EZH1, a homolog of EZH2 encoded by a separate locus , has much less methyltransferase activity and cannot substitute for EZH2 in histone methylation and related biological functions in many tissues . differentiation, PRC2 establishes new H3K27 methylation sites, especially in male germ cells. These new H3K27 methylation marks are introduced into the genome to determine the cell-type-specific transcriptome . In this case, PRC2s recruitment to specific loci for methylation appears to be a more complicated process, potentially involving noncoding RNAs, sequence-specific transcription factors, and/or PRC2-interacting proteins with affinity for Rabbit polyclonal to SP3 CpG-rich DNA elements . Our previous results demonstrate that EED, EZH2, and SUZ12 are dramatically upregulated in pachytene spermatocytes , suggesting the germ cell-specific PRC2 complex has a role in establishing H3K27 methylation during meiotic progression. Therefore, it is crucial to understand how PRC2 can distinguish these two different types of histone methylation coupled with cell proliferation and differentiation. EZH2 may have a major role in determining PRC2s differential methylation roles. It possesses multiple interaction domains for EED and SUZ12, facilitating the methyltransferase activity conveyed by its SET domain [18C20]. EZH1, a homolog of EZH2 encoded by a separate locus , has much less methyltransferase activity and cannot substitute for EZH2 in histone methylation and related biological functions in many tissues . Because other PRC2 subunits only have a subtle effect on EZH2 methyltransferase specificity, we speculate that EZH2s variants themselves diversify PRC2s functional roles in distinct methylation processes during cell proliferation and differentiation. Here, we identified multiple isoforms derived from alternative transcriptional splicing in various tissues and cell types. Expressions of EZH2 variants that include or exclude exon 14 are differentially regulated via cell cycle or meiotic regulators, respectively, during mitosis and meiosis. The EZH2 isoform without exon 14 (ex14D-EZH2has a disrupted CXC domain and is the primary isoform found in spermatocytes. This isoform is responsible for the establishment of H3K27me2, but is less efficient at catalyzing H3K27me3. Moreover, exclusive expression of ex14D-EZH2 in ES cells promotes their differentiation, indicated by precocious and enhanced expression of mesoderm genes. In contrast, the EZH2 isoform with exon 14 (ex14-EZH2) is the most common isoform in proliferating cells and more efficient at catalyzing H3K27me3. Our study suggests that the incorporation of specific EZH2 variants into the PRC2 complex controls the appropriate level and extent IV-23 of H3K27 methylation in polycomb target loci during the establishment and maintenance of these epigenetic marks. Results pre-mRNA splicing is differentially regulated during meiosis and mitosis makes several distinct transcripts due to alternative splicing. Exons 4 and 14 can be skipped and exons 3 and 8 can be truncated (Fig.?1a) . To determine whether different transcripts are cell and tissue type specific, we profiled transcripts in different ages of testes, somatic tissues, embryos, and primary cell IV-23 lines by RT-PCR. The transcripts IV-23 that contain alternative splicing for exon 3 and exon 14 are found in many tissues and cultured cells (Fig.?1b). In contrast, transcripts containing alternative splicing for exons 4 and 8 were barely detected (Additional file 1: Fig. S1). Thus, we focused on transcripts with variations in exons 3 and 14. Open in a separate window Fig.?1 splicing is differentially regulated during meiosis and mitosis. a Schematic structure of the mouse gene and protein. Removal of exon 14 causes the disruption of the CXC domain. b RT-PCR analysis of alternative transcripts in mouse testis at different ages, tissues, embryos, and cell lines. c Quantitation of and transcripts during testis development by qPCR analysis. d IV-23 Quantitation of the transcription of PRC2 core components by qPCR analysis. e Quantitation of and transcripts during the cell cycle progression. f Western blot analysis of PRC2 core components during the cell cycle progression First, we examined and transcript levels during spermatogenesis. transcripts without exon 14 (ex14D-transcripts containing exon 14 (ex14-levels were consistent throughout germ cell development (Fig.?1c), indicating its expression is independent of meiotic differentiation. Because ex14-is abundant in mitotic germ cells and rapidly dividing ES cells and primary MEFs (Fig.?1b), we wanted to determine the dynamics of and transcripts during mitosis. Thus, we synchronized primary MEFs at the G0/G1 phase by serum starvation and then released them into the S and G2/M phases with serum supplementation. In comparison with meiosis, ex14-transcripts with a full exon 3 (ex3-expression decreased.