The role of tumor suppressor menin in IL-6 regulation in mouse islet tumor cells

Menin is a gene product of multiple endocrine neoplasia type1 (Men1), an inherited familial cancer syndrome characterized by tumors of endocrine tissues. To gain insight about how menin performs an endocrine cell-specific tumor suppressor function, we investigated the possibility that menin was integrated in a cancer-associated inflammatory pathway in a cell type-specific manner. Here, we showed that the expression of IL-6, a proinflammatory cytokine, was specifically elevated in mouse islet tumor cells upon depletion of menin and Men^−/− MEF cells, but not in hepatocellular carcinoma cells. Histone H3 lysine (K) 9 methylation, but not H3 K27 or K4 methylation, was involved in menin-dependent IL-6 regulation. Menin occupied the IL-6 promoter and recruited SUV39H1 to induce H3 K9 methylation. Our findings provide a molecular insight that menin-dependent induction of H3 K9 methylation in the cancer-associated interleukin gene might be linked to preventing endocrine-specific tumorigenesis.     

Histone H3 lysine 4 monomethylation (H3K4me1) and H3 lysine 9 monomethylation (H3K9me1): Distribution and their association in regulating gene expression under hyperglycaemic/hyperinsulinemic conditions in 3T3 cells

Hyperglycemia/hyperinsulinemia are leading cause for the induction type 2 diabetes and the role of post-translational histone modifications in dysregulating the expression of genes has emerged as potential important contributor in the progression of disease. The paradoxical nature of histone H3-Lysine 4 and Lysine 9 mono-methylation (H3K4me1 and H3K9me1) in both gene activation and repression motivated us to elucidate the functional relationship of these histone modifications in regulating expression of genes under hyperglycaemic/hyperinsulinemic condition. Chromatin immunoprecipitation–microarray analysis (ChIP-chip) was performed with H3 acetylation, H3K4me1 and H3K9me1 antibody. CLUSTER analysis of ChIP-chip (Chromatin immunoprecipitation–microarray analysis) data showed that mRNA expression and H3 acetylation/H3K4me1 levels on genes were inversely correlated with H3K9me1 levels on the transcribed regions, after 30 min of insulin stimulation under hyperglycaemic condition. Interestingly, we provide first evidence regarding regulation of histone de/acetylases and de/methylases; Myst4, Jmjd2b, Aof1 and Set by H3Ac, H3K4me1 and H3K9me1 under hyperinsulinemic/hyperglycaemic condition. ChIP–qPCR analysis shows association of increased H3Ac/H3K4me1 and decreased levels of H3K9me1 in up regulation of Myst4, Jmjd2, Set and Aof1 genes. We further analyse promoter occupancy of histone modifications by ChIP walking and observed increased occupancy of H3Ac/H3K4me1 on promoter region (−1000 to −1) of active genes and H3K9me1 on inactive genes under hyperglycemic/hyperinsulinemic condition. To best of our knowledge this is the first report that shows regulation of chromatin remodelling genes by alteration in the occupancy of histone H3Ac/H3K4/K9me on both promoter and transcribed regions.     

Epigenetic and genetic variation at the IGF2/H19 imprinting control region on 11p15.5 is associated with cerebellum weight

IGF2 is a paternally expressed imprinted gene with an important role in development and brain function. Allele-specific expression of IGF2 is regulated by DNA methylation at three differentially methylated regions (DMRs) spanning the IGF2/H19 domain on human 11p15.5. We have comprehensively assessed DNA methylation and genotype across the three DMRs and the H19 promoter using tissue from a unique collection of well-characterized and neuropathologically-dissected post-mortem human cerebellum samples (n = 106) and frontal cortex samples (n = 51). We show that DNA methylation, particularly in the vicinity of a key CTCF-binding site (CTCF3) in the imprinting control region (ICR) upstream of H19, is strongly correlated with cerebellum weight. DNA methylation at CTCF3 uniquely explains ∼25% of the variance in cerebellum weight. In addition, we report that genetic variation in this ICR is strongly associated with cerebellum weight in a parental-origin specific manner, with maternally-inherited alleles associated with a 16% increase in cerebellum weight compared with paternally-inherited alleles. Given the link between structural brain abnormalities and neuropsychiatric disease, an understanding of the epigenetic and parent-of-origin specific genetic factors associated with brain morphology provides important clues about the etiology of disorders such as schizophrenia and autism.Key words: epigenetic, DNA methylation, genomic imprinting, cerebellum, IGF2, H19, brain, expression, frontal cortex, genetic, single nucleotide polymorphism     

Genome-wide characterization of menin-dependent H3K4me3 reveals a specific role for menin in the regulation of genes implicated in MEN1-like tumors

Inactivating mutations in the MEN1 gene predisposing to the multiple endocrine neoplasia type 1 (MEN1) syndrome can also cause sporadic pancreatic endocrine tumors. MEN1 encodes menin, a subunit of MLL1/MLL2-containing histone methyltransferase complexes that trimethylate histone H3 at lysine 4 (H3K4me3). The importance of menin-dependent H3K4me3 in normal and transformed pancreatic endocrine cells is unclear. To study the role of menin-dependent H3K4me3, we performed in vitro differentiation of wild-type as well as menin-null mouse embryonic stem cells (mESCs) into pancreatic islet-like endocrine cells (PILECs). Gene expression analysis and genome-wide H3K4me3 ChIP-Seq profiling in wild-type and menin-null mESCs and PILECs revealed menin-dependent H3K4me3 at the imprinted Dlk1-Meg3 locus in mESCs, and all four Hox loci in differentiated PILECs. Specific and significant loss of H3K4me3 and gene expression was observed for genes within the imprinted Dlk1-Meg3 locus in menin-null mESCs and the Hox loci in menin-null PILECs. Given that the reduced expression of genes within the DLK1-MEG3 locus and the HOX loci is associated with MEN1-like sporadic tumors, our data suggests a possible role for menin-dependent H3K4me3 at these genes in the initiation and progression of sporadic pancreatic endocrine tumors. Furthermore, our investigation also demonstrates that menin-null mESCs can be differentiated in vitro into islet-like endocrine cells, underscoring the utility of menin-null mESC-derived specialized cell types for genome-wide high-throughput studies.     

Stress‐associated H3K4 methylation accumulates during postnatal development and aging of rhesus macaque brain

Epigenetic modifications are critical determinants of cellular and developmental states. Epigenetic changes, such as decreased H3K27me3 histone methylation on insulin/IGF1 genes, have been previously shown to modulate lifespan through gene expression regulation. However, global epigenetic changes during aging and their biological functions, if any, remain elusive. Here, we examined the histone modification H3K4 dimethylation (H3K4me2) in the prefrontal cortex of individual rhesus macaques at different ages by chromatin immunoprecipitation, followed by deep sequencing (ChIP‐seq) at the whole genome level. Through integrative analysis of the ChIP‐seq profiles with gene expression data, we found that H3K4me2 increased at promoters and enhancers globally during postnatal development and aging, and those that correspond to gene expression changes in cis are enriched for stress responses, such as the DNA damage response. This suggests that metabolic and environmental stresses experienced by an organism are associated with the progressive opening of chromatin. In support of this, we also observed increased expression of two H3K4 methyltransferases, SETD7 and DPY30, in aged macaque brain.     

Inhibition of the SUV4-20 H1 histone methyltransferase increases frataxin expression in Friedreich's ataxia patient cells

The molecular mechanisms of reduced frataxin (FXN) expression in Friedreich''s ataxia (FRDA) are linked to epigenetic modification of the FXN locus caused by the disease-associated GAA expansion. Here, we identify that SUV4-20 histone methyltransferases, specifically SUV4-20 H1, play an important role in the regulation of FXN expression and represent a novel therapeutic target. Using a human FXN–GAA–Luciferase repeat expansion genomic DNA reporter model of FRDA, we screened the Structural Genomics Consortium epigenetic probe collection. We found that pharmacological inhibition of the SUV4-20 methyltransferases by the tool compound A-196 increased the expression of FXN by ∼1.5-fold in the reporter cell line. In several FRDA cell lines and patient-derived primary peripheral blood mononuclear cells, A-196 increased FXN expression by up to 2-fold, an effect not seen in WT cells. SUV4-20 inhibition was accompanied by a reduction in H4K20me2 and H4K20me3 and an increase in H4K20me1, but only modest (1.4–7.8%) perturbation in genome-wide expression was observed. Finally, based on the structural activity relationship and crystal structure of A-196, novel small molecule A-196 analogs were synthesized and shown to give a 20-fold increase in potency for increasing FXN expression. Overall, our results suggest that histone methylation is important in the regulation of FXN expression and highlight SUV4-20 H1 as a potential novel therapeutic target for FRDA.     

A balance between activating and repressive histone modifications regulates cystic fibrosis transmembrane conductance regulator (CFTR) expression in vivo

The genetic mechanisms that regulate CFTR, the gene responsible for cystic fibrosis, have been widely investigated in cultured cells. However, mechanisms responsible for tissue-specific and time-specific expression are not completely elucidated in vivo. Through the survey of public databases, we found that the promoter of CFTR was associated with bivalent chromatin in human embryonic stem (ES) cells. In this work, we analyzed fetal (at different stages of pregnancy) and adult tissues and showed that, in digestive and lung tissues, which expressed CFTR, H3K4me3 was maintained in the promoter. Histone acetylation was high in the promoter and in two intronic enhancers, especially in fetal tissues. In contrast, in blood cells, which did not express CFTR, the bivalent chromatin was resolved (the promoter was labeled by the silencing mark H3K27me3). Cis-regulatory sequences were associated with lowly acetylated histones. We also provide evidence that the tissue-specific expression of CFTR is not regulated by dynamic changes of DNA methylation in the promoter. Overall, this work shows that a balance between activating and repressive histone modifications in the promoter and intronic enhancers results in the fine regulation of CFTR expression during development, thereby ensuring tissue specificity.