Chromosome condensation is a hallmark of mitosis in eukaryotes and is a prerequisite for faithful segregation of genetic material to daughter cells. Here we show that condensin, which is essential for assembling condensed chromosomes, helps to preclude the detrimental effects of gene transcription on mitotic condensation. ChIP-seq profiling reveals that the fission yeast condensin preferentially binds to active protein-coding genes in a transcription-dependent manner during mitosis. Pharmacological and genetic attenuation of transcription largely rescue bulk chromosome segregation defects observed in condensin mutants. We also demonstrate that condensin is associated with and reduces unwound DNA segments generated by transcription, providing a direct link between an in vitro activity of condensin and its in vivo function. The human condensin isoform condensin I also binds to unwound DNA regions at the transcription start sites of active genes, implying that our findings uncover a fundamental feature of condensin complexes.
Peroxisome proliferator-activated receptor (PPAR)-γ γ γ γ 2, a member of the nuclear hormone receptor superfamily, plays a key role in adipocyte differentiation. Its amino-terminal region carries a ligandindependent gene-activating function, AF-1, and is composed of activation as well as repression domains. We have found PPARγ γ γ γ 2 and its isoform, PPARγ γ γ γ 1, to be modified by small ubiquitin-related modifier (SUMO)-1 in vivo , at a lysine residue in the repression domain. In reporter assays, a sumoylation-defective K107R mutant of PPARγ γ γ γ 2 exhibited much stronger transactivation than the wild-type, comparable with that of a mutant deleted for the repression domain. A close inverse correlation was observed between the levels of sumoylation and transactivation by PPARγ γ γ γ 2, in analyses employing PPARγ γ γ γ 2 forms with mutations in the sumoylation motif and a dominantnegative mutant of the SUMO conjugating enzyme, Ubc9. Studies with phosphorylation-defective mutants suggested that phosphorylation at S112 of PPARγ γ γ γ 2 promotes K107 sumoylation, and this latter exerts the more potent repressive effects. The K107R mutant PPARγ γ γ γ 2, when infected into NIH3T3 cells with a viral vector, promoted differentiation into adipocytes more efficiently than the wild-type. These observations provide evidence that sumoylation is involved in negative regulation of the transactivating function of PPARγ γ γ γ 2.
MCPH1, a protein linked to primary microcephaly, directly modulates condensin II to regulate chromosome condensation and shape.
Condensin II initiates structural reorganization of duplicated chromosomes during S phase to prepare for their proper condensation and segregation in mitosis.
Although ribosomal proteins (RPs) are essential cellular constituents in all living organisms, mechanisms underlying regulation of their gene expression in mammals remain unclear. We have established that 22 out of 79 human RP genes contain sequences similar to the human DREF (DNA replication-related elementbinding factor; hDREF) binding sequence (hDRE) within 200-bp regions upstream of their transcriptional start sites. Electrophoretic gel mobility shift assays and chromatin immunoprecipitation analysis indicated that hDREF binds to hDRE-like sequences in the RP genes both in vitro and in vivo. In addition, transient luciferase assays revealed that hDRE-like sequences act as positive elements for RP gene transcription and cotransfection of an hDREF-expressing plasmid was found to stimulate RP gene promoter activity. Like that of hDREF, expression of RP genes is increased during the late G 1 to S phases, and depletion of hDREF using short hairpin RNA-mediated knockdown decreased RP gene expression and cell proliferation in normal human fibroblasts. Knockdown of the RPS6 gene also resulted in impairment of cell proliferation. These data suggest that hDREF is an important transcription factor for cell proliferation which plays roles in cell cycle-dependent regulation of a number of RP genes.Promoters of Drosophila melanogaster genes related to DNA replication, such as those for the 180-kDa catalytic subunit of DNA polymerase ␣ and proliferating cell nuclear antigen (PCNA), contain a common 8-bp palindromic sequence (5Ј-T ATCGATA-3Ј), named the DNA replication-related element (DRE) (12). These DREs are required for promoter activities both in cultured cells and in flies in vivo (41). We have purified the DRE-binding factor (DREF) from cultured Drosophila cells, consisting of an 86-kDa polypeptide homodimer specifically binding to DRE, and isolated a cDNA (12, 13). The importance of Drosophila DREF in development has been demonstrated from studies using transgenic flies (11,14,44). For example, ectopic expression of Drosophila DREF in eye imaginal disc cells behind the morphogenetic furrow, which are normally postmitotic, induced ectopic DNA synthesis and apoptosis and abolished photoreceptor specifications (11). More recently, we and Hyun et al. have succeeded in knocking down Drosophila DREF expression in various tissues (16,45). Decreased levels of DREF in developing wing and eye imaginal discs were associated with reduction in wing size with smaller cells and drastically aberrant small and rough eyes, respectively. These lines of evidence indicate that the Drosophila DRE/DREF system performs important roles in regulation of cell growth as well as cell proliferation during development.How many and what kind of genes other than those described above are under control of the Drosophila DRE/DREF system? Immunostaining of polytene chromosomes of salivary glands revealed that Drosophila DREF binds to hundreds of loci (8, 10), and recent computational analysis of core promoters in the Drosophila genome showed DRE to be...
Peroxisome proliferator-activated receptors (PPARs) constitute a subfamily of nuclear receptor superfamily. A wide variety of compounds including hypolipidemic agents, antidiabetic drugs, and long-chain fatty acids are the potential ligands of PPARs. To approach the regulatory mechanisms of PPARs, we studied on two subjects in this work. First, we identified a functional PPAR-binding site in the spacer region between the PEX11alpha and perilipin genes, which are arranged in tandem on the mouse genome. By gene reporter assays and in vivo as well as in vitro binding assays, we show that these genes are regulated tissue-selectively through this common binding site: The PEX11alpha gene is activated by PPARalpha in the liver, whereas the perilipin gene by PPARgamma in the adipose tissue. As the second subject, we found that PPARgamma2 is conjugated with small ubiquitin-related modifier (SUMO) at a specific lysine residue in the amino-terminal region. By site-directed mutagenesis combined with gene reporter assays and sumoylation analyses, we show that sumoylation represses the ligand-independent transactivating function carried by this region, and hence negatively regulates the whole transactivating competence of PPARgamma2. In addition, phosphorylation at a specific site in the amino-terminal region represses the transactivation by PPARgamma2 possibly through enhancing sumoylation.
In vertebrates, condensin I and condensin II cooperate to assemble rod-shaped chromosomes during mitosis. Although the mechanism of action and regulation of condensin I have been studied extensively, our corresponding knowledge of condensin II remains very limited. By introducing recombinant condensin II complexes into Xenopus egg extracts, we dissect the roles of its individual subunits in chromosome assembly. We find that one of two HEAT subunits, CAP-D3, plays a crucial role in condensin II-mediated assembly of chromosome axes whereas the other HEAT subunit, CAP-G2, has a very strong negative impact on this process. The SMC ATPase and the basic amino acid clusters of the kleisin subunit CAP-H2 are essential for this process. Deletion of the C-terminal tail of CAP-D3 increases the ability of condensin II to assemble chromosomes and further exposes a hidden function of CAP-G2 in the lateral compaction of chromosomes. Taken together, our results uncover a multilayered regulatory mechanism unique to condensin II, and provide profound implications for the evolution of condensin II.
Eight horse breeds-Hokkaido, Kiso, Misaki, Noma, Taishu, Tokara, Miyako and Yonaguniare native to Japan. Although Japanese native breeds are believed to have originated from ancient Mongolian horses imported from the Korean Peninsula, the phylogenetic relationships among these breeds are not well elucidated. In the present study, we compared genetic diversity among 32 international horse breeds previously evaluated by the Equine Genetic Diversity Consortium, the eight Japanese native breeds and Japanese Thoroughbreds using genome-wide SNP genotype data. The proportion of polymorphic loci and expected heterozygosity showed that the native Japanese breeds, with the exception of the Hokkaido, have relatively low diversity compared to the other breeds sampled. Phylogenetic and cluster analyses demonstrated relationships among the breeds that largely reflect their geographic distribution in Japan. Based on these data, we suggest that Japanese horses originated from Mongolian horses migrating through the Korean Peninsula. The Japanese Thoroughbreds were distinct from the native breeds, and although they maintain similar overall diversity as Thoroughbreds from outside Japan, they also show evidence of uniqueness relative to the other Thoroughbred samples. This is the first study to place the eight native Japanese breeds and Japanese Thoroughbred in context with an international sample of diverse breeds.
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