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 chromatinlike 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 B23⌬C, 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 B23⌬C 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.

SummaryCell separation in Bacillus subtilis depends on specific activities of DL-endopeptidases CwlS, LytF and LytE. Immunofluorescence microscopy (IFM) indicated that the localization of LytF depended on its N-terminal LysM domain. In addition, we revealed that the LysM domain efficiently binds to peptidoglycan (PG) prepared by chemically removing wall teichoic acids (WTAs) from the B. subtilis cell wall. Moreover, increasing amounts of the LysM domain bound to TagB-or TagO-depleted cell walls. These results strongly suggested that the LysM domain specifically binds to PG, and that the binding may be prevented by WTAs. IFM with TagB-, TagF-or TagO-reduced cells indicated that LytF-6xFLAG was observed not only at cell separation site and poles but also as a helical pattern along the sidewall. Moreover, we found that LytF was localizable on the whole cell surface in TagB-, TagF-or TagO-depleted cells. These results strongly suggest that WTAs inhibit the sidewall localization of LytF. Furthermore, the helical LytF localization was observed on the lateral cell surface in MreB-depleted cells, suggesting that cell wall modification by WTAs along the sidewall might be governed by an actin-like cytoskeleton homologue, MreB.

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.

Nucleophosmin (NPM1/B23) is a nucleolar protein implicated in growth-associated functions, in which the RNA binding activity of B23 plays essential roles in ribosome biogenesis. The C-terminal globular domain (CTD) of B23 has been believed to be the RNA binding domain because the splicing variant B23.2 lacking the CTD binds considerably less efficiently to RNA. However, the recognition of target RNAs by B23 remains poorly understood. Herein, we report a novel mechanism by which B23 recognizes specific RNA targets. We observed that the nucleolar retention of B23.3 lacking the basic region of B23.1 was lower than that of B23.1 because of its low RNA binding activity. Circular dichroism measurements indicated that the basic region and adjacent acidic regions of B23 are intrinsically disordered regions (IDRs). Biochemical analyses revealed that the basic IDR alone strongly binds to RNA with low specificity. The excessive RNA binding activity of the basic IDR was restrained by intra-molecular interaction with the acidic IDR of B23. Chemical cross-linking experiments and fluorescent labeling of bipartite tetracysteine-tagged proteins suggested that the inter- and intra-molecular interactions between the two IDRs contribute to the regulation of the RNA binding activity of CTD to control the cellular localization and functions of B23.

In adenoviral virions, the genome is organized into a chromatin-like structure by viral basic core proteins. Consequently viral DNAs must be replicated, chromatinized and packed into progeny virions in infected cells. Although viral DNA replication centers can be visualized by virtue of viral and cellular factors, the spatiotemporal regulation of viral genomes during subsequent steps remains to be elucidated. In this study, we used imaging analyses to examine the fate of adenoviral genomes and to track newly replicated viral DNA as well as replication-related factors. We show de novo formation of a subnuclear domain, which we termed Virus-induced Post-Replication (ViPR) body, that emerges concomitantly with or immediately after disintegration of initial replication centers. Using a nucleoside analogue, we show that viral genomes continue being synthesized in morphologically distinct replication compartments at the periphery of ViPR bodies and are then transported inward. In addition, we identified a nucleolar protein Mybbp1a as a molecular marker for ViPR bodies, which specifically associated with viral core protein VII. In conclusion, our work demonstrates the formation of previously uncharacterized viral DNA replication compartments specific for late phases of infection that produce progeny viral genomes accumulating in ViPR bodies. 1 Like cellular chromatin, the resulting chromatin structure plays a critical role in genome functions, eg, as template for transcription or replication. 1 Additionally, viral genome/chromatin has to be remodeled from an 'intracellular' to an 'intravirion' form to be packed into viral particles before or during virion assembly and maturation. Despite these essential steps, for many DNA viruses, the timing, the mechanistic details and the subcellular environment where these transitions take place remain largely unknown. During the first few hours of infection, the chromatin structure of the viral genome changes. We have shown that cellular histones are deposited onto incoming viral genomes. 5 We also reported that newly replicated viral DNAs associate with cellular histones. 6 Our analyses on histone deposition are consistent with other independent biochemical studies. 7-10 The deposition of cellular

Histone chaperones regulate the density of incorporated histone proteins around DNA transcription sites and therefore constitute an important site-specific regulatory mechanism for the control of gene expression. At present, the targeting mechanism conferring this site specificity is unknown. We previously reported that the histone chaperone B23/nucleophosmin associates with rRNA chromatin (r-chromatin) to stimulate rRNA transcription. Here, we report on the mechanism for site-specific targeting of B23 to the r-chromatin. We observed that, during mitosis, B23 was released from chromatin upon inactivation of its RNA binding activity by cdc2 kinase-mediated phosphorylation. The phosphorylation status of B23 was also shown to strongly affect its chromatin binding activity. We further found that r-chromatin binding of B23 was a necessary condition for B23 histone chaperone activity in vivo. In addition, we found that depletion of upstream binding factor (UBF; an rRNA transcription factor) decreased the chromatin binding affinity of B23, which in turn led to an increase in histone density at the r-chromatin. These two major strands of evidence suggest a novel cell cycle-dependent mechanism for the site-specific regulation of histone density via joint RNA-and transcription factor-mediated recruitment of histone chaperones to specific chromosome loci.

The adenovirus (Ad) genome is believed to be packaged into the virion by forming a chromatin‐like structure. The replicated viral genome is likely to be condensed through binding with viral core proteins before encapsidation. Replicated viral genomes accumulate in the central region of the nucleus, which we termed virus‐induced postreplication (ViPR) body. However, the molecular mechanism by which the nuclear structure is reorganized and its functional significance in virus production are currently not understood. In this study, we found that viral packaging protein IVa2, but not capsid proteins, accumulated in the ViPR body. In addition, nucleolar chromatin regulatory proteins, nucleophosmin 1 (NPM1), upstream binding factor, and nucleolin accumulated in the ViPR body in late‐stage Ad infection. NPM1 depletion increased the nuclease‐resistant viral genome and delayed the ViPR body formation. These results suggested that structural changes in the infected cell nucleus depend on the formation of viral chromatin by host chromatin regulatory proteins. Because NPM1 depletion decreases production of the infectious virion, we propose that host factor‐mediated viral chromatin remodeling and concomitant ViPR body formation are prerequisites for efficient encapsidation of Ad chromatin.

Linker histones play important roles in the genomic organization of mammalian cells. Of the linker histone variants, H1.X shows the most dynamic behavior in the nucleus. Recent research has suggested that the linker histone variants H1.X and H1.0 have different chromosomal binding site preferences. However, it remains unclear how the dynamics and binding site preferences of linker histones are determined. Here, we biochemically demonstrated that the DNA/nucleosome and histone chaperone binding activities of H1.X are significantly lower than those of other linker histones. This explains why H1.X moves more rapidly than other linker histones in vivo. Domain swapping between H1.0 and H1.X suggests that the globular domain (GD) and C-terminal domain (CTD) of H1.X independently contribute to the dynamic behavior of H1.X. Our results also suggest that the N-terminal domain (NTD), GD, and CTD cooperatively determine the preferential binding sites, and the contribution of each domain for this determination is different depending on the target genes. We also found that linker histones accumulate in the nucleoli when the nucleosome binding activities of the GDs are weak. Our results contribute to understanding the molecular mechanisms of dynamic behaviors, binding site selection, and localization of linker histones. Histones, including linker histones, are highly basic proteins that randomly bind to DNA. To maintain genomic integrity, DNA binding of histones should be strictly controlled. It has been established that random DNA binding of histones abrogates the sequential nucleosome assembly process (1). Histone chaperones directly bind to histones and transfer them to DNA to assemble the chromatin structure. In addition, histone chaperones have been suggested to play an important role in inhibiting the unfavorable DNA binding of histones (1). Our previous studies identified template activating factor I (TAF-I), nucleosome assembly protein 1 (NAP1), and nucleophosmin/B23 as factors involved in regulating the transcription and replication of adenovirus chromatin (2, 3). Recently, all three proteins, TAF-I, NAP1, and B23, were found to function as linker histone chaperones (4-7). A nucleolar protein, nucleolin (NCL), and prothymosin ␣ (ProT␣) were shown to regulate linker histone-chromatin binding (8-10).Linker histones are comprised of three regions: the N-terminal domain (NTD), central globular domain (GD), and C-terminal domain (CTD). The GDs of about 80 amino acids are able to reside on the DNA entry and exit sites of a nucleosome (11). Recent structural analysis of the GD of chicken linker histone H5 demonstrated that the GD makes contact with two extended DNA strands from the nucleosome and the nucleosome dyad (12). The CTD of linker histones are generally basic and rich in lysines, alanines, and prolines. The CTD plays a critical role in the condensation of DNA through its random DNA binding activity (13,14). The GD and CTD show distinct DNA binding activity (15), but neither is sufficient to stably associate with nu...