue of polycomb. Our data indicate that PcG-mediated repression of gene activity involves histone deacetylation. This mechanistic link between two distinct, global gene repression systems is accomplished through the interaction of HDAC proteins with a particular PcG protein, EED.