RBR-2 controls the enhancer signature of the lin-11 locus As ChIP-seq analysis of the rbr-2(tm3141) allele identified increased levels of H3K4me3 in regions upstream the TSSs (cluster 3 and 6), where distal enhancer elements are generally found (Bulger and Groudine, 2010), we postulated that RBR-2 could positively regulate the rate of transcription by controlling enhancer signatures and activities, as also recently proposed for RBR-2 mammalian homologues (Kidder et al., 2014; Outchkourov et al., 2013). Active enhancers are characterized by the presence of high levels of H3K4me1 and H3K27ac, low level of H3K4me3 and by the binding of CBP-1 (CBP/p300 homologue), responsible for H3K27 acetylation (Calo and Wysocka, 2013; Heintzman and Ren, 2007; Rada-Iglesias et al., 2011). In support of our hypothesis, we found that loss of rbr-2 results in a significant reduction of global level of H3K4me1 and of H3K27ac (Fig. 5A). To validate this hypothesis in vivo, we took advantage of the role of RBR-2 in vulva development and we analyzed the histone modifications in wild- type and rbr-2(tm3141) animals at the lin-11 locus, encoding a transcription factor previously implicated in vulva formation and regulated, at least in part, by LIN-12/Notch signaling (Gupta and Sternberg, 2002; Gupta et al., 2003; Newman et al., 1999). The selection of this gene was based on the evidence that its expression is reduced in rbr-2(tm3141) worms (as observed by RNA-seq, qPCR and marker analysis) and on the fact that an enhancer element (called here lin-11 enh) of 625 bp, specifically required for lin-11 expression in VPCs, was previously identified (Gupta and Sternberg, 2002; Gupta et al., 2003; Marri and Gupta, 2009; Newman et al., 1999). In wild-type animals, RBR-2 (Fig. 5B) and H3K4me1 and H3K27ac (Fig. 5C) are enriched at lin-11 enh when compared with control regions, confirming at the molecular level the regulatory role of this enhancer element. Strikingly, in rbr-2 (tm3141) animals, H3K4me1 and H3K27ac in the lin-11 enh region seem reduced to background levels, as compared with wild type, with concomitant increased H3K4me3 (Fig. 5C). As expression of EGL-17 in vulC and vulD is reduced in a subset of rbr-2(tm3141) animals (Fig. 2D,E), we analyzed the egl-17 enhancer that drives the expression of egl-17 in vulC and vulD cells (Cui and Han, 2003) and obtained similar results for H3K4 methylation (Fig. S7A,B). Of note, when two genes, unc-54 and myo-3, unrelated to vulva development and with well-described enhancer regions (Jantsch- Plunger and Fire, 1994; Okkema et al., 1993), were analyzed (Fig. S7C-E), we did not observe any enrichment of RBR-2::GFP at their enhancer regions, nor changes in H3K4me1 and H3K27ac levels in rbr-2 animals, compared with wild type. In agreement, the expression levels of UNC-54 and MYO-3 seem to be unchanged in rbr-2 animals.
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The median age of onset of MN is in the early 50s, an MN has been established as the most important cause of the nephrotic syndrome in elderly patients (aged > 65 years). Accumulating evidence also suggests that aging is associated with systemic and vascular inflammation . Circulating inflammatory cytokines are elevated in some older adults [26, 27], and chronic exposure to inflammation is thought to be closely related to the development and progression of renal injury [28–30]. Previous studies have shown that the LPS-mediated NF-kB signaling pathway, which plays a critical role in the inflammatory response, is in- volved in H3K4 me3 redistribution in monocyte-derived dendritic cells [31, 32]. Silencing of MLL inhibits both H3K4 me3 enrichment and miRNA expression, and affects TNF-α production [31, 32]. Our data using a co- cultured system with podocytes and peritoneal macro- phages suggests that cytokines derived from macrophages stimulated with LPS cause increased H3K4 me3 in podo- cytes. Thus, MN pathogenesis may involve the induction of H3K4 me3 by inflammatory cytokines, leading to an in- creased expression of cathepsin L and a decreased expres- sion of synaptopodin. These changes were observed to induce the deterioration of renal function and proteinuria in LPS-treated mice, and were consistent with the findings in patients with MN.
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cells, and SMYD3 is abundant in cancer cells compared with that in normal cells, thereby suggesting that SMYD3-mediated methylation at H34K signi ﬁ cantly changed the downstream target gene expressions levels. In our analyses, cell proliferation, migration, invasion, and colony formation were positively regulated by SMYD3, and apoptosis was negatively regulated by SMYD3. Further study will be needed to analyze the target genes of SMYD3 in cancer development, will be important to understand the regulatory basis of cancer cell behavior, and may be a key point to demonstrate that SMYD3-mediated apoptosis signaling regulates BLAC cell migration, proliferation, and invasion. 1.5
Mixed-lineage leukemia (MLL) is a proto-oncogene frequently involved in chromosomal translocations associ- ated with acute leukemia. These chromosomal translocations commonly result in MLL fusion proteins that dysregulate transcription. Recent data suggest that the MYB proto-oncogene, which is an important regulator of hematopoietic cell development, has a role in leukemogenesis driven by the MLL-ENL fusion protein, but exactly how is unclear. Here we have demonstrated that c-Myb is recruited to the MLL histone methyl transfer- ase complex by menin, a protein important for MLL-associated leukemic transformation, and that it contrib- utes substantially to MLL-mediated methylation of histone H3 at lysine 4 (H3K4). Silencing MYB in human leukemic cell lines and primary patient material evoked a global decrease in H3K4 methylation, an unexpected decrease in HOXA9 and MEIS1 gene expression, and decreased MLL and menin occupancy in the HOXA9 gene locus. This decreased occupancy was associated with a diminished ability of an MLL-ENL fusion protein to transform normal mouse hematopoietic cells. Previous studies have shown that MYB expression is regulated by Hoxa9 and Meis1, indicating the existence of an autoregulatory feedback loop. The finding that c-Myb has the ability to direct epigenetic marks, along with its participation in an autoregulatory feedback loop with genes known to transform hematopoietic cells, lends mechanistic and translationally relevant insight into its role in MLL-associated leukemogenesis.
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ABSTRACT Histone post-translational modiﬁcations (PTMs) alter chromatin structure by promoting the interaction of chromatin- modifying complexes with nucleosomes. The majority of chromatin-modifying complexes contain multiple domains that preferentially interact with modi ﬁ ed histones, leading to speculation that these domains function in concert to target nucleosomes with distinct combinations of histone PTMs. In Saccharomyces cerevisiae , the NuA3 histone acetyltransferase complex contains three domains, the PHD ﬁ nger in Yng1, the PWWP domain in Pdp3, and the YEATS domain in Taf14; which in vitro bind to H3K4 methylation, H3K36 methylation, and acetylated and crotonylated H3K9, respectively. While the in vitro binding has been well characterized, the relative in vivo contributions of these histone PTMs in targeting NuA3 is unknown. Here, through genome-wide colocalization and by mutational interrogation, we demonstrate that the PHD ﬁnger of Yng1, and the PWWP domain of Pdp3 independently target NuA3 to H3K4 and H3K36 methylated chromatin, respectively. In contrast, we ﬁnd no evidence to support the YEATS domain of Taf14 functioning in NuA3 recruitment. Collectively our results suggest that the presence of multiple histone PTM binding domains within NuA3, rather than restricting it to nucleosomes containing distinct combinations of histone PTMs, can serve to increase the range of nucleosomes bound by the complex. Interestingly, however, the simple presence of NuA3 is insufﬁcient to ensure acetylation of the associated nucleosomes, suggesting a secondary level of acetylation regulation that does not involve control of HAT-nucleosome interactions.
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Higher metazoans possess additional H3K4 methylases, the mixed lineage leukemia (MLL) class of proteins, which act through distinct complexes similar to COMPASS (reviewed by Eissenberg and Shilatifard 2010). The MLL proteins (MLL1–5) are required at limited but important subsets of gene targets, such as homeotic and hormone re- sponse genes (J. Lee et al. 2008; S. Lee 2008; Wang et al. 2009; reviewed by Ansari and Mandal 2010; Eissenberg and Shilatifard 2010). H3K4 methylases identiﬁed in Drosophila melanogaster to date include Trx (homologous to MLL1–2), Trr (homologous to MLL3–4), and Ash1 (Beisel et al. 2002; Byrd and Shearn 2003; Sedkov et al. 2003; Smith et al. 2004). Ash1 and Trx are members of the Trithorax group of proteins that antagonize Polycomb group-mediated gene silencing (Klymenko and Muller 2004). In addition, Trx methylates H3K4 at heat-shock loci upon induction and appears to be required for mediating stress responses to heat stimuli (Smith et al. 2004). Trr is recruited to and required for H3K4 methylation at gene targets responsive to the in- sect nuclear hormone ecdysone (Sedkov et al. 2003). Al- though these HMTs are known to catalyze H3K4 methylation and are widely believed to act as the main global H3K4 methylases in Drosophila (Beisel et al. 2002; Byrd and Shearn 2003; Sedkov et al. 2003; Smith et al. 2004), the functional roles of the Drosophila ortholog of Set1 (dSet1) have remained undeﬁned, largely because its location within centric heterochromatin makes genetic and molecular analysis particularly challenging.
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Because the DVL3 but not DVL1 and DVL2 level was reduced by PRMT5 inhibition (Figure 9, A–C and data not shown), whereas overexpression of PRMT5 increased the DVL3 level (Figure 9D), we explored the mechanism by which PRMT5 positively affected DVL3 level. K562 cells were pretreated with the proteasome inhibitor MG132 (1.0 μM) for 2 hours, followed by PJ-68 (15.0 μM or 20.0 μM) treatment for 24 hours. MG132 did not prevent PJ-68–mediated reduction of DVL3 protein (Supplemental Figure 7F). By contrast, PRMT5 activated DVL3 gene transcription, and PJ-68 treatment greatly inhibited the DVL3 mRNA level in K562 cells (Figure 9, H and I). Furthermore, we set out to examine whether the promoter of DVL3 was epigenetically regulated by PRMT5. Given that PRMT5 mediates symmetric dimethylation of histone H3R2 (H3R2SDM) and recruits WDR5 to promote H3K4 methylation and gene activa- tion (40, 41), we determined the level of H3R2SDM and found that it was significantly decreased after PRMT5 knockdown or PJ-68 treat- ment in K562 cells (Figure 9J) or CML CD34 + cells (Supplemental
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previously demonstrated (Krogan et al. 2002; Mueller and Jaehning 2002; Squazzo et al. 2002), all ﬁve deletion mu- tants were viable, but showed different mitotic growth rates (data not shown). Among them, the paf1 and ctr9 deletion mutants grew poorly in the vegetative phase and entered meiosis inefﬁciently and asynchronously (H. Sasanuma, un- published results), consistent with previous reports (Foreman and Davis 1996; Shi et al. 1996; Koch et al. 1999). Therefore, we focused our further studies on the three remaining de- letion mutants, rtf1, cdc73, and leo1. First, we checked levels of the two types of histone H3 methylation promoted by Paf1C, H3K4me (me1, me2, and me3) and H3K79me (me3), in cell lysates by Western blotting (Figure 1A). As a control, we also included a set1 deletion mutant, which is defective in H3K4 methylation. In wild-type cell lysates, lev- els of both H3K4me3 and H3K79me3 were almost constant during meiosis with similar levels observed during vegetative growth, which corresponds to 0-hr time point of the meiotic time course. On the other hand, levels of H3K4me1 and me2 increased slightly 2 hr after induction of meiosis in wild-type cells. The set1 deletion completely abolished H3K4 methyl- ation, but did not affect levels of H3K79me3. As reported previously (Jaehning 2010), the rtf1 mutation abolished detectable levels of the H3K4me3/me2/me1 and strongly reduced H3K79me3 compared to the levels detected in wild-type cells. Defective H3K4 methylation in the rtf1 mu- tant might be due to reduced Set1/COMPASS binding to the promoter (Krogan et al. 2003; Ng et al. 2003). The cdc73 mutation decreased H3K4me3 to about two-thirds of the wild-type level, while intensities of H3K4me1, H3K4me2, and H3K79me3 were reduced by half (Figure S1). The de- letion of the LEO1 gene did not affect either H3K4 or H3K79 methylation (Figure S1).
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We report here the effect of somatic mutations in the catalytic domain of MLL3. Our data show that mutations in the SET domain of MLL3 may lead to tumorigenesis through two converse mechanisms. The N4848S mutation leads to a loss of the catalytic activity of MLL3, which re- sembles the effect of other loss of function mutations in MLL3, like frame shifts or loss of expression. Such muta- tions may lead to the loss of H3K4 methylation at target genes and inhibit the expression of tumor suppressor genes. The Y4884C mutation leads to a change in the sub- strate specificity and product pattern of MLL3. This may result in the deposition of aberrant H3K4 trimethylation at enhancers leading to their conversion to promoters and the expression of oncogenes similarly as observed after knockdown of the Kdm5c H3K4me3 demethylase . Hence, MLL3 inhibitors are promising therapeutic options for cancers containing Y4884C mutations, but they might even be harmful in cancers with N4848S mutations. These data illustrate that individual cancer mutations even in
Humans are auxotrophic for both folate and methionine. (Although humans can synthesize methionine from homo- cysteine, humans lack genes required for de novo homocys- teine synthesis.) In addition to the methionine taken from diet, 5-methyltetrahydrofolate is still required for proper methionine homeostasis in humans, through the remethyla- tion of homocysteine produced by methylation reactions. Moreover, many histone methylations are conserved from yeast to humans; for instance, H3K4 methylation in humans is placed by human SET-domain proteins and is enriched at transcriptionally active loci (Ruthenburg et al. 2007), just as in yeast species. In addition, humans have well-described histone methylations on lysines K9, K27, K36, and K79 of histone H3, and lysine K20 of histone H4 (Barski et al. 2007; Mikkelsen et al. 2007), all of which are shared with S.cerevisiae, S. pombe, or other fungal species (Smith et al. 2008). H3K9 methylation is enriched in heterochromatin (Barski et al. 2007), making it functionally distinct from H3K4 methyla- tion. To determine whether the effects observed in S. cerevisiae and S. pombe pertained to human cells, histone methylation levels in human K562 cells were tested at a range of folate and methionine concentrations that are within the range of concentrations in human serum (Friso et al. 2002; Kimura et al. 2004).
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Yeasts have only one H3K4 methyltransferase (Briggs et al., 2001), originally called the Set1 complex (Set1C) (Roguev et al., 2001; Krogan et al., 2002; Noma and Grewal, 2002; Roguev et al., 2003), and the distinction between mono-, di- and trimethylated H3K4 sites is mediated by regulation of its enzymatic SET domain (Kim et al., 2013) or by opposing demethylases (Seward et al., 2007; Kooistra and Helin, 2012). Yeast Set1C contains eight subunits and was the first linkage between H3K4 methylation and Trithorax group (Trx-G) action (Roguev et al., 2001). This linkage was based on two observations: (1) one of the eight Set1C subunits, Bre2, is the yeast ortholog of the fly Trx-G protein Ash2; (2) among the diversity of SET methyltransferase domains, the SET domain of yeast Set1 is almost identical to that of Drosophila Trx. Because yeast Set1C is an H3K4 methyltransferase, it was likely that Drosophila Trx and the mammalian orthologs Mll1 and Mll2 would also be H3K4 methyltransferases, as was subsequently proven (Milne et al., 2002).
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cell lines SGC7901 and BGC823, respectively[9-10, 17]. As shown in Figure 1a and b, the survival rates of sensitive cells SGC7901 and BGC823 showed a dose-dependent decline in response to cisplatin. However, the resistant cells SGC7901/DDP and BGC823/DDP still maintained a high survival rate under the effect of cisplatin. We first examined the methylation level of H3K4 in drug-resistant cells and -sensitive cells, and found that the overall methylation level of H3K4 especially di-methylation and tri- methylation levels were decreased in both drug- resistant cells, and the most significantly decreased was tri-methylation (Figure 1c). To elucidate the relevance of H3K4 methylation to drug resistance, we used the methyltransferase inhibitor MM-102 and the demethylase inhibitor JIB-04 to alter intracellular H3K4 methylation (Supplementary Figure 1a and b). The results showed that the inhibition of H3K4 methylation by MM-102 significantly increased the survival rates of sensitive cells SGC7901 and BGC823 under the treatment of cisplatin (Figure 1d and e). Conversely, the use of JIB-04 to promote methylation of H3K4 significantly reduced the survival rates of drug-resistant cells under cisplatin treatment (Figure 1f and g). In summary, these results suggested that demethylation of H3K4 could promote the drug resistance of cancer cells.
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The Set1 histone methyltransferase and the Bre1 E3 ubiquitin ligase have well-established roles in regulating gene expression (Shukla et al. 2006; Mutiu et al. 2007). Nonetheless, several lines of evidence suggest that the phe- notypes that we have detected are due to reduced replica- tion activity as opposed to altered gene expression. First, many chromatin-modifying enzymes showed no synthetic growth phenotype when combined with the cdc6-1 muta- tion. Some of these non-interacting genes include those with much more profound effects on patterns of gene expression than Set1, suggesting that replication phenotypes are not a general outcome of perturbed gene expression control. In- terestingly, even the Rpd3 histone deacetylase that regulates origin ﬁring time within S phase (Knott et al. 2009) did not genetically interact with cdc6-1 (Table 3), implying that the synthetic growth phenotypes observed here are relatively speciﬁc for origin function and not origin ﬁring time. Sec- ond, a genome-wide analysis identiﬁed only 55 transcripts that changed signiﬁcantly in a set1 D strain compared to a wild-type strain, and none of those genes are predicted to directly affect origin activity (Lenstra et al. 2011). Third, the mini-chromosome maintenance phenotypes associated with loss of H3K4 di-methylation were largely suppressed by the addition of extra origins to the test plasmid, indicat- ing that the phenotypes are closely tied to origin function and not to other biological parameters. Fourth, we directly detected H3K4 di- and tri-methylation at two yeast origins. Moreover, our analysis of a published genome-wide H3K4 tri-methylation data set identiﬁed peaks of methylation dis- tinct from nearby transcription-associated peaks (Radman- Livaja et al. 2010 and our unpublished observations). Finally, our observation that loss of H3K4 methylation exacerbates poor growth of a replication hypomorphic strain but suppresses the poor growth of an origin-ﬁring hypermorphic strain indi- cates that the replication phenotypes reported here are most likely direct positive effects of H3K4 di-methylation at origins. Taken together, these data provide strong evidence that the role of H3K4 di-methylation in origin function is direct and separate from any indirect transcriptional effects.
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with CLL (P=0.028 and P=0.03, respectively) and the global histone H3K9 methylation in patients with CLL were significantly increased compared with controls (P=0.02), while there was no significant difference in the global histone H3K4 methylation between the two groups. The level of SIRT1 and EZH2 mRNA expression was upregulated in patients with CLL (P=0.03 and P=0.02, respectively), which increased significantly with progression from Binet stage A to stage C (P=0.015 and P=0.01, respectively) and Rai good to high risk stage (P=0.007 and P=0.008, respectively). The level of HDAC1 and HDAC7 mRNA expression was significantly increased (P=0.02 and P=0.008, respectively) and HDAC2 and P300 mRNA expression was reduced in patients with CLL (P=0.002 and P=0.001, respectively). In conclusion, it is observed that the aberrant histone modification plays an important role in the pathogenesis of CLL. Keywords: histone methylation, histone acetylation, SIRT1, EZH2, CLL
The requirement for histone methylation in opening the chromatin structure is both less understood and more complex as multiple methyl groups can be added to either arginine (R) residues or lysine (K) residues. Methylation of arginine residues is associated with gene activation (Bauer, Daujat et al. 2002), but methylation of lysine residues has been historically associated with gene inactivation as methylation was thought to be both silencing and irreversible (Jenuwein and Allis 2001; Nakayama, Rice et al. 2001; Berger 2002; Lehnertz, Ueda et al. 2003; Plath, Fang et al. 2003; Shilatifard 2006; Sparmann and van Lohuizen 2006). Some of the first histone methylating events identified were silencing in nature. Histone H3 dimethylated at lysine 9 (H3K9me2) interacts with heterochromatin protein 1to facilitate chromatin silencing (Lachner, O'Carroll et al. 2001; Nakayama, Rice et al. 2001; Jacobs and Khorasanizadeh 2002) and histone H3 trimethylated at lysine 27 (H3K27me3) has been associated with a variety of processes including X chromosome inactivation and imprinting (Plath, Fang et al. 2003; Sparmann and van Lohuizen 2006). However with the discovery of histone demethylases, lysine methylation is now known to be dynamic and certain methylation events are intrinsic for gene expression as modifications such as the di- and trimethylation (hypermethylation) of H3 at lysine (K) 4 and K36 are found at actively transcribed genes (Bernstein, Humphrey et al. 2002; Santos-Rosa, Schneider et al. 2002; Bannister, Schneider et al. 2005; Shilatifard 2006; Tsukada, Fang et al. 2006; Whetstine, Nottke et al. 2006). Histone H3K36 methylation is localized within gene coding sequence and functions in transcriptional memory to prevent inappropriate transcription initiation by recruiting histone deacetylases (HDACs) in the wake of elongating RNA polymerase II (Carrozza, Li et al. 2005; Joshi and Struhl 2005; Lee and Shilatifard 2007). In addition
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Particularly striking in comparison to the majority of (HPV-associated) cervical squamous cell carcin- omas within the reference collection was the rather flat copy number profile of our case, hinting at a po- tential defect in DNA repair causing point mutations (not detectable with the methylation array) rather than being driven by a virus. It is, however, not en- tirely to be excluded that this unusual type of cancer described here evolved through infection with a rare, undetectable HPV genotype.
Overexpression of glucosylceramide synthase (GCS) increases multidrug resistance (MDR) in many cancer cells. However, its mechanism is unknown. The aim of the present study is to detect the association of methylation at the GCS gene promoter with its expression and MDR in invasive ductal breast cancer. 40 cases GCS-positive and 40 cases GCS-negative primary breast carcinoma samples, three drug-sensitive breast cancer cell lines and one multidrug-resistant breast cancer cell line were used. Immunohistochemistry, methylation-specific PCR (MSP), quantitative real-time (qPCR), westernblot and cytotoxicity assay techniques were employed. Thwe results revealed that there was a statistically negative correlation between GCS CpG islands methylation and GCS phenotype in patients with breast cancer. GCS CpG islands methylation was negatively associated with high ER, meanwhile positively with high HER-2 status. Similar results were obtained from the analysis of breast cancer cell lines. Treatment with the demethylating agent 5-aza-2′-deoxycytidine (5-Aza-dc) changed the GCS promoter methylation pattern in three sensitive cells and also caused increased drug resistance of them. These results suggested that the changes of DNA methylation status of the GCS promoter correlates with multidrug resistance in breast cancer.
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The application of next-generation sequencing technologies in the field of cancer genomics has substantially increased our under- standing of cancer biology. Detection of germline and somatic mutations in specific tumor types not only expands the current repertoire of driver mutations and downstream effectors in tumor- igenesis, but also sheds light on how oncometabolites may exert their oncogenic roles. For example, the identification of mutually exclusive mutations in IDH1 and TET2 in AML led to the char- acterization of TET2 as a major pathological target of D-2HG (34, 110). Additionally, the discovery of somatic CUL3, SIRT1, and NRF2 mutations in sporadic PRCC2 converges with FH mutation in HLRCC, in which NRF2 activation is a consequence of fuma- rate-mediated succination of KEAP1, indicating the functional prominence of the NRF2 pathway in PRCC2 (73). In light of this, the identification of somatic mutations in genes encoding the chromatin-modifying enzymes histone H3K36 methyltransferase (SETD2), histone H3K4 demethylase JARID1C (KDM5C), histone H3K27 demethylase UTX (KDM6A), and the SWI/SNF chromatin remodelling complex gene PBRM1 in clear cell renal cell carcino- ma (111–113) highlights the importance of epigenetic modulation in human cancer and raises the potential for systematic testing in other types of tumors such as those associated with FH mutations.
Histone methylation is one of several epigenetic layers for transcriptional regulation. Most studies on the importance of this histone modification in regulating fungal secondary metabolite gene expression and pathogenicity have focussed on the role of histone methyltransferases, while few studies have focussed on the role of histone demethylases that catalyse the reversal of the modification. Epichloë festucae (Ascomycota) is an endophyte that forms a mutualistic interaction with perennial ryegrass. The fungus contributes to the symbiosis by the production of several classes of secondary metabolites, these have anti-insect and/or anti-mammalian activity. The EAS and LTM clusters in E. festucae are located subtelomerically and contain the biosynthetic genes for two of these important metabolites which are only synthesised in planta. Thus, in the host plant these genes are highly expressed, but they are tightly silenced in culture conditions. Previous study has shown that histone H3K9 and H3K27 methylation and their corresponding histone methyltransferases are important for this process. In this study, the role of histone lysine demethylases (KDMs) in regulating these genes and the symbiotic interaction is described. Eight candidate histone demethylases (Jmj1-Jmj8) were identified in E. festucae, among these proteins are homologues of mammalian KDM4, KDM5, KDM8, JMDJ7, and N. crassa Dmm-1. The genes for the proteins were overexpressed in E. festucae and histone methylation levels were determined in the strains. Overexpression of the genes was not observed to cause any change to the culture and symbiotic phenotypes of the fungus. Western blot analysis subsequently identified one of the proteins, KdmB, as the histone H3K4me3 demethylase. Further analysis by ChIP- and RT-qPCR showed that demethylation of H3K4me3 by KdmB at the eas/ltm genes is crucial for the activation of these genes in planta. The full expression of several other telomeric genes was similarly found to require KdmB. On the other hand, the COMPASS H3K4 methyltransferase complex subunit CclA that is required for H3K4 trimethylation in E. festucae represses the eas/ltm genes in culture conditions by maintaining H3K4me3 levels at the loci.
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One criticism of the present study regards the number of GC specimens used. Although the results obtained on two GC patients were roughly similar, to be confident that a mechanism underlying GKN1 gene silencing is also based on the up-regulation of histone methylation H3K4me3 by SUV39H1 and deacetyla- tion mediated by HDAC1, a larger number of samples should be analyzed. This aspect is not easy to afford because of the difficulty in the recruitment of GC pa- tients, in the quality and quantity of tissues specimens that can be obtained after surgery. Moreover, also the intrinsic difficulty of the ChiP assay experimental procedure should be taken into consideration.
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