Identification of JmjC domain-containing UTX and JMJD3 as histone H3 lysine 27 demethylases

Covalent modifications of histones, such as acetylation and methylation, play important roles in the regulation of gene expression. Histone lysine methylation has been implicated in both gene activation and repression, depending on the specific lysine (K) residue that becomes methylated and the state of methylation (mono-, di-, or trimethylation). Methylation on K4, K9, and K36 of histone H3 has been shown to be reversible and can be removed by site-specific demethylases. However, the enzymes that antagonize methylation on K27 of histone H3 (H3K27), an epigenetic mark important for embryonic stem cell maintenance, Polycomb-mediated gene silencing, and X chromosome inactivation have been elusive. Here we show the JmjC domain-containing protein UTX (ubiquitously transcribed tetratricopeptide repeat, X chromosome), as well as the related JMJD3 (jumonji domain containing 3), specifically removes methyl marks on H3K27 in vitro. Further, the demethylase activity of UTX requires a catalytically active JmjC domain. Finally, overexpression of UTX and JMJD3 leads to reduced di- and trimethylation on H3K27 in cells, suggesting that UTX and JMJD3 may function as H3K27 demethylases in vivo. The identification of UTX and JMJD3 as H3K27-specific demethylases provides direct evidence to indicate that similar to methylation on K4, K9, and K36 of histone H3, methylation on H3K27 is also reversible and can be dynamically regulated by site-specific histone methyltransferases and demethylases.     

Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27)

Trimethylation of histone H3 on lysine 27 (H3K27me3) is a repressive posttranslational modification mediated by the histone methyltransferase EZH2. EZH2 is a component of the polycomb repressive complex 2 and is overexpressed in many cancers. In B-cell lymphomas, its substrate preference is frequently altered through somatic mutation of the EZH2 Y641 residue. Herein, we identify mutation of EZH2 A677 to a glycine (A677G) among lymphoma cell lines and primary tumor specimens. Similar to Y641 mutant cell lines, an A677G mutant cell line revealed aberrantly elevated H3K27me3 and decreased monomethylated H3K27 (H3K27me1) and dimethylated H3K27 (H3K27me2). A677G EZH2 possessed catalytic activity with a substrate specificity that was distinct from those of both WT EZH2 and Y641 mutants. Whereas WT EZH2 displayed a preference for substrates with less methylation [unmethylated H3K27 (H3K27me0):me1:me2 k(cat)/K(m) ratio = 9:6:1] and Y641 mutants preferred substrates with greater methylation (H3K27me0:me1:me2 k(cat)/K(m) ratio = 1:2:13), the A677G EZH2 demonstrated nearly equal efficiency for all three substrates (H3K27me0:me1:me2 k(cat)/K(m) ratio = 1.1:0.6:1). When transiently expressed in cells, A677G EZH2, but not WT EZH2, increased global H3K27me3 and decreased H3K27me2. Structural modeling of WT and mutant EZH2 suggested that the A677G mutation acquires the ability to methylate H3K27me2 through enlargement of the lysine tunnel while preserving activity with H3K27me0/me1 substrates through retention of the Y641 residue that is crucial for orientation of these smaller substrates. This mutation highlights the interplay between Y641 and A677 residues in the substrate specificity of EZH2 and identifies another lymphoma patient population that harbors an activating mutation of EZH2.     

Caenorhabditis elegans chromatin-associated proteins SET-2 and ASH-2 are differentially required for histone H3 Lys 4 methylation in embryos and adult germ cells

Methylation of histone H3 lysine 4 (H3K4me), a mark associated with gene activation, is mediated by SET1 and the related mixed lineage leukemia (MLL) histone methyltransferases (HMTs) across species. Mammals contain seven H3K4 HMTs, Set1A, Set1B, and MLL1-MLL5. The activity of SET1 and MLL proteins relies on protein-protein interactions within large multisubunit complexes that include three core components: RbBP5, Ash2L, and WDR5. It remains unclear how the composition and specificity of these complexes varies between cell types and during development. Caenorhabditis elegans contains one SET1 protein, SET-2, one MLL-like protein, SET-16, and single homologs of RbBP5, Ash2L, and WDR5. Here we show that SET-2 is responsible for the majority of bulk H3K4 methylation at all developmental stages. However, SET-2 and absent, small, or homeotic discs 2 (ASH-2) are differentially required for tri- and dimethylation of H3K4 (H3K4me3 and -me2) in embryos and adult germ cells. In embryos, whereas efficient H3K4me3 requires both SET-2 and ASH-2, H3K4me2 relies mostly on ASH-2. In adult germ cells by contrast, SET-2 serves a major role whereas ASH-2 is dispensable for H3K4me3 and most H3K4me2. Loss of SET-2 results in progressive sterility over several generations, suggesting an important function in the maintenance of a functional germ line. This study demonstrates that individual subunits of SET1-related complexes can show tissue specificity and developmental regulation and establishes C. elegans as a model to study SET1-related complexes in a multicellular organism.     

Defective megakaryopoiesis and abnormal erythroid development in Fli-1 gene-targeted mice

Mouse embryos homozygous for a targeted disruption in the Fli-1 gene show hemorrhage into the neural tube and brain on embryonic day (E)11.0 and die shortly thereafter. Livers from the mutant embryos contain drastically reduced numbers of pronormoblasts, basophilic normoblasts, and colony-forming cells. To determine the nature of impaired hematopoiesis, we carried out cell culture studies of mutant embryonic stem (ES) cells and cells from the aorta-gonad-mesonephros (AGM) region of E10.0 mutant embryos. There was a striking reduction in the number of megakaryocytes in cultures of mutant AGM cells compared with cultures of AGM cells from wild-type or heterozygous embryos. Furthermore, Fli-1 mutant ES cells failed to produce megakaryocyte colonies and multilineage colonies containing megakaryocytes. Consistent with the observed defect in megakaryopoiesis, we also demonstrated the down-regulation of c-mpl in the AGM of mutant embryos. The percentages of pronormoblasts and basophilic normoblasts were significantly reduced in cultures of mutant AGM embryos, which contained primarily polychromatophilic and orthochromatic normoblasts. These results provide further evidence for the role of Fli-1 in the regulation of hematopoiesis and for c-mpl as a Fli-1 target gene.     

Extracellular signal-regulated kinase 2 is necessary for mesoderm differentiation

The extracellular signal-regulated kinase (ERK) is a component of the mitogen-activated protein kinase cascade. Exon 2 of erk2 was deleted by homologous recombination and resulted in embryonic lethality at embryonic day 6.5. erk2 mutant embryos did not form mesoderm and showed increased apoptosis but comparable levels of BrdUrd incorporation, indicating a defect in differentiation. erk2 null embryonic stem (ES) cells exhibited reduced total ERK activity upon serum stimulation, augmented ERK1 phosphorylation, and decreased downstream p90Rsk phosphorylation and activity; yet ES cell proliferation was unaffected. Mutant ES cells were capable of forming mesoderm; however, treatment of mutant ES cells with the mitogen-activated protein kinase kinase inhibitor PD184352 decreased total ERK activity and expression of the mesodermal marker brachyury, suggesting that ERK1 can compensate for ERK2 in vitro. Normal embryos at embryonic day 6.5 expressed activated ERK1/2 in the extraembryonic ectoderm, whereas erk2 mutant embryos had no detectable activated ERK1/2 in this region, suggesting that activated ERK1 was not expressed, and therefore cannot compensate for loss of ERK2 in vivo. These data indicate that ERK2 plays an essential role in mesoderm differentiation during embryonic development.     

Epigenetic Dysfunction in Turner Syndrome Immune Cells

Turner syndrome (TS) is a chromosomal condition associated with partial or complete absence of the X chromosome that involves characteristic findings in multiple organ systems. In addition to well-known clinical characteristics such as short stature and gonadal failure, TS is also associated with T cell immune alterations and chronic otitis media, suggestive of a possible immune deficiency. Recently, ubiquitously transcribed tetratricopeptide repeat on the X chromosome (UTX), a histone H3 lysine 27 (H3K27) demethylase, has been identified as a downregulated gene in TS immune cells. Importantly, UTX is an X-linked gene that escapes X-chromosome inactivation and thus is haploinsufficient in TS. Mice with T cell-specific UTX deficiency have impaired clearance of chronic viral infection due to decreased frequencies of T follicular helper (Tfh) cells, which are critical for B cell antibody generation. In parallel, TS patients have decreased Tfh frequencies in peripheral blood. Together, these findings suggest that haploinsufficiency of the X-linked UTX gene in TS T cells underlies an immune deficit, which may manifest as increased predisposition to chronic otitis media.     

Somatic mutations at EZH2 Y641 act dominantly through a mechanism of selectively altered PRC2 catalytic activity, to increase H3K27 trimethylation

Next-generation sequencing of follicular lymphoma and diffuse-large B-cell lymphoma has revealed frequent somatic, heterozygous Y641 mutations in the histone methyltransferase EZH2. Heterozygosity and the presence of equal quantities of both mutant and wild-type mRNA and expressed protein suggest a dominant mode of action. Surprisingly, B-cell lymphoma cell lines and lymphoma samples harboring heterozygous EZH2(Y641) mutations have increased levels of histone H3 Lys-27-specific trimethylation (H3K27me3). Expression of EZH2(Y641F/N) mutants in cells with EZH2(WT) resulted in an increase of H3K27me3 levels in vivo. Structural modeling of EZH2(Y641) mutants suggests a "Tyr/Phe switch" model whereby structurally neutral, nontyrosine residues at position 641 would decrease affinity for unmethylated and monomethylated H3K27 substrates and potentially favor trimethylation. We demonstrate, using in vitro enzyme assays of reconstituted PRC2 complexes, that Y641 mutations result in a decrease in monomethylation and an increase in trimethylation activity of the enzyme relative to the wild-type enzyme. This represents the first example of a disease-associated gain-of-function mutation in a histone methyltransferase, whereby somatic EZH2 Y641 mutations in lymphoma act dominantly to increase, rather than decrease, histone methylation. The dominant mode of action suggests that allele-specific EZH2 inhibitors should be a future therapeutic strategy for this disease.     

Dependency on the polycomb gene Ezh2 distinguishes fetal from adult hematopoietic stem cells

Polycomb-group (PcG) proteins are essential regulators of hematopoietic stem cells (HSCs). In contrast to Bmi1, a component of Polycomb repressive complex 1 (PRC1), the role of PRC2 and its components in hematopoiesis remains elusive. Here we show that Ezh2, a core component of PRC2, is essential for fetal, but not adult, HSCs. Ezh2-deficient embryos died of anemia because of insufficient expansion of HSCs/progenitor cells and defective erythropoiesis in fetal liver. Deletion of Ezh2 in adult BM, however, did not significantly compromise hematopoiesis, except for lymphopoiesis. Of note, Ezh2-deficient fetal liver cells showed a drastic reduction in trimethylation of histone H3 at lysine 27 (H3K27me3) accompanied by derepression of a large cohort of genes, whereas on homing to BM, they acquired a high level of H3K27me3 and long-term repopulating capacity. Quantitative RT-PCR revealed that Ezh1, the gene encoding a backup enzyme, is highly expressed in HSCs/progenitor cells in BM compared with those in fetal liver, whereas Ezh2 is ubiquitously expressed. These findings suggest that Ezh1 complements Ezh2 in the BM, but not in the fetal liver, and reveal that the reinforcement of PcG-mediated gene silencing occurs during the transition from proliferative fetal HSCs to quiescent adult HSCs.     

Influence of sex chromosome constitution on the genomic imprinting of germ cells

Germ cells in XY male mice establish site-specific methylation on imprinted genes during spermatogenesis, whereas germ cells in XX females establish their imprints in growing oocytes. We showed previously that in vitro, sex-specific methylation patterns of pluripotent stem cell lines derived from germ cells were influenced more by the sex chromosome constitution of the cells themselves than by the gender of the embryo from which they had been derived. To see whether the same situation would prevail in vivo, we have now determined the methylation status of H19 expressed from the maternal allele, and the expression and methylation status of a paternally expressed gene Peg3, in germ cells from sex-reversed and control embryos. For these imprinted genes, we conclude that the female imprint is a response of the germ cells to undergoing oogenesis, rather than to their XX chromosome constitution. Similarly, both our XY and our sex-reversed XX male germ cells clearly showed a male rather than a female pattern of DNA methylation; here, however, the sex chromosome constitution had a significant effect, with XX male germ cells less methylated than the XY controls.