07-441 Sigma-AldrichAnti-dimethyl-Histone H3 (Lys9) Antibody

RNA interference (RNAi) is a widespread gene-silencing mechanism and is required for heterochromatin assembly in a variety of organisms. The RNA-induced transcriptional silencing complex (RITS), composed of Ago1, Tas3 and Chp1, is a key component of RNAi machinery in fission yeast that connects short interference RNA (siRNA) and heterochromatin formation. However, the process by which RITS is assembled is not well understood. Here, we identified Sgf73, a subunit of the SAGA co-transcriptional complex, is required for pericentromeric heterochromatin silencing and the generation of siRNA. This novel role of Sgf73 is independent of enzymatic activities or structural integrity of SAGA. Instead, Sgf73 is physically associated with Ago1 and Chp1. The interactions among the subunits of the RITS, including those between Tas3 and Chp1, between Chp1 and Ago1, between Ago1 and Tas3, were all impaired by the deletion of sgf73(+). Consistently, the recruitment of Ago1 and Chp1 to the pericentromeric region was abolished in sgf73Δ cells. Our study unveils a moonlighting function of a SAGA subunit. It suggests Sgf73 is a novel factor that promotes assembly of RITS and RNAi-mediated heterochromatin formation.

Emerging evidence has led to considerable interest in the role of Traf2- and Nck-interacting kinase (TNIK) in polycystic ovary syndrome (PCOS) development. However, the epigenetic mechanism regulating TNIK transcription remains largely unknown. Here, we show that (i) TNIK mRNA expression is significantly increased in PCOS ovarian tissues, compared to normal ovarian tissues; (ii) PCOS ovarian tissues exhibit a hypermethylation pattern at the cg10180092 site, (iii) and cg10180092 is the critical site for the transcriptional regulation of TNIK. Mechanistically, hypermethylated cg10180092 site-mediated loss of holocarboxylase synthetase (HLCS)-related H3K9me enrichment activated TNIK transcription in PCOS ovarian tissues. Notably, a substantial body of evidence indicates that DNA hypermethylation is an alternative mechanism for gene inactivation, and a new role for DNA hypermethylationmediated TNIK activating was observed in this study. This may improve our understanding of divergent transcriptional regulation in the initiation and progression of TNIK-related PCOS.

The monophyletic carnivorous genus Genlisea (Lentibulariaceae) is characterized by a bi-directional genome size evolution resulting in a 25-fold difference in nuclear DNA content. This is one of the largest ranges found within a genus so far and makes Genlisea an interesting subject to study mechanisms of genome and karyotype evolution. Genlisea nigrocaulis, with 86 Mbp one of the smallest plant genomes, and the 18-fold larger genome of G. hispidula (1,550 Mbp) possess identical chromosome numbers (2n = 40) but differ considerably in chromatin organization, nuclear and cell size. Interphase nuclei of G. nigrocaulis and of related species with small genomes, G. aurea (133 Mbp, 2n ≈ 104) and G. pygmaea (179 Mbp, 2n = 80), are hallmarked by intensely DAPI-stained chromocenters, carrying typical heterochromatin-associated methylation marks (5-methylcytosine, H3K9me2), while in G. hispidula and surprisingly also in the small genome of G. margaretae (184 Mbp, 2n = 38) the heterochromatin marks are more evenly distributed. Probes of tandem repetitive sequences together with rDNA allow the unequivocal discrimination of 13 out of 20 chromosome pairs of G. hispidula. One of the repetitive sequences labeled half of the chromosome set almost homogenously supporting an allopolyploid status of G. hispidula and its close relative G. subglabra (1,622 Mbp, 2n = 40). In G. nigrocaulis 11 chromosome pairs could be individualized using a combination of rDNA and unique genomic probes. The presented data provide a basis for future studies of karyotype evolution within the genus Genlisea.

Histone lysine methylation contributes to transcriptional regulation by serving as a platform for the recruitment of various cofactors. Intense studies have been conducted for elucidating the functional meaning of H3K79 methylation, and to date, the only known HMTase responsible for the modification was DOT1L. In this study, we report that the MMSET isoform RE-IIBP has HMTase activity for H3K79. It was uncovered that RE-IIBP up-regulates MEIS1 transcription through H3K79 methylation via recruitment to the MEIS1 promoter. By means of proteomic and biochemical analysis, association of RE-IIBP with the E3 ubiquitin ligase RNF20 was demonstrated for synergistic activation of MEIS1 transcription via H3K79 HMTase activity. Furthermore, It was observed that RE-IIBP induces MEIS1-mediated apoptosis, which was dependent on H2BK120 ubiquitination by RNF20. These findings suggest RE-IIBP as another candidate for further studies to elucidate the mechanism of H3K79 methylation and its biological functions.

Alternative splicing is the main source of proteome diversity. Here, we have investigated how alternative splicing affects the function of two human histone methyltransferases (HMTase): G9A and SUV39H2. We show that exon 10 in G9A and exon 3 in SUV39H2 are alternatively included in a variety of tissues and cell lines, as well as in a different species. The production of these variants is likely tightly regulated because both constitutive and alternative splicing factors control their splicing profiles. Based on this evidence, we have assessed the link between the inclusion of these exons and the activity of both enzymes. We document that these HMTase genes yield several protein isoforms, which are likely issued from alternative splicing regulation. We demonstrate that inclusion of SUV39H2 exon 3 is a determinant of the stability, the sub-nuclear localization, and the HMTase activity. Genome-wide expression analysis further revealed that alternative inclusion of SUV39H2 exon 3 differentially modulates the expression of target genes. Our data also suggest that a variant of G9A may display a function that is independent of H3K9 methylation. Our work emphasizes that expression and function of genes are not collinear; therefore alternative splicing must be taken into account in any functional study.

Kdm3b is a Jumonji C domain-containing protein that demethylates mono- and di-methylated lysine 9 of histone H3 (H3K9me1 and H3K9me2). Although the enzyme activity of Kdm3b is well characterized in vitro, its genetic and physiological function remains unknown. Herein, we generated Kdm3b knockout (Kdm3bKO) mice and observed restricted postnatal growth and female infertility in these mice. We found that Kdm3b ablation decreased IGFBP-3 expressed in the kidney by 53% and significantly reduced IGFBP-3 in the blood, which caused an accelerated degradation of IGF-1 and a 36% decrease in circulating IGF-1 concentration. We also found Kdm3b was highly expressed in the female reproductive organs including ovary, oviduct and uterus. Knockout of Kdm3b in female mice caused irregular estrous cycles, decreased 45% of the ovulation capability and 47% of the fertilization rate, and reduced 44% of the uterine decidual response, which were accompanied with a more than 50% decrease in the circulating levels of the 17beta-estradiol. Importantly, these female reproductive phenotypes were associated with significantly increased levels of H3K9me1/2/3 in the ovary and uterus. These results demonstrate that Kdm3b-mediated H3K9 demethylation plays essential roles in maintenance of the circulating IGF-1, postnatal somatic growth, circulating 17beta-estradiol, and female reproductive function.

Histone H3K9 methyltransferase (HMTase) G9a-mediated transcriptional repression is a major epigenetic silencing mechanism. UHRF1 (ubiquitin-like with PHD and ring finger domains 1) binds to hemimethylated DNA and plays an essential role in the maintenance of DNA methylation. Here, we provide evidence that UHRF1 is transcriptionally downregulated by H3K9 HMTase G9a. We found that increased expression of G9a along with transcription factor YY1 specifically represses UHRF1 transcription during TPA-mediated leukemia cell differentiation. Using ChIP analysis, we found that UHRF1 was among the transcriptionally silenced genes during leukemia cell differentiation. Using a DNA methylation profiling array, we discovered that the UHRF1 promoter was hypomethylated in samples from leukemia patients, further supporting its overexpression and oncogenic activity. Finally, we showed that G9a regulates UHRF1-mediated H3K23 ubiquitination and proper DNA replication maintenance. Therefore, we propose that H3K9 HMTase G9a is a specific epigenetic regulator of UHRF1.

Recent advances in rice flowering studies have shown that the accurate control of flowering by photoperiod is regulated by key mechanisms that involve the regulation of flowering genes including Heading date1 (Hd1), Early hd1 (Ehd1), Hd3a, and RFT1. The chromatin mechanism involved in the regulation of rice flowering genes is presently not well known. Here we show that the rice enhancer of zeste [E(z)] genes SDG711 and SDG718, which encode the polycomb repressive complex2 (PRC2) key subunit that is required for trimethylation of histone H3 lysine 27 (H3K27me3), are respectively, involved in long day (LD) and short day (SD) regulation of key flowering genes. The expression of SDG711 and SDG718 is induced by LD and SD, respectively. Over-expression and down-regulation of SDG711 respectively, repressed and promoted flowering in LD, but had no effect in SD. By contrast, down-regulation of SDG718 had no effect in LD but delayed flowering in SD. SDG711 and SDG718 repressed OsLF (a repressor of Hd1) respectively in LD and SD, leading to a higher expression of Hd1 thus late flowering in LD and early flowering in SD. SDG711 was also found to be involved in the repression of Ehd1 in LD. SDG711 was shown to directly target to OsLF and Ehd1 loci to mediate H3K27me3 and gene repression. The function of the rice E(z) genes in LD repression and SD promotion of flowering suggests that PRC2-mediated epigenetic repression of gene expression is involved in the accurate photoperiod control of rice flowering.

Epigenetic factors have been implicated in the regulation of CD4(+) T-cell differentiation. Jmjd3 plays a role in many biological processes, but its in vivo function in T-cell differentiation remains unknown. Here we report that Jmjd3 ablation promotes CD4(+) T-cell differentiation into Th2 and Th17 cells in the small intestine and colon, and inhibits T-cell differentiation into Th1 cells under different cytokine-polarizing conditions and in a Th1-dependent colitis model. Jmjd3 deficiency also restrains the plasticity of the conversion of Th2, Th17 or Treg cells to Th1 cells. The skewing of T-cell differentiation is concomitant with changes in the expression of key transcription factors and cytokines. H3K27me3 and H3K4me3 levels in Jmjd3-deficient cells are correlated with altered gene expression through interactions with specific transcription factors. Our results identify Jmjd3 as an epigenetic factor in T-cell differentiation via changes in histone methylation and target gene expression.

DNA methylation and dimethylation of lysine 9 of histone H3 (H3K9me2) are two chromatin modifications that can be associated with gene expression or recombination rate. The maize genome provides a complex landscape of interspersed genes and transposons. The genome-wide distribution of DNA methylation and H3K9me2 were investigated in seedling tissue for the maize inbred B73 and compared to patterns of these modifications observed in Arabidopsis thaliana. Most maize transposons are highly enriched for DNA methylation in CG and CHG contexts and for H3K9me2. In contrast to findings in Arabidopsis, maize CHH levels in transposons are generally low but some sub-families of transposons are enriched for CHH methylation and these families exhibit low levels of H3K9me2. The profile of modifications over genes reveals that DNA methylation and H3K9me2 is quite low near the beginning and end of genes. Although elevated CG and CHG methylation are found within gene bodies, CHH and H3K9me2 remain low. Maize has much higher levels of CHG methylation within gene bodies than observed in Arabidopsis and this is partially attributable to the presence of transposons within introns for some maize genes. These transposons are associated with high levels of CHG methylation and H3K9me2 but do not appear to prevent transcriptional elongation. Although the general trend is for a strong depletion of H3K9me2 and CHG near the transcription start site there are some putative genes that have high levels of these chromatin modifications. This study provides a clear view of the relationship between DNA methylation and H3K9me2 in the maize genome and how the distribution of these modifications is shaped by the interplay of genes and transposons.