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.

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 HP1b 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 b-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 HP1b 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.

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. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

NPM1/Nucleophosmin/B23, also termed NO38 or numatrin, is an acidic nucleolar protein that plays multiple roles in cell growth and proliferation. In general, the expression level of B23 is proportional to the cell growth rate, suggesting that it plays a positive role(s) in cell growth and proliferation. It is important to note that the deletion of the B23 gene and expression of an aberrant type of this gene--caused by gene conversion via translocation or reading-frame shift via nucleotides insertion-have been observed in diverse haematopoietic malignancies. Thus, it is important to understand the function of B23 in the regulation of cell growth and proliferation. In addition, B23 has been reported to undergo a variety of post-translational modifications such as phosphorylation, ubiquitination, SUMOylation, acetylation and poly-(ADP-ribosyl)ation. In this review, the basic structure and functions of B23 as well as the regulation of these functions are summarized.

Nucleophosmin/B23 is a nucleolar phosphoprotein. It has been shown that B23 binds to nucleic acids, digests RNA, and is localized in nucleolar granular components from which preribosomal particles are transported to cytoplasm. The intracellular localization of B23 is significantly changed during the cell cycle. Here, we have examined the cellular localization of B23 proteins and the effect of mitotic phosphorylation of B23.1 on its RNA binding activity. Two splicing variants of B23 proteins, termed B23.1 and B23.2, were complexed both in vivo and in vitro. The RNA binding activity of B23.1 was impaired by hetero-oligomer formation with B23.2. Both subtypes of B23 proteins were phosphorylated during mitosis by cyclin B/cdc2. The RNA binding activity of B23.1 was repressed through cyclin B/cdc2-mediated phosphorylation at specific sites in B23. Thus, the RNA binding activity of B23.1 is stringently modulated by its phosphorylation and subtype association. Interphase B23.1 was mainly localized in nucleoli, whereas B23.2 and mitotic B23.1, those of which were incapable of binding to RNA, were dispersed throughout the nucleoplasm and cytoplasm, respectively. These results suggest that nucleolar localization of B23.1 is mediated by its ability to associate with RNA. INTRODUCTIONThe cell nucleolus is the place for ribosome biogenesis, which is the synthesis and processing of a precursor rRNA (pre-rRNA), and the assembly of ribosomal proteins on rRNAs to form premature ribosome. rRNA is first transcribed by RNA polymerase I as a pre-rRNA of ϳ13,500 nucleotides. To produce mature rRNAs (18S, 5.8S, and 28S rRNA), external and internal spacer sequences (ETS and ITS, respectively) in the long pre-rRNA are removed sequentially while nucleotide modifications such as pseudouridylation and 2Ј-O-methylation occur concurrently (Maden, 1990). For such complex processes, it is suggested that nonribosomal proteins and small nucleolar RNAs are multifunctional (Srivastava and Pollard, 1999;Peculis, 2000). One such nucleolar protein is the well-characterized and abundant nucleophosmin/B23. It is highly conserved in vertebrates. In rat and human cells, at least two isoforms of nucleophosmin/B23, termed B23.1 and B23.2, are expressed (Chang and Olson, 1990;Okuwaki et al., 2001a). These two proteins are identical except that the C-terminal 35 amino acids observed in B23.1 are absent in B23.2. Nucleophosmin/B23 functions to bind nucleic acids (Dumbar et al., 1989), to endonucleolytically cleave RNA preferentially within ITS2 in pre-rRNA (Savkur and Olson, 1998), to suppress the aggregation of proteins (Szebeni and Olson, 1999), and to bind peptides containing nuclear localization signals for their nuclear import (Szebeni et al., 1995). Recently, phosphorylation of nucleophosmin/ B23 by cyclin E/cdk2, a G1 cyclin-dependent kinase, was shown to be essential for centrosome duplication in fibroblast cells (Okuda et al., 2000). Furthermore, it is shown that B23 protein binds a wide diversity of proteins, such as nucleolar phosphoproteins, nucl...

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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.

To study the mechanisms of replication and transcription on chromatin, we have been using the adenovirus DNA complexed with viral basic core proteins, called Ad core. We have identified template activating factor (TAF)-I from uninfected HeLa cells as the factor that stimulates replication and transcription from the Ad core. The nuclease sensitivity assays have revealed that TAF-I remodels the Ad core, thereby making transcription and replication apparatus accessible to the template DNA. To examine whether TAF-I remodels the chromatin consisting of histones, the chromatin structure was reconstituted on the DNA fragment with core histones by the salt dialysis method. The transcription from the reconstituted chromatin was completely repressed, while TAF-I remodeled the chromatin and stimulated the transcription. TAF-I was found to interact with histones. Furthermore, it was shown that TAF-I is capable not only of disrupting the chromatin structure but also of preventing the formation of DNA-histone aggregation and transferring histones to naked DNA. The possible function of TAF-I in conjunction with a histone chaperone activity is discussed.