Ryoji Fujiki

TET2 promotes histone O-GlcNAcylation during gene transcription

Ten eleven translocation (TET) enzymes, including TET1, TET2 and TET3, convert 5-methylcytosine to 5-hydroxymethylcytosine and regulate gene transcription. However, the molecular mechanism by which TET family enzymes regulate gene transcription remains elusive. Using protein affinity purification, here we search for functional partners of TET proteins, and find that TET2 and TET3 associate with O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT), an enzyme that by itself catalyses the addition of O-GlcNAc onto serine and threonine residues (O-GlcNAcylation) in vivo. TET2 directly interacts with OGT, which is important for the chromatin association of OGT in vivo. Although this specific interaction does not regulate the enzymatic activity of TET2, it facilitates OGT-dependent histone O-GlcNAcylation. Moreover, OGT associates with TET2 at transcription start sites. Downregulation of TET2 reduces the amount of histone 2B Ser 112 GlcNAc marks in vivo, which are associated with gene transcription regulation. Taken together, these results reveal a TET2-dependent O-GlcNAcylation of chromatin. The double epigenetic modifications on both DNA and histones by TET2 and OGT coordinate together for the regulation of gene transcription.

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

GlcNAcylation of histone H2B facilitates its monoubiquitination

Chromatin reorganization is governed by multiple post-translational modifications of chromosomal proteins and DNA. These histone modifications are reversible, dynamic events that can regulate DNA-driven cellular processes. However, the molecular mechanisms that coordinate histone modification patterns remain largely unknown. In metazoans, reversible protein modification by O-linked N-acetylglucosamine (GlcNAc) is catalysed by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). However, the significance of GlcNAcylation in chromatin reorganization remains elusive. Here we report that histone H2B is GlcNAcylated at residue S112 by OGT in vitro and in living cells. Histone GlcNAcylation fluctuated in response to extracellular glucose through the hexosamine biosynthesis pathway (HBP). H2B S112 GlcNAcylation promotes K120 monoubiquitination, in which the GlcNAc moiety can serve as an anchor for a histone H2B ubiquitin ligase. H2B S112 GlcNAc was localized to euchromatic areas on fly polytene chromosomes. In a genome-wide analysis, H2B S112 GlcNAcylation sites were observed widely distributed over chromosomes including transcribed gene loci, with some sites co-localizing with H2B K120 monoubiquitination. These findings suggest that H2B S112 GlcNAcylation is a histone modification that facilitates H2BK120 monoubiquitination, presumably for transcriptional activation.

GlcNAcylation of a histone methyltransferase in retinoic-acid-induced granulopoiesis

The post-translational modifications of histone tails generate a ‘histone code’ that defines local and global chromatin states. The resultant regulation of gene function is thought to govern cell fate, proliferation and differentiation. Reversible histone modifications such as methylation are under mutual controls to organize chromosomal events. Among the histone modifications, methylation of specific lysine and arginine residues seems to be critical for chromatin configuration and control of gene expression. Methylation of histone H3 lysine 4 (H3K4) changes chromatin into a transcriptionally active state. Reversible modification of proteins by β-N-acetylglucosamine (O-GlcNAc) in response to serum glucose levels regulates diverse cellular processes. However, the epigenetic impact of protein GlcNAcylation is unknown. Here we report that nuclear GlcNAcylation of a histone lysine methyltransferase (HKMT), MLL5, by O-GlcNAc transferase facilitates retinoic-acid-induced granulopoiesis in human HL60 promyelocytes through methylation of H3K4. MLL5 is biochemically identified in a GlcNAcylation-dependent multi-subunit complex associating with nuclear retinoic acid receptor RARα (also known as RARA), serving as a mono- and di-methyl transferase to H3K4. GlcNAcylation at Thr 440 in the MLL5 SET domain evokes its H3K4 HKMT activity and co-activates RARα in target gene promoters. Increased nuclear GlcNAcylation by means of O-GlcNAc transferase potentiates retinoic-acid-induced HL60 granulopoiesis and restores the retinoic acid response in the retinoic-acid-resistant HL60-R2 cell line. Thus, nuclear MLL5 GlcNAcylation triggers cell lineage determination of HL60 through activation of its HKMT activity.

Ligand-induced transrepression by VDR through association of WSTF with acetylated histones

We have previously shown that the novel ATP‐dependent chromatin‐remodeling complex WINAC is required for the ligand‐bound vitamin D receptor (VDR)‐mediated transrepression of the 25(OH)D3 1α‐hydroxylase (1α(OH)ase) gene. However, the molecular basis for VDR promoter association, which does not involve its binding to specific DNA sequences, remains unclear. To address this issue, we investigated the function of WSTF in terms of the association between WINAC and chromatin for ligand‐induced transrepression by VDR. Results of in vitro experiments using chromatin templates showed that the association of unliganded VDR with the promoter required physical interactions between WSTF and both VDR and acetylated histones prior to VDR association with chromatin. The acetylated histone‐interacting region of WSTF was mapped to the bromodomain, and a WSTF mutant lacking the bromodomain served as a dominant‐negative mutant in terms of ligand‐induced transrepression of the 1α(OH)ase gene. Thus, our findings indicate that WINAC associates with chromatin through a physical interaction between the WSTF bromodomain and acetylated his tones, which appears to be indispensable for VDR/promoter association for ligand‐induced transrepression of 1α(OH)ase gene expression.

A histone chaperone, DEK, transcriptionally coactivates a nuclear receptor

Chromatin reorganization is essential for transcriptional control by sequence-specific transcription factors. However, the molecular link between transcriptional control and chromatin reconfiguration remains unclear. By colocalization of the nuclear ecdysone receptor (EcR) on the ecdysone-induced puff in the salivary gland, Drosophila DEK (dDEK) was genetically identified as a coactivator of EcR in both insect cells and intact flies. Biochemical purification and characterization of the complexes containing fly and human DEKs revealed that DEKs serve as histone chaperones via phosphorylation by forming complexes with casein kinase 2. Consistent with the preferential association of the DEK complex with histones enriched in active epigenetic marks, dDEK facilitated H3.3 assembly during puff formation. In some human myeloid leukemia patients, DEK was fused to CAN by chromosomal translocation. This mutation significantly reduced formation of the DEK complex, which is required for histone chaperone activity. Thus, the present study suggests that at least one histone chaperone can be categorized as a type of transcriptional coactivator for nuclear receptors.

RETRACTED: 1α,25(OH)2D3-induced DNA methylation suppresses the human CYP27B1 gene

CYP27B 1 is a critical enzyme of Vitamin D biosynthesis that hydroxylates 25(OH)D 3 at the final step of the biosynthetic pathway. The CYP27B 1 gene is expressed primarily in kidney and negatively controlled by Vitamin D receptor. We have characterized the negative vitamin D response element and its binding protein, a bHLH transcription factor. This factor directly binds to the lanVDRE and activates transcription, but its transcriptional activity is suppressed by the ligand-activated Vitamin D receptor through recruitment of histone deacetylase. We have shown that histone deacetylation is a critical step for chromatin structure remodeling in suppression of the CYP27B 1 gene. We have further demonstrated that, in addition to histone acetylation, this transrepression by VDR requires DNA methylation in the CYP27B 1 gene promoter. Thus, transcriptional regulation of the CYP27B 1 gene appears to be mediated by dual epigenetic modifications.

Nuclear receptor coregulators merge transcriptional coregulation with epigenetic regulation.

Members of the nuclear steroid/thyroid hormone receptor (NR) gene superfamily are DNA-binding transcription factors that regulate target genes in a spatiotemporal manner, depending on the promoter context. In vivo observations of ligand responses in NR-mediated gene regulation led to the identification of ligand-dependent coregulators that directly interact with NRs. Functional dissection of NR coregulators revealed that their transcriptional coregulation was linked to histone acetylation. However, recent work in the fields of reversible histone modification and chromatin remodeling indicates that histone-modifying enzymes, including histone methylases and chromatin remodelers, are potential transcriptional coregulators that interact directly and indirectly with NRs.

Distinct function of 2 chromatin remodeling complexes that share a common subunit, Williams syndrome transcription factor (WSTF)

A number of nuclear complexes modify chromatin structure and operate as functional units. However, the in vivo role of each component within the complexes is not known. ATP-dependent chromatin remodeling complexes form several types of protein complexes, which reorganize chromatin structure cooperatively with histone modifiers. Williams syndrome transcription factor (WSTF) was biochemically identified as a major subunit, along with 2 distinct complexes: WINAC, a SWI/SNF-type complex, and WICH, an ISWI-type complex. Here, WSTF−/− mice were generated to investigate its function in chromatin remodeling in vivo. Loss of WSTF expression resulted in neonatal lethality, and all WSTF−/− neonates and ≈10% of WSTF+/− neonates suffered cardiovascular abnormalities resembling those found in autosomal-dominant Williams syndrome patients. Developmental analysis of WSTF−/− embryos revealed that Gja5 gene regulation is aberrant from E9.5, conceivably because of inappropriate chromatin reorganization around the promoter regions where essential cardiac transcription factors are recruited. In vitro analysis in WSTF−/− mouse embryonic fibroblast (MEF) cells also showed impaired transactivation functions of cardiac transcription activators on the Gja5 promoter, but the effects were reversed by overexpression of WINAC components. Likewise in WSTF−/− MEF cells, recruitment of Snf2h, an ISWI ATPase, to PCNA and cell survival after DNA damage were both defective, but were ameliorated by overexpression of WICH components. Thus, the present study provides evidence that WSTF is shared and is a functionally indispensable subunit of the WICH complex for DNA repair and the WINAC complex for transcriptional control.

Retraction: ‘A cell cycle‐dependent co‐repressor mediates photoreceptor cell‐specific nuclear receptor function’

Photoreceptor cell‐specific nuclear receptor (PNR) (NR2E3) acts as a sequence‐specific repressor that controls neuronal differentiation in the developing retina. We identified a novel PNR co‐repressor, Ret‐CoR, that is expressed in the developing retina and brain. Biochemical purification of Ret‐CoR identified a multiprotein complex that included E2F/Myb‐associated proteins, histone deacetylases (HDACs) and NCoR/HDAC complex‐related components. Ret‐CoR appeared to function as a platform protein for the complex, and interacted with PNR via two CoRNR motifs. Purified Ret‐CoR complex exhibited HDAC activity, co‐repressed PNR transrepression function in vitro, and co‐repressed PNR function in PNR target gene promoters, presumably in the retinal progenitor cells. Notably, the appearance of Ret‐CoR protein was cell‐cycle‐stage‐dependent (from G1 to S). Therefore, Ret‐CoR appears to act as a component of an HDAC co‐repressor complex that supports PNR repression function in the developing retina, and may represent a co‐regulator class that supports transcriptional regulator function via cell‐cycle‐dependent expression.