Tatsuhiko Kodama

A wave of nascent transcription on activated human genes.

Genome-wide studies reveal that transcription by RNA polymerase II (Pol II) is dynamically regulated. To obtain a comprehensive view of a single transcription cycle, we switched on transcription of five long human genes (>100 kbp) with tumor necrosis factor-α (TNFα) and monitored (using microarrays, RNA fluorescence in situ hybridization, and chromatin immunoprecipitation) the appearance of nascent RNA, changes in binding of Pol II and two insulators (the cohesin subunit RAD21 and the CCCTC-binding factor CTCF), and modifications of histone H3. Activation triggers a wave of transcription that sweeps along the genes at ≈3.1 kbp/min; splicing occurs cotranscriptionally, a major checkpoint acts several kilobases downstream of the transcription start site to regulate polymerase transit, and Pol II tends to stall at cohesin/CTCF binding sites.

Obesity and metabolic syndrome in histone demethylase JHDM2a-deficient mice.

Histone H3 lysine 9 (H3K9) methylation is a crucial epigenetic mark of heterochromatin formation and transcriptional silencing. Recent studies demonstrated that most covalent histone lysine modifications are reversible and the jumonji C (JmjC)‐domain‐containing proteins have been shown to possess such demethylase activities. However, there is little information available on the biological roles of histone lysine demethylation in intact animal model systems. JHDM2A (JmjC‐domain‐containing histone demethylase 2A, also known as JMJD1A) catalyses removal of H3K9 mono‐ and dimethylation through iron and α‐ketoglutarate dependent oxidative reactions. Here, we demonstrate that JHDM2a also regulates metabolic genes related to energy homeostasis including anti‐adipogenesis, regulation of fat storage, glucose transport and type 2 diabetes. Mice deficient in JHDM2a (JHDM2a−/−) develop adult onset obesity, hypertriglyceridemia, hypercholesterolemia, hyperinsulinemia and hyperleptinemia, which are hallmarks of metabolic syndrome. JHDM2a−/− mice furthermore exhibit fasted induced hypothermia indicating reduced energy expenditure and also have a higher respiratory quotient indicating less fat utilization for energy production. These observations may explain the obesity phenotype in these mice. Thus, H3K9 demethylase JHDM2a is a crucial regulator of genes involved in energy expenditure and fat storage, which suggests it is a previously unrecognized key regulator of obesity and metabolic syndrome.

Dynamic Change of Chromatin Conformation in Response to Hypoxia Enhances the Expression of GLUT3 (SLC2A3) by Cooperative Interaction of Hypoxia-Inducible Factor 1 and KDM3A

ABSTRACT Hypoxia-inducible factor 1 (HIF1) is a master regulator of adaptive gene expression under hypoxia. However, a role for HIF1 in the epigenetic regulation remains unknown. Genome-wide analysis of HIF1 binding sites (chromatin immunoprecipitation [ChIP] with deep sequencing) of endothelial cells clarified that HIF1 mainly binds to the intergenic regions distal from transcriptional starting sites under both normoxia and hypoxia. Next, we examined the temporal profile of gene expression under hypoxic conditions by using DNA microarrays. We clarified that early hypoxia-responsive genes are functionally associated with glycolysis, including GLUT3 (SLC2A3). Acetylated lysine 27 of histone 3 covered the HIF1 binding sites, and HIF1 functioned as an enhancer of SLC2A3 by interaction with lysine (K)-specific demethylase 3A (KDM3A). Knockdown of HIF1α and KDM3A showed that glycolytic genes are regulated by both HIF1 and KDM3A and respond to hypoxia in a manner independent of cell type specificity. We elucidated that both the chromatin conformational structure and histone modification change under hypoxic conditions and enhance the expression of SLC2A3 based on the combined results of chromatin conformation capture (3C) and ChIP assays. KDM3A is recruited to the SLC2A3 locus in an HIF1-dependent manner and demethylates H3K9me2 so as to upregulate its expression. These findings provide novel insights into the interaction between HIF1 and KDM3A and also the epigenetic regulation of HIF1.

Global Mapping of Cell Type–Specific Open Chromatin by FAIRE-seq Reveals the Regulatory Role of the NFI Family in Adipocyte Differentiation

Identification of regulatory elements within the genome is crucial for understanding the mechanisms that govern cell type–specific gene expression. We generated genome-wide maps of open chromatin sites in 3T3-L1 adipocytes (on day 0 and day 8 of differentiation) and NIH-3T3 fibroblasts using formaldehyde-assisted isolation of regulatory elements coupled with high-throughput sequencing (FAIRE-seq). FAIRE peaks at the promoter were associated with active transcription and histone modifications of H3K4me3 and H3K27ac. Non-promoter FAIRE peaks were characterized by H3K4me1+/me3-, the signature of enhancers, and were largely located in distal regions. The non-promoter FAIRE peaks showed dynamic change during differentiation, while the promoter FAIRE peaks were relatively constant. Functionally, the adipocyte- and preadipocyte-specific non-promoter FAIRE peaks were, respectively, associated with genes up-regulated and down-regulated by differentiation. Genes highly up-regulated during differentiation were associated with multiple clustered adipocyte-specific FAIRE peaks. Among the adipocyte-specific FAIRE peaks, 45.3% and 11.7% overlapped binding sites for, respectively, PPARγ and C/EBPα, the master regulators of adipocyte differentiation. Computational motif analyses of the adipocyte-specific FAIRE peaks revealed enrichment of a binding motif for nuclear family I (NFI) transcription factors. Indeed, ChIP assay showed that NFI occupy the adipocyte-specific FAIRE peaks and/or the PPARγ binding sites near PPARγ, C/EBPα, and aP2 genes. Overexpression of NFIA in 3T3-L1 cells resulted in robust induction of these genes and lipid droplet formation without differentiation stimulus. Overexpression of dominant-negative NFIA or siRNA–mediated knockdown of NFIA or NFIB significantly suppressed both induction of genes and lipid accumulation during differentiation, suggesting a physiological function of these factors in the adipogenic program. Together, our study demonstrates the utility of FAIRE-seq in providing a global view of cell type–specific regulatory elements in the genome and in identifying transcriptional regulators of adipocyte differentiation.

Active RNA Polymerases: Mobile or Immobile Molecular Machines?

Although it is widely assumed that active RNA polymerase tracks along its template, we find that DNA, not the polymerase, moves, suggesting that polymerase works by reeling in the template.

Inhibition of Histone Demethylase JMJD1A Improves Anti-Angiogenic Therapy and Reduces Tumor-Associated Macrophages

Antiangiogenic strategies can be effective for cancer therapy, but like all therapies resistance poses a major clinical challenge. Hypoxia and nutrient starvation select for aggressive qualities that may render tumors resistant to antiangiogenic attack. Here, we show that hypoxia and nutrient starvation cooperate to drive tumor aggressiveness through epigenetic regulation of the histone demethylase JMJD1A (JHDM2A; KDM3A). In cancer cells rendered resistant to long-term hypoxia and nutrient starvation, we documented a stimulation of AKT phosphorylation, cell morphologic changes, cell migration, invasion, and anchorage-independent growth in culture. These qualities associated in vivo with increased angiogenesis and infiltration of macrophages into tumor tissues. Through expression microarray analysis, we identified a cluster of functional drivers such as VEGFA, FGF18, and JMJD1A, the latter which was upregulated in vitro under conditions of hypoxia and nutrient starvation and in vivo before activation of the angiogenic switch or the prerefractory phase of antiangiogenic therapy. JMJD1A inhibition suppressed tumor growth by downregulating angiogenesis and macrophage infiltration, by suppressing expression of FGF2, HGF, and ANG2. Notably, JMJD1A inhibition enhanced the antitumor effects of the anti-VEGF compound bevacizumab and the VEGFR/KDR inhibitor sunitinib. Our results form the foundation of a strategy to attack hypoxia- and nutrient starvation-resistant cancer cells as an approach to leverage antiangiogenic treatments and limit resistance to them.

Severe dermatitis with loss of epidermal Langerhans cells in human and mouse zinc deficiency

Zinc deficiency can be an inherited disorder, in which case it is known as acrodermatitis enteropathica (AE), or an acquired disorder caused by low dietary intake of zinc. Even though zinc deficiency diminishes cellular and humoral immunity, patients develop immunostimulating skin inflammation. Here, we have demonstrated that despite diminished allergic contact dermatitis in mice fed a zinc-deficient (ZD) diet, irritant contact dermatitis (ICD) in these mice was more severe and prolonged than that in controls. Further, histological examination of ICD lesions in ZD mice revealed subcorneal vacuolization and epidermal pallor, histological features of AE. Consistent with the fact that ATP release from chemically injured keratinocytes serves as a causative mediator of ICD, we found that the severe ICD response in ZD mice was attenuated by local injection of soluble nucleoside triphosphate diphosphohydrolase. In addition, skin tissue from ZD mice with ICD showed increased levels of ATP, as did cultured wild-type keratinocytes treated with chemical irritants and the zinc-chelating reagent TPEN. Interestingly, numbers of epidermal Langerhans cells (LCs), which play a protective role against ATP-mediated inflammatory signals, were decreased in ZD mice as well as samples from ZD patients. These findings suggest that upon exposure to irritants, aberrant ATP release from keratinocytes and impaired LC-dependent hydrolysis of nucleotides may be important in the pathogenesis of AE.

Epigenetically coordinated GATA2 binding is necessary for endothelium-specific endomucin expression.

GATA2 is well recognized as a key transcription factor and regulator of cell‐type specificity and differentiation. Here, we carried out comparative chromatin immunoprecipitation with comprehensive sequencing (ChIP‐seq) to determine genome‐wide occupancy of GATA2 in endothelial cells and erythroids, and compared the occupancy to the respective gene expression profile in each cell type. Although GATA2 was commonly expressed in both cell types, different GATA2 bindings and distinct cell‐specific gene expressions were observed. By using the ChIP‐seq with epigenetic histone modifications and chromatin conformation capture assays; we elucidated the mechanistic regulation of endothelial‐specific GATA2‐mediated endomucin gene expression, that was regulated by the endothelial‐specific chromatin loop with a GATA2‐associated distal enhancer and core promoter. Knockdown of endomucin markedly attenuated endothelial cell growth, migration and tube formation. Moreover, abrogation of GATA2 in endothelium demonstrated not only a reduction of endothelial‐specific markers, but also induction of mesenchymal transition promoting gene expression. Our findings provide new insights into the correlation of endothelial‐expressed GATA2 binding, epigenetic modification, and the determination of endothelial cell specificity.

GATA factor switching from GATA2 to GATA1 contributes to erythroid differentiation

Transcription factor GATA2 is highly expressed in hematopoietic stem cells and progenitors, whereas its expression declines after erythroid commitment of progenitors. In contrast, the start of GATA1 expression coincides with the erythroid commitment and increases along with the erythroid differentiation. We refer this dynamic transition of GATA factor expression to as the ‘GATA factor switching’. Here, we examined contribution of the GATA factor switching to the erythroid differentiation. In Gata1‐knockdown embryos that concomitantly express Gata2‐GFP reporter, high‐level expression of GFP reporter was detected in accumulated immature hematopoietic cells with impaired differentiation, demonstrating that GATA1 represses Gata2 gene expression in hematopoietic progenitors in vivo. We have conducted chromatin immunoprecipitation (ChIP) on microarray analyses of GATA2 and GATA1, and results indicate that the GATA1‐binding sites widely overlap with the sites pre‐occupied by GATA2 before the GATA1 expression. Importantly, erythroid genes harboring GATA boxes bound by both GATA1 and GATA2 tend to be expressed in immature erythroid cells, whereas those harboring GATA boxes to which GATA1 binds highly but GATA2 binds only weakly are important for the mature erythroid cell function. Our results thus support the contention that preceding binding of GATA2 helps the following binding of GATA1 and thereby secures smooth expression of the transient‐phase genes.

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.