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Identification of proteins stably associated with G9a/GLP (EHMT2/EHMT1) complex of histone methyltransferases

ABSTRACT: G9a (EHMT2) and the G9a-like protein GLP (EHMT1) form a stable G9a/GLP heterodimer in embryonic stem cells and function cooperatively to establish and maintain the abundant repressive H3K9me2 modification, in addition to modifying several non-histone proteins. The G9a-dependent H3K9me2 is implicated in lineage-specific gene silencing and covers large chromosomal domains. While the mechanism of H3K9me2maintenance by G9a/GLP is known, how new patterns of this modification are established is not well understood. With this in mind, we used FLAG affinity purification of G9a under two different stringency conditions (150 and 300 mM NaCl) coupled with mass spectrometry to identify proteins stably associated with G9a/GLP, which could serve as potential recruiters of the complex to unmodified chromatin.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture, Embryonic Stem Cell

SUBMITTER: Irina Stancheva

LAB HEAD: Dr.Irina Stancheva

PROVIDER: PXD003466 | Pride | 2016-03-16

REPOSITORIES: Pride

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			Action	DRS
	130918_01_Orbi2_FLA_CO_130_IS_Burak_S1.mgf	Mgf
	130918_01_Orbi2_FLA_CO_130_IS_Burak_S1.raw	Raw
	130918_02_Orbi2_FLA_CO_130_IS_Burak_S2.mgf	Mgf
	130918_02_Orbi2_FLA_CO_130_IS_Burak_S2.raw	Raw
	130918_03_Orbi2_FLA_CO_130_IS_Burak_S3.mgf	Mgf

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GLP is critical for oogenesis exhibiting a G9A-independent role in transcriptional repression

Project description:GLP (EHMT1) functions as an H3K9me1 and H3K9me2 methyltransferase through its reportedly obligatory dimerization with G9A (EHMT2). Here, we investigated the role of GLP in oocyte and embryo development in comparison to G9A using oocyte-specific conditional knockout mouse models (G9a cKO, Glp cKO, G9a-Glp cDKO). Loss of GLP in oogenesis severely impairs oocyte maturation, fertilization and embryo development, resulting in lethality before embryonic day E12.5. In contrast, loss of G9A has a milder effect with a proportion of embryos producing viable offspring. The Glp cKO also showed loss of G9A protein and, hence, was phenotypically very similar to the G9a-Glp cDKO. H3K9me2 was equally depleted in all cKO genotypes, whereas H3K9me1 was decreased only in Glp cKO and G9a-Glp cDKO oocytes. Furthermore, the transcriptome, DNA methylome and proteome were markedly more affected in G9a-Glp cDKO than G9a cKO oocytes, demonstrating that in the absence of GLP there are widespread epigenetic and gene expression changes in the oocyte independent of H3K9me2. Gene dysregulation with coupled changes in DNA methylation suggest localised loss of chromatin repression, resulting in upregulated protein expression. Together, our findings demonstrate that GLP can function independently of G9A in the oocyte and is required for oocyte developmental competence.

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Investigation of the role of histone modification propagating activity of GLP

Project description:We use ChIP-Seq and RNA-Seq technology to profile the H3K9me2 modification and transcription under different conditions of GLP activity. GLP and G9a are major H3K9 dimethylases, and are essential for mouse early embryonic development. Here we report that GLP and G9a possess intrinsic histone methylation propagating activities. The histone methyltransferase activities of GLP and G9a are stimulated by neighboring nucleosomes pre-methylated at H3K9. These stimulation events function in cis and are dependent on H3K9 methylation binding activities of ankyrin repeats domains in GLP and G9a. In mouse embryonic stem cells (ESCs) harboring a mutant GLP lacking H3K9 methylation propagating activity, pluripotent genes display a delayed kinetics in establishing H3K9 methylation and gene silencing during differentiation. Disruption of the H3K9 methylation propagating activity of GLP in mice causes growth retardation of the embryos, ossification defects of calvaria and early postnatal lethality. We propose that GLP¡¯s ability to rapidly propagate H3K9 methylation is required for efficient gene silencing during programmed cell fate transition. H3K9me2 and H3K9me1 are ChIPped and sequenced in WT mESC and GLP-mutant mESCs, and RNA-Seq was done for those cells as well.

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Multi-omics analyses demonstrate a critical role for EHMT1 methyltransferase in transcriptionalrepression during oogenesis

Project description:EHMT1 (also known as GLP) is a multifunctional protein, best known for its role as an H3K9me1 and H3K9me2 methyltransferase through its reportedly obligatory dimerization with EHMT2 (also known as G9A). Here, we investigated the role of EHMT1 in the oocyte in comparison to EHMT2 using oocyte-specific conditional knockout mouse models (Ehmt2 cKO, Ehmt1cKO, Ehmt1/2 cDKO), with ablation from the early phase of oocyte growth. Loss of EHMT1 in Ehmt1 cKO and Ehmt1/2 cDKO oocytes recapitulated meiotic defects observed in the Ehmt2 cKO; however, there was a significant impairment in oocyte maturation and developmental competence in Ehmt1 cKO and Ehmt1/2 cDKO oocytes beyond that observed in the Ehmt2cKO. Consequently, loss of EHMT1 in oogenesis results, upon fertilization, in mid-gestation embryonic lethality. To identify H3K9 methylation and other meaningful biological changes in each mutant to explore the molecular functions of EHMT1 and EHMT2, we performed immunofluorescence imaging, multi-omics sequencing, and mass spectrometry (MS)–based proteome analyses in cKO oocytes. Although H3K9me1 was depleted only upon loss of EHMT1, H3K9me2 was decreased, and H3K9me2-enriched domains were eliminated equally upon loss of EHMT1 or EHMT2. Furthermore, there were more significant changes in the transcriptome, DNA methylome, and proteome in Ehmt1/2 cDKO than Ehmt2 cKO oocytes, withtranscriptional derepression leading to increased protein abundance and local changes in genic DNA methylation in Ehmt1/2 cDKO oocytes. Together, our findings suggest that EHMT1 contributes to local transcriptional repression in the oocyte, partially independent of EHMT2, and is critical for oogenesis and oocyte developmental competence

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G9a/GLP-dependent H3K9me2 patterning alters chromatin structure at CpG islands in hematopoietic progenitors.

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