Takeya Nakagawa

The chromatin-remodeling complex WINAC targets a nuclear receptor to promoters and is impaired in Williams syndrome

S phase progression. WINAC mediates the recruitment Hirochika Kitagawa,1,2 Ryoji Fujiki,1 Kimihiro Yoshimura,1 Yoshihiro Mezaki,1 Yoshikatsu Uematsu,1 Daisuke Matsui,1 Satoko Ogawa,1 Kiyoe Unno,1,3 Mataichi Okubo,3 Akifumi Tokita,3 Takeya Nakagawa,4 Takashi Ito,4 Yukio Ishimi,5 of unliganded VDR to VDR target sites in promoters, Hiromichi Nagasawa,6 Toshio Matsumoto,2 while subsequent binding of coregulators requires liJunn Yanagisawa,1,7 and Shigeaki Kato1,7,* gand binding. This recruitment order exemplifies that Institute of Molecular and Cellular Biosciences an interaction of a sequence-specific regulator with a University of Tokyo chromatin-remodeling complex can organize nucleo1-1-1 Yayoi somal arrays at specific local sites in order to make Bunkyo-ku promoters accessible for coregulators. Furthermore, Tokyo 113-0032 overexpression of WSTF could restore the impaired Japan recruitment of VDR to vitamin D regulated promoters 2 First Department of Internal Medicine in fibroblasts from Williams syndrome patients. This University of Tokushima School of Medicine suggests that WINAC dysfunction contributes to 3-18-15 Kuramoto-cho Williams syndrome, which could therefore be considTokushima 770-8503 ered, at least in part, a chromatin-remodeling factor Japan disease. 3 Department of Pediatrics

Deubiquitylation of histone H2A activates transcriptional initiation via trans-histone cross-talk with H3K4 di- and trimethylation

Transcriptional initiation is a key step in the control of mRNA synthesis and is intimately related to chromatin structure and histone modification. Here, we show that the ubiquitylation of H2A (ubH2A) correlates with silent chromatin and regulates transcriptional initiation. The levels of ubH2A vary during hepatocyte regeneration, and based on microarray expression data from regenerating liver, we identified USP21, a ubiquitin-specific protease that catalyzes the hydrolysis of ubH2A. When chromatin is assembled in vitro, ubH2A, but not H2A, specifically represses the di- and trimethylation of H3K4. USP21 relieves this ubH2A-specific repression. In addition, in vitro transcription analysis revealed that ubH2A represses transcriptional initiation, but not transcriptional elongation, by inhibiting H3K4 methylation. Notably, ubH2A-mediated repression was not observed when H3 Lys 4 was changed to arginine. Furthermore, overexpression of USP21 in the liver up-regulates a gene that is normally down-regulated during hepatocyte regeneration. Our studies revealed a novel mode of trans-histone cross-talk, in which H2A ubiquitylation controls the di- and trimethylation of H3K4, resulting in regulation of transcriptional initiation.

Dual Roles of p300 in Chromatin Assembly and Transcriptional Activation in Cooperation with Nucleosome Assembly Protein 1 In Vitro

ABSTRACT In a yeast two-hybrid screen to identify proteins that bind to the KIX domain of the coactivator p300, we obtained cDNAs encoding nucleosome assembly protein 1 (NAP-1), a 60-kDa histone H2A-H2B shuttling protein that promotes histone deposition. p300 associates preferentially with the H2A-H2B-bound form of NAP-1 rather than with the unbound form of NAP-1. Formation of NAP-1-p300 complexes was found to increase during S phase, suggesting a potential role for p300 in chromatin assembly. In micrococcal nuclease and supercoiling assays, addition of p300 promoted efficient chromatin assembly in vitro in conjunction with NAP-1 and ATP-utilizing chromatin assembly and remodeling factor; this effect was dependent in part on the intrinsic histone acetyltransferase activity of p300. Surprisingly, NAP-1 potently inhibited acetylation of core histones by p300, suggesting that efficient assembly requires acetylation of either NAP-1 or p300 itself. As p300 acted cooperatively with NAP-1 in stimulating transcription from a chromatin template in vitro, our results suggest a dual role of NAP-1-p300 complexes in promoting chromatin assembly and transcriptional activation.

The USP21 Short Variant (USP21SV) Lacking NES, Located Mostly in the Nucleus In Vivo, Activates Transcription by Deubiquitylating ubH2A In Vitro

USP21 is a deubiquitylase that catalyzes isopeptide bond hydrolysis between ubiquitin and histone H2A. Since ubiqutylated H2A (ubH2A) represses transcription, USP21 plays a role in transcriptional activation. On the other hand, the localization of USP21 suggests it has an additional function in the cytoplasm. Here, we identified a USP21 short variant (USP21SV) lacking a nuclear export signal (NES). Differential localization of USP21SV, more in the nucleus than the USP21 long variant (USP21LV), suggests they have redundant roles in the cell. Ectopic expression of both USP21 variants decreased ubH2A in the nucleus. Furthermore, both recombinant USP21 variants activate transcription by deubiquitylating ubH2A in vitro. These data suggest multiple roles for USP21 in the ubiquitylation-deubiquitylation network in the cell.

SMARCAD1 is an ATP-dependent stimulator of nucleosomal H2A acetylation via CBP, resulting in transcriptional regulation

Histone acetylation plays a pivotal role in transcriptional regulation, and ATP-dependent nucleosome remodeling activity is required for optimal transcription from chromatin. While these two activities have been well characterized, how they are coordinated remains to be determined. We discovered ATP-dependent histone H2A acetylation activity in Drosophila nuclear extracts. This activity was column purified and demonstrated to be composed of the enzymatic activities of CREB-binding protein (CBP) and SMARCAD1, which belongs to the Etl1 subfamily of the Snf2 family of helicase-related proteins. SMARCAD1 enhanced acetylation by CBP of H2A K5 and K8 in nucleosomes in an ATP-dependent fashion. Expression array analysis of S2 cells having ectopically expressed SMARCAD1 revealed up-regulated genes. Using native genome templates of these up-regulated genes, we found that SMARCAD1 activates their transcription in vitro. Knockdown analysis of SMARCAD1 and CBP indicated overlapping gene control, and ChIP-seq analysis of these commonly controlled genes showed that CBP is recruited to the promoter prior to SMARCAD1. Moreover, Drosophila genetic experiments demonstrated interaction between SMARCAD1/Etl1 and CBP/nej during development. The interplay between the remodeling activity of SMARCAD1 and histone acetylation by CBP sheds light on the function of chromatin and the genome-integrity network.

Dzip3 regulates developmental genes in mouse embryonic stem cells by reorganizing 3D chromatin conformation

In mouse embryonic stem (mES) cells, ubiquitylation of histone H2A lysine 119 represses a large number of developmental genes and maintains mES cell pluripotency. It has been suggested that a number of H2A ubiquitin ligases as well as deubiquitylases and related peptide fragments contribute to a delicate balance between self-renewal and multi-lineage differentiation in mES cells. Here, we tested whether known H2A ubiquitin ligases and deubiquitylases are involved in mES cell regulation and discovered that Dzip3, the E3 ligase of H2AK119, represses differentiation-inducible genes, as does Ring1B. The two sets of target genes partially overlapped but had different spectra. We found that Dzip3 represses gene expression by orchestrating changes in 3D organization, in addition to regulating ubiquitylation of H2A. Our results shed light on the epigenetic mechanism of transcriptional regulation, which depends on 3D chromatin reorganization to regulate mES cell differentiation.

Enhancer of Acetyltransferase Chameau (EAChm) Is a Novel Transcriptional Co-Activator

Acetylation of nucleosomal histones by diverse histone acetyltransferases (HAT) plays pivotal roles in many cellular events. Discoveries of novel HATs and HAT related factors have provided new insights to understand the roles and mechanisms of histone acetylation. In this study, we identified prominent Histone H3 acetylation activity in vitro and purified its activity, showing that it is composed of the MYST acetyltransferase Chameau and Enhancer of the Acetyltransferase Chameau (EAChm) family. EAChm is a negatively charged acidic protein retaining aspartate and glutamate. Furthermore, we identified that Chameau and EAChm stimulate transcription in vitro together with purified general transcription factors. In addition, RNA-seq analysis of Chameu KD and EAChm KD S2 cells suggest that Chameau and EAChm regulate transcription of common genes in vivo. Our results suggest that EAChm regulates gene transcription in Drosophila embryos by enhancing Acetyltransferase Chameau activity.

Enhancer function regulated by combinations of transcription factors and cofactors

Regulation of the expression of diverse genes is essential for making possible the complexity of higher organisms, and the temporal and spatial regulation of gene expression allows for the alteration of cell types and growth patterns. A critical component of this regulation is the DNA sequence‐specific binding of transcription factors (TFs). However, most TFs do not independently participate in gene transcriptional regulation, because they lack an effector function. Instead, TFs are thought to work by recruiting cofactors, including Mediator complex (Mediator), chromatin‐remodeling complexes (CRCs), and histone‐modifying complexes (HMCs). Mediator associates with the majority of transcribed genes and acts as an integrator of multiple signals. On the other hand, CRCs and HMCs are selectively recruited by TFs. Although all the pairings between TFs and CRCs or HMCs are not fully known, there are a growing number of established TF–CRC and TF–HMC combinations. In this review, we focused on the most important of these pairings and discuss how they control gene expression.

Defects in centromeric/pericentromeric histone H2A T120 phosphorylation by hBUB1 cause chromosome missegregation producing multinucleated cells

Histone H2A phosphorylation plays a role both in chromatin condensation during mitosis and in transcriptional activation during the G1/S transition. Bub1 and NHK1/VRK1 have been identified as histone H2A kinases. However, little is known about the importance of histone H2A phosphorylation in chromosome segregation. Here, we expressed recombinant hBUB1 and confirmed that it phosphorylates histone H2A T120 in the in vitro‐assembled nucleosome. Knockdown (KD) of BUB1 decreases bulk H2A T120 phosphorylation in HeLa cells, whereas hBUB1 is upregulated during mitosis, which corresponds with H2A T120 phosphorylation. ChIP‐qPCR of the DXZ1 centromeric and γ‐ALR pericentromeric region showed that BUB1 localizes to this region and increases local H2A T120 phosphorylation during M phase. BUB1 KD did not induce apoptosis but increased the M phase cell population, as detected by flow cytometry. BUB1 KD also caused an abnormal metaphase and telophase, resulting in multinucleated cells and impaired cancer cell growth both in vitro and in vivo. Over‐expression of the histone H2A T120D or T120E mutations, which mimic phosphorylated threonine, decreased the number of multinucleated cells caused by BUB1 KD. These results strengthen the apparent importance of BUB1‐mediated H2A T120 phosphorylation in normal mitosis.

MicroRNA-3662 expression correlates with antiviral drug resistance in adult T-cell leukemia/lymphoma cells

Interferon regulatory factor (IRF) 4 and the proto-oncogene c-Rel cooperate in growth and antiviral drug resistance of adult T-cell leukemia/lymphoma (ATLL). To elucidate the target of IRF4 and c-Rel in ATLL, we determined the simultaneous binding sites of IRF4 and c-Rel using ChIP-seq technology. Nine genes were identified within 2 kb of binding sites, including MIR3662. Expression of miR-3662 was regulated by IRF4, and to a lesser extent by c-Rel. Cell proliferation was inhibited by knockdown of miR-3662 and expression of miR-3662 was correlated with antiviral drug resistance in ATLL cell lines. Thus, miR-3662 represents a target for therapies against ATLL.