Mitsuru Okuwaki

Epigenetic control of rDNA loci in response to intracellular energy status

Intracellular energy balance is important for cell survival. In eukaryotic cells, the most energy-consuming process is ribosome biosynthesis, which adapts to changes in intracellular energy status. However, the mechanism that links energy status and ribosome biosynthesis is largely unknown. Here, we describe eNoSC, a protein complex that senses energy status and controls rRNA transcription. eNoSC contains Nucleomethylin, which binds histone H3 dimethylated Lys9 in the rDNA locus, in a complex with SIRT1 and SUV39H1. Both SIRT1 and SUV39H1 are required for energy-dependent transcriptional repression, suggesting that a change in the NAD(+)/NADH ratio induced by reduction of energy status could activate SIRT1, leading to deacetylation of histone H3 and dimethylation at Lys9 by SUV39H1, thus establishing silent chromatin in the rDNA locus. Furthermore, eNoSC promotes restoration of energy balance by limiting rRNA transcription, thus protecting cells from energy deprivation-dependent apoptosis. These findings provide key insight into the mechanisms of energy homeostasis in cells.

Function of nucleophosmin/B23, a nucleolar acidic protein, as a histone chaperone

We previously identified and purified a nucleolar phosphoprotein, nucleophosmin/B23, as a stimulatory factor for replication from the adenovirus chromatin. We show here that nucleophosmin/B23 functions as a histone chaperone protein such as nucleoplasmin, TAF‐I, and NAP‐I. Nucleophosmin/B23 was shown to bind to histones, preferentially to histone H3, to mediate formation of nucleosome, and to decondense sperm chromatin. These activities of B23 were dependent on its acidic regions as other histone chaperones, suggesting that B23/nucleophosmin is a member of histone chaperone proteins.

Structural basis of HP1/PXVXL motif peptide interactions and HP1 localisation to heterochromatin

HP1 family proteins are adaptor molecules, containing two related chromo domains that are required for chromatin packaging and gene silencing. Here we present the structure of the chromo shadow domain from mouse HP1β bound to a peptide containing a consensus PXVXL motif found in many HP1 binding partners. The shadow domain exhibits a novel mode of peptide recognition, where the peptide binds across the dimer interface, sandwiched in a β‐sheet between strands from each monomer. The structure allows us to predict which other shadow domains bind similar PXVXL motif‐containing peptides and provides a framework for predicting the sequence specificity of the others. We show that targeting of HP1β to heterochromatin requires shadow domain interactions with PXVXL‐containing proteins in addition to chromo domain recognition of Lys‐9‐methylated histone H3. Interestingly, it also appears to require the simultaneous recognition of two Lys‐9‐methylated histone H3 molecules. This finding implies a further complexity to the histone code for regulation of chromatin structure and suggests how binding of HP1 family proteins may lead to its condensation.

Transcription Regulation of the rRNA Gene by a Multifunctional Nucleolar Protein, B23/Nucleophosmin, through Its Histone Chaperone Activity

ABSTRACT It is well established that the transcription rate of the rRNA gene is closely associated with profound alterations in the cell growth rate. Regulation of rRNA gene transcription is likely to be dependent on the dynamic conversion of the chromatin structure. Previously, we identified B23/nucleophosmin, a multifunctional nucleolar phosphoprotein, as a component of template activating factor III that remodels the chromatin-like structure of the adenovirus genome complexed with viral basic proteins. It has also been shown that B23 has histone chaperone activity. Here, we examined the effect of B23 on rRNA gene transcription. B23 was found to be associated with the rRNA gene chromatin. Small-interfering-RNA-mediated down-regulation of the B23 expression level resulted in reduction of the transcription rate of the rRNA gene. We constructed a B23 mutant termed B23ΔC, which lacks the domain essential for the histone chaperone activity and inhibited the histone binding activity of B23 in a dominant-negative manner. Expression of B23ΔC decreased rRNA gene transcription and the rate of cell proliferation. These results suggest that B23 is involved in the transcription regulation of the rRNA gene as a nucleolar histone chaperone.

NAP‐I is a functional homologue of TAF‐I that is required for replication and transcription of the adenovirus genome in a chromatin‐like structure

Background: For the activation of replication and transcription from DNA in a chromatin structure, a variety of factors are thought to be needed that alter the chromatin structure. Template activating factor‐I (TAF‐I) has been identified as such a host factor required for replication of the adenovirus (Ad) genome complexed with viral basic core proteins (Ad core). TAF‐I also stimulates transcription from the Ad core DNA.

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 Template-Activating Factor I/SET in Transcription of Adenovirus Early Genes as a Positive-Acting Factor

ABSTRACT The adenovirus genome complexed with viral core protein VII (adenovirus DNA-protein VII complex) at least is the bona fide template for transcription of adenovirus early genes. It is believed that the highly basic protein VII, like cellular histones, is a negative regulator for genome functions. Analyses with in vitro replication and transcription systems using the adenovirus DNA-protein VII complex have revealed that remodeling of the complex is crucial for efficient DNA replication and transcription. We identified host acidic proteins, template-activating factor I (TAF-I), TAF-II, and TAF-III as stimulatory factors for replication from the adenovirus DNA-protein VII complex. Recently, it was reported that the adenovirus DNA interacts with TAF-I and pp32, another host acidic protein (Y. Xue, J. S. Johnson, D. A. Ornelles, J. Lieberman, and D. A. Engel, J. Virol. 79:2474-2483, 2005). We found that TAF-I interacts and colocalizes with protein VII in adenovirus-infected cells during the early phases of infection, but pp32 does not. Although pp32 had the potential ability to interact with protein VII, pp32 did not remodel the adenovirus DNA-protein VII complex in vitro. Small interfering RNA-mediated knockdown of TAF-I expression leads to the delay of the transcription timing of early genes. These results provide evidence that TAF-I plays an important role in the early stages of the adenovirus infection cycle.

Ternary complex formation between DNA-adenovirus core protein VII and TAF-Iβ/SET, an acidic molecular chaperone

The adenovirus (Ad) genome complexed with viral core proteins designated Ad core is the template for transcription of early genes and the first round of replication in Ad‐infected cells. A cellular protein designated template‐activating factor‐I (TAF‐I) is found to be involved in remodeling of the Ad core in vitro. Here we found that TAF‐I interacts with the Ad DNA through core protein VII in infected cells in early phases of infection. In vitro binding assays using recombinant proteins showed that TAF‐I forms ternary complexes with DNA–protein VII complexes.

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.

Function of homo- and hetero-oligomers of human nucleoplasmin/nucleophosmin family proteins NPM1, NPM2 and NPM3 during sperm chromatin remodeling

Sperm chromatin remodeling after oocyte entry is the essential step that initiates embryogenesis. This reaction involves the removal of sperm-specific basic proteins and chromatin assembly with histones. In mammals, three nucleoplasmin/nucleophosmin (NPM) family proteins–NPM1, NPM2 and NPM3–expressed in oocytes are presumed to cooperatively regulate sperm chromatin remodeling. We characterized the sperm chromatin decondensation and nucleosome assembly activities of three human NPM proteins. NPM1 and NPM2 mediated nucleosome assembly independently of other NPM proteins, whereas the function of NPM3 was largely dependent on formation of a complex with NPM1. Maximal sperm chromatin remodeling activity of NPM2 required the inhibition of its non-specific nucleic acid-binding activity by phosphorylation. Furthermore, the oligomer formation with NPM1 elicited NPM3 nucleosome assembly and sperm chromatin decondensation activity. NPM3 also suppressed the RNA-binding activity of NPM1, which enhanced the nucleoplasm–nucleolus shuttling of NPM1 in somatic cell nuclei. Our results proposed a novel mechanism whereby three NPM proteins cooperatively regulate chromatin disassembly and assembly in the early embryo and in somatic cells.