Find the principal on amount 11800 at 6

Abstract

The Polycomb group of proteins is required for the proper orchestration of gene expression due to its role in maintaining transcriptional silencing. It is composed of several chromatin modifying complexes, including Polycomb Repressive Complex 2 [PRC2], which deposits H3K27me2/3. Here, we report the identification of a cofactor of PRC2, EZHIP [EZH1/2 Inhibitory Protein], expressed predominantly in the gonads. EZHIP limits the enzymatic activity of PRC2 and lessens the interaction between the core complex and its accessory subunits, but does not interfere with PRC2 recruitment to chromatin. Deletion of Ezhip in mice leads to a global increase in H3K27me2/3 deposition both during spermatogenesis and at late stages of oocyte maturation. This does not affect the initial number of follicles but is associated with a reduction of follicles in aging. Our results suggest that mature oocytes Ezhip−/− might not be fully functional and indicate that fertility is strongly impaired in Ezhip−/− females. Altogether, our study uncovers EZHIP as a regulator of chromatin landscape in gametes.

Introduction

Early in development, cells commit to specific lineages and acquire precise identities that require maintenance throughout the lifespan of the organism. Polycomb group proteins play an important role in this process by maintaining transcriptional repression through the regulation of chromatin structure. In mammals, this machinery is composed of two main complexes: Polycomb Repressive Complex 1 and 2 [PRC1 and 2]. The core PRC2 complex is composed of four subunits: the catalytic subunit EZH1/2, SUZ12, EED, and RbAp46/48. PRC2 catalyzes the di- and tri-methylation of lysine 27 on histone H3 [H3K27me2/3], an enzymatic activity which is required for its function. Indeed, the mutation of lysine 27 of histone H3 to arginine leads to loss of gene repression, and mutant flies display a phenotype similar to deletion of PRC2 components. H3K27me3 is generally enriched around the promoter of transcriptionally silent genes and contributes to the recruitment of PRC1. H3K27me2 is widely distributed, covering 50–70% of histones, and its role is less defined but may be to prevent aberrant enhancer activation.

The question of how PRC2 is targeted to chromatin and how its enzymatic activity is controlled has received ongoing attention. Cumulative evidence suggests that PRC2 may not be actively recruited to chromatin and that, instead its activity, is promoted by the recognition of its own mark H3K27me3, ubiquitination of lysine 119 of H2A, GC-richness, or by condensed chromatin. Conversely, some histone modifications negatively influence PRC2 function, particularly those associated with active transcription, such as H3K4me3 and H3K36me3. PRC2 binding to chromatin may also be inhibited by DNA methylation, although other reports suggest that PRC2 is compatible with DNA methylation.

A number of accessory subunits have now been shown to influence PRC2 function. Recent comprehensive proteomic analyses suggest that they might form around two main PRC2 subtypes, PRC2.1 and PRC2.2. The subunit SUZ12 plays a central role by orchestrating the cofactor interactions. PRC2.1 includes one of the three Polycomb-like proteins [PHF1, MTF2 or PHF19] together with the recently identified PRC2 partners EPOP and PALI1,. The three Polycomb-like proteins harbor one Tudor domain and two PHD finger domains each. Their Tudor domain is able to recognize H3K36me3 decorated genes, which could be important for PRC2 association with transcribed targets. The function of EPOP remains ambiguous since, in vitro, it stimulates PRC2 catalytic activity while, in vivo, it limits PRC2 binding, likely through interaction with Elongin BC. In contrast, PALI1 is required for H3K27me3 deposition both in vitro and in vivo. The other complex, PRC2.2, includes JARID2 and AEBP2 subunits in equal stoichiometry,. Both are able to stimulate PRC2 catalytic activity in vitro with JARID2 being also able to bind nucleosomes. JARID2 also appears to be necessary for PRC2 targeting at its loci, possibly through its DNA-binding domain or as a result of its methylation by PRC2. AEBP2 binds to DNA in vitro, but appears to negatively modulate PRC2 in vivo,. Of note, AEBP2 was reported to stimulate PRC2 through a mechanism independent of PRC2 allosteric activation,. While we now have a good picture of the accessory subunits interacting with PRC2, their precise roles are only partially understood. This might be due to compensatory mechanisms, such that interfering both with PRC2.1 and PRC2.2 is required to inhibit PRC2 recruitment as observed upon loss of SUZ12.

The regulation of chromatin structure in germ cells is pivotal, as these cells are the bridge between generations and therefore potential vector of epigenetic information. In particular, H3K27me3 has been shown to be involved in parental imprinting,,. Yet, in contrast to the extensive characterization of PRC2 in models, such as mouse embryonic stem cells [ESC], much less is known about the regulation of its enzymatic activity in germ cells. Deletion of PRC2 core components during spermatogenesis results in the progressive loss of germ cells, indicating that its activity is required for this process,. At later stages of spermatogenesis, when round spermatids differentiate into mature sperm, histones are progressively replaced by protamines. A variable fraction of the genome retains a nucleosomal structure [1% in mice, 10–15% in human], with histones carrying posttranslational modifications, including H3K27me3. During oogenesis, histones are maintained and H3K27me3 is detected throughout this process,,. However, H3K27me3 displays a peculiar pattern of enrichment in the growing oocyte, showing broad enrichment in intergenic regions and gene deserts [reviewed in refs. ,]. Genetic interference with PRC2 function in growing oocytes does not prevent their maturation, but has been linked to a postnatal overgrowth phenotype in the progeny possibly through the control of imprinting.

Here, we report the identification of a tissue-specific cofactor of PRC2, EZHIP. In human and mouse, we show that this cofactor is expressed primarily in gonads and that it limits PRC2-mediated H3K27me3 deposition. Inactivation of this cofactor in mice results in excessive deposition of this mark both during spermatogenesis and oogenesis. We further provide evidences that mutant oocytes with this altered epigenetic content are not fully functional, and that mouse female fertility is impaired.

Results

Identification of a cofactor of PRC2 in the gonad

PRC2 recruitment and enzymatic activity is controlled by a set of cofactors interacting in a partially mutually exclusive manner with the core subunit SUZ12, but little is known about its regulation in germ cells. To tackle this question, we first focused on testes [more abundant material than ovaries], and took advantage of knock-in mouse models expressing an N-terminal Flag-tagged version of either EZH1 or EZH2 from their respective endogenous locus [this study and]. We verified the expression of the tagged-EZH1 by western blot on mouse testis nuclear extract, and were able to detect the presence of a slowly migrating polypeptide, which is specifically pulled down by Flag-Immunoprecipitation [Flag-IP] [Supplementary Fig. ]. We then isolated nuclei from adult mouse testes [WT control, EZH2-Flag or EZH1-Flag], performed Flag-IP, and subjected the samples to mass spectrometry. The results of three independent IPs are represented as volcano plots [Fig. ; Supplementary Fig. ]. As expected, both EZH1 and EZH2 proteins interact with the other PRC2 core components and with known accessory subunits: AEBP2, JARID2, PHF1, and MTF2 [Fig. ; Supplementary Fig. ]. Interestingly, our experiments also reveal the existence of an additional partner, the uncharacterized protein AU022751 [ENSMUST00000117544; NM_001166433.1], which we retrieved in both EZH1 and EZH2 pulldowns. We referred to this cofactor as “EZHIP” for EZH1/2 inhibitory protein. Of note, this protein was previously identified in PRC2 interactomes of mouse embryonic stem cells, but its function was not further investigated,,. In order to confirm this interaction, we overexpressed Flag-tagged versions of the mouse and human homologs in HeLa-S3 cells [Supplementary Fig. ], and performed IP followed by mass spectrometry. These reverse IPs confirmed the interaction between PRC2 and EZHIP [Fig. ; Supplementary Fig. ]. Additional putative partners were identified in both IPs, but with the exception of USP7, they were not common to both homologs. We therefore did not pursue their study further. Importantly, these reverse IPs also indicate that EZHIP interacts with both PRC2 complex subtypes.

Fig. 1

EZHIP interacts with PRC2 in gonads. a Volcano plot of EZH2 interactome from EZH2-Flag mice testis IP compared with WT. Core complex subunits are in red, in green the cofactors and in blue EZHIP, n = 3. b Volcano plot representation of EZHIP interactome after Flag-IP from HeLa-S3 overexpressing EZHIP compare with WT. Same color codes as in [a], n = 3. c Schematic representation of EZHIP protein sequence from Mus Musculus [upper part] and Homo Sapiens [middle part]. Serine-rich region is colored in beige, and conserved amino acid stretch in green. The conserved sequence stretch is displayed as well as protein residues conservation between the two sequences in green [Sequence Homology determined using Genious software]. d Ezhip and Ezh2 mRNA relative abundance normalized to Tbp in various mice tissues [mean, n = 2]. e EZHIP and EZH2 IHC staining on human adult seminiferous tubules sections. Representative result, n ≥ 5. Scale bars, 50 μm. f EZHIP and EZH2 IHC staining on human adult ovaries sections, black arrows indicate the follicles. Representative result, n ≥ 5. Scale bars, 30 μm

Full size image

EZHIP is located on the X chromosome. In most species, it is a monoexonic gene—that may indicate that it was generated by retroposition—but in the mouse, splicing also creates a shorter isoform. Using phylogenetic analysis by maximum likelihood [PAML], we observed that EZHIP homologs are present across Eutheria, but we did not identify any homologs outside of this clade based on either sequence conservation or on synteny. EZHIP genes have rapidly evolved both at the nucleotide and amino acid levels, the rodent homologs being particularly distant from the rest [Fig. ; Supplementary Fig. ]. This contrasts with the other PRC2 components, such as EZH2, which are highly conserved across mammals [Supplementary Fig. ]. No known protein domain was predicted for EZHIP, and the only distinguishing feature is a serine-rich region [Fig. in green], including a short amino acid stretch that is fully conserved in all orthologs identified [Fig. in purple]. To characterize Ezhip expression, we performed RT-qPCR on various tissues [3-month-old females and males]. Ezhip mRNA expression was particularly high in ovaries; it was also expressed in testes, and much less in other tissues [Fig. ]. Of note, Ezhip transcript level appears at least tenfold higher than any PRC2 core components or cofactors in oocytes [Supplementary Fig. ]. Ezhip’s pattern of expression is distinct from that of Ezh2, which is expressed tissue wide, with the strongest expression observed in the spleen. Analysis of public gene expression data sets from fetal gonads indicates Ezhip is preferentially expressed in E13.5 primordial germ cells [PGCs] compared with somatic cells, correlating with germ cell markers, such as Piwil2 or Prdm14 [Supplementary Fig. ]. Interestingly, Ezhip belongs to a set of genes referred to as “germline-reprogramming-responsive” that become active following PGC DNA demethylation, as they are associated with strong CpG island promoters. Similarly, in humans EZHIP is highly transcribed in male and female PGCs from week 5 until week 9 of pregnancy, while almost absent in ESCs and somatic cells [Supplementary Fig. ]. We confirmed this observation at the protein level by performing immunohistochemistry on sections of testes and ovaries of human origin. hEZHIP protein was detected in male germ cells inside the seminiferous tubules, especially in spermatogonia and round spermatids [Fig. ]. In ovaries, EZHIP antibody stained primordial follicles and oocytes [red arrows], but not the external follicle cells in contrast to EZH2 antibody, which stained both zones [Fig. ]. To summarize, EZHIP is a genuine cofactor of PRC2 in placental mammals. It is a fast-evolving protein with no known protein domain, it is expressed primarily in PGCs during development and remains present in the adult gonad.

EZHIP is a negative regulator of PRC2 activity

To study the molecular role of EZHIP, we sought a model cell line that would express this factor endogenously. The EZHIP transcript is undetectable from most cell lines, with the exception of U2OS, an osteosarcoma-derived cell line [Supplementary Fig. ]. We used genome editing to generate U2OS clonal cells that were knockout for EZHIP or for EED as a control for PRC2 inactivation [U2OS EZHIP−/− and U2OS EED−/−, respectively]. Both cell lines were viable, and had not overt phenotype. Western blot showed that deletion of EED destabilized the other PRC2 core components, such as EZH2, while inactivation of EZHIP had no discernible effect on the accumulation of these proteins [Fig. ]. We then assessed H3K27 methylation and observed a robust increase in H3K27me2/3 upon EZHIP deletion, while H3K27me1 was stable and H3K27ac slightly reduced [Fig. ]. Interestingly, H3K27me3 level was very low in U2OS compared with extract prepared from HEK-293T cells, which do not express EZHIP [Supplementary Fig. ]. To confirm that EZHIP deletion was directly responsible for the increased H3K27me3 in U2OS, we stably restored its expression using either full-length [FL] or deletion mutants [Fig. ] as verified by western blot [WB] and RT-q-PCR [Supplementary Fig. ]. Upon re-expression of FL and mutant EZHIP, H3K27me3 returned to basal levels [Fig. ] with the notable exception of mutant M5 that lacks the conserved amino acids stretch [Fig. ]. Given that such deletion abolishes EZHIP interaction with PRC2 in co-IP [mutant 6 vs. mutant 7; Supplementary Fig. ], it stands to reason that EZHIP likely regulates H3K27me3 deposition through direct interference with PRC2 activity.

Fig. 2

EZHIP inhibits H3K27me3 deposition. a Western blot analysis of PRC2 core complex subunits [SUZ12, RBAP48, EED, and EZH2], EZHIP and HDAC1 [loading control] on U2OS nuclear extracts WT, EED−/−, and EZHIP−/−. b Western blot analysis of H3K27 methylation: H3K27me1, H3K27me2, H3K27me3, H3K27ac and, H3 and H4 [loading controls]. c Scheme representing EZHIP mutants [left panel] stably reintroduced in U2OS EZHIP−/− line. The red lambda indicates the epitope recognized by antibody detecting EZHIP protein. Western blot analysis [right panel] of cell lines expressing EZHIP FL or mutants probed with antibodies indicated on the left. d Mean-difference plot showing average log2 counts per million [logCPM] vs. log2 fold-change [logFC] expression between U2OS WT and EED−/− or U2OS WT and EZHIP−/− [right]. Right corner: the number of genes significantly differentially expressed [FDR  1 in at least two samples. Raw count data were normalized with the TMM method and transformed to log2-CPM. A linear model was fit to the normalized data, adjusting for batch effects, and empirical Bayes statistics were computed. Differentially expressed genes for each KO vs. WT were identified from the linear fit after adjusting for multiple testing and filtered to include those with FDR  30.

For differential expression analysis, genes were filtered to include those with CPM > 1 in two or more samples. Raw count data were normalized with the TMM method and converted to log2-CPM. A linear model was fit to the normalized data, and empirical Bayes statistics were computed for each comparison. Differentially expressed genes for each comparison were identified from the linear fit after adjusting for multiple testing and filtered to include those with FDR