Kohsuke Kato

Assembly and Disassembly of Nucleosome Core Particles Containing Histone Variants by Human Nucleosome Assembly Protein I

ABSTRACT Histone variants play important roles in the maintenance and regulation of the chromatin structure. In order to characterize the biochemical properties of the chromatin structure containing histone variants, we investigated the dynamic status of nucleosome core particles (NCPs) that were assembled with recombinant histones. We found that in the presence of nucleosome assembly protein I (NAP-I), a histone chaperone, H2A-Barr body deficient (H2A.Bbd) confers the most flexible nucleosome structure among the mammalian histone H2A variants known thus far. NAP-I mediated the efficient assembly and disassembly of the H2A.Bbd-H2B dimers from NCPs. This reaction was accomplished more efficiently when the NCPs contained H3.3, a histone H3 variant known to be localized in the active chromatin, than when the NCPs contained the canonical H3. These observations indicate that the histone variants H2A.Bbd and H3.3 are involved in the formation and maintenance of the active chromatin structure. We also observed that acidic histone binding proteins, TAF-I/SET and B23.1, demonstrated dimer assembly and disassembly activity, but the efficiency of their activity was considerably lower than that of NAP-I. Thus, both the acidic nature of NAP-I and its other functional structure(s) may be essential to mediate the assembly and disassembly of the dimers in NCPs.

Involvement of Nucleocytoplasmic Shuttling of Yeast Nap1 in Mitotic Progression

ABSTRACT Nucleosome assembly protein 1 (Nap1) is widely conserved from yeasts to humans and facilitates nucleosome formation in vitro as a histone chaperone. Nap1 is generally localized in the cytoplasm, except that subcellular localization of Drosophila melanogaster Nap1 is dynamically regulated between the cytoplasm and nucleus during early development. The cytoplasmic localization of Nap1 is seemingly incompatible with the proposed role of Nap1 in nucleosome formation, which should occur in the nucleus. Here, we have examined the roles of a putative nuclear export signal (NES) sequence in yeast Nap1 (yNap1). yNap1 mutants lacking the NES-like sequence were localized predominantly in the nucleus. Deletion of NAP1 in cells harboring a single mitotic cyclin gene is known to cause mitotic delay and temperature-sensitive growth. A wild-type NAP1 complemented these phenotypes while nap1 mutant genes lacking the NES-like sequence or carboxy-terminal region did not. These and other results suggest that yNap1 is a nucleocytoplasmic shuttling protein and that its shuttling is important for yNap1 function during mitotic progression. This study also provides a possible explanation for Nap1s involvement in nucleosome assembly and/or remodeling in the nucleus.

Regulation of histone acetylation and nucleosome assembly by transcription factor JDP2.

Jun dimerization protein-2 (JDP2) is a component of the AP-1 transcription factor that represses transactivation mediated by the Jun family of proteins. Here, we examine the functional mechanisms of JDP2 and show that it can inhibit p300-mediated acetylation of core histones in vitro and in vivo. Inhibition of histone acetylation requires the N-terminal 35 residues and the DNA-binding region of JDP2. In addition, we demonstrate that JDP2 has histone-chaperone activity in vitro. These results suggest that the sequence-specific DNA-binding protein JDP2 may control transcription via direct regulation of the modification of histones and the assembly of chromatin.

Functional characterization of human nucleosome assembly protein 1-like proteins as histone chaperones.

Nucleosome Assembly Protein 1 (NAP1) is a highly conserved histone chaperone protein suspected to be involved in the dynamical regulation of the histone H2A‐H2B hetero‐dimer. However, the exact mechanism by which NAP1‐like proteins act is currently unknown. In this work, we characterized the biochemical properties of two human NAP1‐like proteins, hNAP1L1 and hNAP1L4, including a previously uncharacterized subtype, with the aim of determining their exact mechanistic role. Both hNAP1L1 and hNAP1L4 were found to be localized mainly to the cytoplasm and a minor population of them was suggested to be in the nucleus. Biochemical analyses demonstrated that both hNAP1L1 and hNAP1L4 mediated nucleosome formation. In addition, hNAP1L1 was shown to possess a significantly greater nucleosome disassembly activity than hNAP1L4, suggesting that hNAP1L1 and hNAP1L4 may play distinct roles in the regulation of histone dynamics. Building upon this initial discovery we also found that histone H2A‐H2B and various histone H2A variants‐H2B dimers were found to associate with both hNAP1L1 and hNAP1L4 in cell extracts. These results suggest that human NAP1‐like proteins play overlapping roles in transport and deposition of histone H2A‐H2B or H2A variants‐H2B dimers on chromatin and nonoverlapping roles in nucleosome disassembly.

Role of Template Activating Factor-I as a chaperone in linker histone dynamics.

Linker histone H1 is a fundamental chromosomal protein involved in the maintenance of higher-ordered chromatin organization. The exchange dynamics of histone H1 correlates well with chromatin plasticity. A variety of core histone chaperones involved in core histone dynamics has been identified, but the identity of the linker histone chaperone in the somatic cell nucleus has been a long-standing unanswered question. Here we show that Template Activating Factor-I (TAF-I, also known as protein SET) is involved in histone H1 dynamics as a linker histone chaperone. Among previously identified core histone chaperones and linker histone chaperone candidates, only TAF-I was found to be associated specifically with histone H1 in mammalian somatic cell nuclei. TAF-I showed linker histone chaperone activity in vitro. Fluorescence recovery after photobleaching analyses revealed that TAF-I is involved in the regulation of histone H1 dynamics in the nucleus. Therefore, we propose that TAF-I is a key molecule that regulates linker histone-mediated chromatin assembly and disassembly.

Histone acetylation-independent transcription stimulation by a histone chaperone

Histone chaperones are thought to be important for maintaining the physiological activity of histones; however, their exact roles are not fully understood. The physiological function of template activating factor (TAF)-I, one of the histone chaperones, also remains unclear; however, its biochemical properties have been well studied. By performing microarray analyses, we found that TAF-I stimulates the transcription of a sub-set of genes. The transcription of endogenous genes that was up-regulated by TAF-I was found to be additively stimulated by histone acetylation. On performing an experiment with a cell line containing a model gene integrated into the chromosome, TAF-I was found to stimulate the model gene transcription in a histone chaperone activity-dependent manner additively with histone acetylation. TAF-I bound to the core histones and remodeled the chromatin structure independent of the N-terminal histone tail and its acetylation level in vitro. These results suggest that TAF-I remodel the chromatin structure through its interaction with the core domain of the histones, including the histone fold, and this mechanism is independent of the histone acetylation status.

Synergistic action of MLL, a TRX protein with template activating factor-I, a histone chaperone.

MLL is involved in the process of gene activity maintenance. It is shown that the amino‐terminal region of MLL (MLLN) interacts with TAF‐Iβ/SET. In this study, using yeast two‐hybrid assays, we have found that the acidic region of TAF‐Iβ is essential for its binding to MLLN. Pull‐down assays using GST‐MLLN demonstrated that TAF‐Iβ and histones interact with GST‐MLLN. MLLN and TAF‐Iβ synergistically upregulated the transcription level of Hoxa9 and co‐immunoprecipitated in chromatin containing the Hoxa9 promoter region. These results suggest that TAF‐Iβ plays an important role in MLL‐mediated transcription and possibly chromatin maintenance.

Glucocorticoids facilitate the transcription from the human cytomegalovirus major immediate early promoter in glucocorticoid receptor- and nuclear factor-I-like protein-dependent manner

Human cytomegalovirus (HCMV) is a common and usually asymptomatic virus agent in healthy individuals. Initiation of HCMV productive infection depends on expression of the major immediate early (MIE) genes. The transcription of HCMV MIE genes is regulated by a diverse set of transcription factors. It was previously reported that productive HCMV infection is triggered probably by elevation of the plasma hydroxycorticoid level. However, it is poorly understood whether the transcription of MIE genes is directly regulated by glucocorticoid. Here, we found that the dexamethasone (DEX), a synthetic glucocorticoid, facilitates the transcription of HCMV MIE genes through the MIE promoter and enhancer in a glucocorticoid receptor (GR)-dependent manner. By competitive EMSA and reporter assays, we revealed that an NF-I like protein is involved in DEX-mediated transcriptional activation of the MIE promoter. Thus, this study supports a notion that the increased level of hydroxycorticoid in the third trimester of pregnancy reactivates HCMV virus production from the latent state.

Mitotic phosphorylation of CCCTC‐binding factor (CTCF) reduces its DNA binding activity

During mitosis, higher order chromatin structures are disrupted and chromosomes are condensed to achieve accurate chromosome segregation. CCCTC‐binding factor (CTCF) is a highly conserved and ubiquitously expressed C2H2‐type zinc finger protein which is considered to be involved in epigenetic memory through regulation of higher order chromatin architecture. However, the regulatory mechanism of CTCF in mitosis is still unclear. Here we found that the DNA‐binding activity of CTCF is regulated in a phosphorylation‐dependent manner during mitosis. The linker domains of the CTCF zinc finger domain were found to be phosphorylated during mitosis. The phosphorylation of linker domains impaired the DNA‐binding activity in vitro. Mutation analyses showed that amino acid residues (Thr289, Thr317, Thr346, Thr374, Ser402, Ser461, and Thr518) located in the linker domains were phosphorylated during mitosis. Based on these results, we propose that the mitotic phosphorylation of the linker domains of CTCF is important for the dissociation of CTCF from mitotic chromatin.

Reprogramming Antagonizes the Oncogenicity of HOXA13‐Long Noncoding RNA HOTTIP Axis in Gastric Cancer Cells

Reprogramming of cancer cells into induced pluripotent stem cells (iPSCs) is a compelling idea for inhibiting oncogenesis, especially through modulation of homeobox proteins in this reprogramming process. We examined the role of various long noncoding RNAs (lncRNAs)‐homeobox protein HOXA13 axis on the switching of the oncogenic function of bone morphogenetic protein 7 (BMP7), which is significantly lost in the gastric cancer cell derived iPS‐like cells (iPSLCs). BMP7 promoter activation occurred through the corecruitment of HOXA13, mixed‐lineage leukemia 1 lysine N‐methyltransferase, WD repeat‐containing protein 5, and lncRNA HoxA transcript at the distal tip (HOTTIP) to commit the epigenetic changes to the trimethylation of lysine 4 on histone H3 in cancer cells. By contrast, HOXA13 inhibited BMP7 expression in iPSLCs via the corecruitment of HOXA13, enhancer of zeste homolog 2, Jumonji and AT rich interactive domain 2, and lncRNA HoxA transcript antisense RNA (HOTAIR) to various cis‐element of the BMP7 promoter. Knockdown experiments demonstrated that HOTTIP contributed positively, but HOTAIR regulated negatively to HOXA13‐mediated BMP7 expression in cancer cells and iPSLCs, respectively. These findings indicate that the recruitment of HOXA13–HOTTIP and HOXA13–HOTAIR to different sites in the BMP7 promoter is crucial for the oncogenic fate of human gastric cells. Reprogramming with octamer‐binding protein 4 and Jun dimerization protein 2 can inhibit tumorigenesis by switching off BMP7. Stem Cells 2017;35:2115–2128