Ryo Nakaki

H3K4/H3K9me3 Bivalent Chromatin Domains Targeted by Lineage-Specific DNA Methylation Pauses Adipocyte Differentiation.

Bivalent H3K4me3 and H3K27me3 chromatin domains in embryonic stem cells keep active developmental regulatory genes expressed at very low levels and poised for activation. Here, we show an alternative and previously unknown bivalent modified histone signature in lineage-committed mesenchymal stem cells and preadipocytes that pairs H3K4me3 with H3K9me3 to maintain adipogenic master regulatory genes (Cebpa and Pparg) expressed at low levels yet poised for activation when differentiation is required. We show lineage-specific gene-body DNA methylation recruits H3K9 methyltransferase SETDB1, which methylates H3K9 immediately downstream of transcription start sites marked with H3K4me3 to establish the bivalent domain. At the Cebpa locus, this prevents transcription factor C/EBPβ binding, histone acetylation, and further H3K4me3 deposition and is associated with pausing of RNA polymerase II, which limits Cebpa gene expression and adipogenesis.

JMJD1A is a signal-sensing scaffold that regulates acute chromatin dynamics via SWI/SNF association for thermogenesis

Histone 3 lysine 9 (H3K9) demethylase JMJD1A regulates β-adrenergic-induced systemic metabolism and body weight control. Here we show that JMJD1A is phosphorylated at S265 by protein kinase A (PKA), and this is pivotal to activate the β1-adrenergic receptor gene (Adrb1) and downstream targets including Ucp1 in brown adipocytes (BATs). Phosphorylation of JMJD1A by PKA increases its interaction with the SWI/SNF nucleosome remodelling complex and DNA-bound PPARγ. This complex confers β-adrenergic-induced rapid JMJD1A recruitment to target sites and facilitates long-range chromatin interactions and target gene activation. This rapid gene induction is dependent on S265 phosphorylation but not on demethylation activity. Our results show that JMJD1A has two important roles in regulating hormone-stimulated chromatin dynamics that modulate thermogenesis in BATs. In one role, JMJD1A is recruited to target sites and functions as a cAMP-responsive scaffold that facilitates long-range chromatin interactions, and in the second role, JMJD1A demethylates H3K9 di-methylation.

The FBXL10/KDM2B Scaffolding Protein Associates with Novel Polycomb Repressive Complex-1 to Regulate Adipogenesis

Background: Polycomb repressive complex PRC1 is an epigenetic regulator of cellular differentiation. However, its function during adipogenesis is unknown. Results: FBXL10/KDM2B recruited noncanonical PRC1 complex in F-box and LRR motifs dependent on cell cycle-related genes and Pparg genes and repressed 3T3-L1 adipogenesis. Conclusion: Noncanonical PRC1 complex containing FBXL10/KDM2B regulates adipogenesis. Significance: Our findings revealed a novel epigenetic mechanism of adipogenesis. Polycomb repressive complex 1 (PRC1) plays an essential role in the epigenetic repression of gene expression during development and cellular differentiation via multiple effector mechanisms, including ubiquitination of H2A and chromatin compaction. However, whether it regulates the stepwise progression of adipogenesis is unknown. Here, we show that FBXL10/KDM2B is an anti-adipogenic factor that is up-regulated during the early phase of 3T3-L1 preadipocyte differentiation and in adipose tissue in a diet-induced model of obesity. Interestingly, inhibition of adipogenesis does not require the JmjC demethylase domain of FBXL10, but it does require the F-box and leucine-rich repeat domains, which we show recruit a noncanonical polycomb repressive complex 1 (PRC1) containing RING1B, SKP1, PCGF1, and BCOR. Knockdown of either RING1B or SKP1 prevented FBXL10-mediated repression of 3T3-L1 preadipocyte differentiation indicating that PRC1 formation mediates the inhibitory effect of FBXL10 on adipogenesis. Using ChIP-seq, we show that FBXL10 recruits RING1B to key specific genomic loci surrounding the key cell cycle and the adipogenic genes Cdk1, Uhrf1, Pparg1, and Pparg2 to repress adipogenesis. These results suggest that FBXL10 represses adipogenesis by targeting a noncanonical PRC1 complex to repress key genes (e.g. Pparg) that control conversion of pluripotent cells into the adipogenic lineage.

NFIA co-localizes with PPARγ and transcriptionally controls the brown fat gene program

Brown fat dissipates energy as heat and protects against obesity. Here, we identified nuclear factor I-A (NFIA) as a transcriptional regulator of brown fat by a genome-wide open chromatin analysis of murine brown and white fat followed by motif analysis of brown-fat-specific open chromatin regions. NFIA and the master transcriptional regulator of adipogenesis, PPARγ, co-localize at the brown-fat-specific enhancers. Moreover, the binding of NFIA precedes and facilitates the binding of PPARγ, leading to increased chromatin accessibility and active transcription. Introduction of NFIA into myoblasts results in brown adipocyte differentiation. Conversely, the brown fat of NFIA-knockout mice displays impaired expression of the brown-fat-specific genes and reciprocal elevation of muscle genes. Finally, expression of NFIA and the brown-fat-specific genes is positively correlated in human brown fat. These results indicate that NFIA activates the cell-type-specific enhancers and facilitates the binding of PPARγ to control the brown fat gene program.

Novel lnc RNA regulated by HIF‐1 inhibits apoptotic cell death in the renal tubular epithelial cells under hypoxia

Chronic tubulointerstitial hypoxia plays an important role as the final common pathway to end‐stage renal disease. HIF‐1 (hypoxia‐inducible factor‐1) is a master transcriptional factor under hypoxia, regulating downstream target genes. Genome‐wide analysis of HIF‐1 binding sites using high‐throughput sequencers has clarified various kinds of downstream targets and made it possible to demonstrate the novel roles of HIF‐1. Our aim of this study is to identify novel HIF‐1 downstream epigenetic targets which may play important roles in the kidney. Immortalized tubular cell lines (HK2; human kidney‐2) and primary cultured cells (RPTEC; renal proximal tubular cell lines) were exposed to 1% hypoxia for 24–72 h. We performed RNA‐seq to clarify the expression of mRNA and long non‐coding RNA (lncRNA). We also examined ChIP‐seq to identify HIF‐1 binding sites under hypoxia. RNA‐seq identified 44 lncRNAs which are up‐regulated under hypoxic condition in both cells. ChIP‐seq analysis demonstrated that HIF‐1 also binds to the lncRNAs under hypoxia. The expression of novel lncRNA, DARS‐AS1 (aspartyl‐tRNA synthetase anti‐sense 1), is up‐regulated only under hypoxia and HIF‐1 binds to its promoter region, which includes two hypoxia‐responsive elements. Its expression is also up‐regulated with cobalt chloride exposure, while it is not under hypoxia when HIF‐1 is knocked down by siRNA. To clarify the biological roles of DARS‐AS1, we measured the activity of caspase 3/7 using anti‐sense oligo of DARS‐AS1. Knockdown of DARS‐AS1 deteriorated apoptotic cell death. In conclusion, we identified the novel lncRNAs regulated by HIF‐1 under hypoxia and clarified that DARS‐AS1 plays an important role in inhibiting apoptotic cell death in renal tubular cells.

Dynamically and epigenetically coordinated GATA/ETS/SOX transcription factor expression is indispensable for endothelial cell differentiation

Abstract Although studies of the differentiation from mouse embryonic stem (ES) cells to vascular endothelial cells (ECs) provide an excellent model for investigating the molecular mechanisms underlying vascular development, temporal dynamics of gene expression and chromatin modifications have not been well studied. Herein, using transcriptomic and epigenomic analyses based on H3K4me3 and H3K27me3 modifications at a genome-wide scale, we analysed the EC differentiation steps from ES cells and crucial epigenetic modifications unique to ECs. We determined that Gata2, Fli1, Sox7 and Sox18 are master regulators of EC that are induced following expression of the haemangioblast commitment pioneer factor, Etv2. These master regulator gene loci were repressed by H3K27me3 throughout the mesoderm period but rapidly transitioned to histone modification switching from H3K27me3 to H3K4me3 after treatment with vascular endothelial growth factor. SiRNA knockdown experiments indicated that these regulators are indispensable not only for proper EC differentiation but also for blocking the commitment to other closely aligned lineages. Collectively, our detailed epigenetic analysis may provide an advanced model for understanding temporal regulation of chromatin signatures and resulting gene expression profiles during EC commitment. These studies may inform the future development of methods to stimulate the vascular endothelium for regenerative medicine.

Integrative analysis of transcription factor occupancy at enhancers and disease risk loci in noncoding genomic regions

Noncoding regions of the human genome possess enhancer activity and harbor risk loci for heritable diseases. Whereas the binding profiles of multiple transcription factors (TFs) have been investigated, integrative analysis with the large body of public data available so as to provide an overview of the function of such noncoding regions has remained a challenge. Here we have fully integrated public ChIP-seq and DNase-seq data (n ~ 70,000), including those for 743 human transcription factors (TFs) with 97 million binding sites, and have devised a data-mining platform —designated ChIP-Atlas—to identify significant TF-genome, TF-gene, and TF-TF interactions. Using this platform, we found that TFs enriched at macrophage or T-cell enhancers also accumulated around risk loci for autoimmune diseases, whereas those enriched at hepatocyte or macrophage enhancers were preferentially detected at loci associated with HDL-cholesterol levels. Of note, we identified “hotspots” around such risk loci that accumulated multiple TFs and are therefore candidates for causal variants. Integrative analysis of public chromatin-profiling data is thus able to identify TFs and tissues associated with heritable disorders.

Diminished nuclear RNA decay upon Salmonella infection upregulates antibacterial noncoding RNAs

Cytoplasmic mRNA degradation controls gene expression to help eliminate pathogens during infection. However, it has remained unclear whether such regulation also extends to nuclear RNA decay. Here, we show that 145 unstable nuclear RNAs, including enhancer RNAs (eRNAs) and long noncoding RNAs (lncRNAs) such as NEAT1v2, are stabilized upon Salmonella infection in HeLa cells. In uninfected cells, the RNA exosome, aided by the Nuclear EXosome Targeting (NEXT) complex, degrades these labile transcripts. Upon infection, the levels of the exosome/NEXT components, RRP6 and MTR4, dramatically decrease, resulting in transcript stabilization. Depletion of lncRNAs, NEAT1v2, or eRNA07573 in HeLa cells triggers increased susceptibility to Salmonella infection concomitant with the deregulated expression of a distinct class of immunity‐related genes, indicating that the accumulation of unstable nuclear RNAs contributes to antibacterial defense. Our results highlight a fundamental role for regulated degradation of nuclear RNA in the response to pathogenic infection.

Dynamics of chromatin accessibility during TGF- β- induced EMT of Ras-transformed mammary gland epithelial cells

Epithelial-mesenchymal transition (EMT) is induced by transforming growth factor (TGF)-β and facilitates tumor progression. We here performed global mapping of accessible chromatin in the mouse mammary gland epithelial EpH4 cell line and its Ras-transformed derivative (EpRas) using formaldehyde-assisted isolation of regulatory element (FAIRE)-sequencing. TGF-β and Ras altered chromatin accessibility either cooperatively or independently, and AP1, ETS, and RUNX binding motifs were enriched in the accessible chromatin regions of EpH4 and EpRas cells. Etv4, an ETS family oncogenic transcription factor, was strongly expressed and bound to more than one-third of the accessible chromatin regions in EpRas cells treated with TGF-β. While knockdown of Etv4 and another ETS family member Etv5 showed limited effects on the decrease in the E-cadherin abundance and stress fiber formation by TGF-β, gene ontology analysis showed that genes encoding extracellular proteins were most strongly down-regulated by Etv4 and Etv5 siRNAs. Accordingly, TGF-β-induced expression of Mmp13 and cell invasiveness were suppressed by Etv4 and Etv5 siRNAs, which were accompanied by the reduced chromatin accessibility at an enhancer region of Mmp13 gene. These findings suggest a mechanism of transcriptional regulation during Ras- and TGF-β-induced EMT that involves alterations of accessible chromatin, which are partly regulated by Etv4 and Etv5.

Genome-wide analysis revealed that DZNep reduces tubulointerstitial fibrosis via down-regulation of pro-fibrotic genes.

Tubulointerstitial fibrosis has been recently reported to be caused by the collapse of the epigenetic regulation of kidney diseases. We examined whether pharmacological inhibition of histone modification is effective against renal fibrosis. DZNep (3-deazaneplanocin A) was originally developed as an anti-cancer drug to inhibit the repressive histone mark, H3K27me3. We used a model of chronic tubulointerstitial fibrosis induced by unilateral ischaemia/reperfusion and administered DZNep intravenously to the mice for 8 weeks. We found DZNep contributes to the reduction of tubulointerstitial fibrosis. We selected only tubular cells from in vivo samples using laser-capture microdissection because epigenetic regulation is specific to the cell types, and we focused on the changes in the tubular cells. We performed a genome-wide analysis of tubular cells using high-throughput sequencing (RNA-seq) to identify novel epigenetic factors associated with renal fibrosis. We found that pro-fibrotic genes such as COL3A1 (collagen type 3a1) and TIMP2 (tissue inhibitor of metalloproteinase 2) were suppressed by DZNep in vivo. In addition, pro-fibrotic genes such as COL4A1 (collagen type 4a1), TIMP2 and MMP14 were down-regulated by DZNep in vitro. In conclusion, we found that pharmacological epigenetic modification by DZNep decreased the expression levels of fibrogenic genes in tubular cells and inhibited tubulointerstitial fibrosis.