Role In Nucleosome Assembly Research Articles

Cloning and expression of retinoblastoma-binding protein 4 gene in embryo diapause termination and in response to salinity stress from brine shrimp Artemia sinica

Retinoblastoma binding protein 4 (RBBP4) is a nuclear protein with four WD-repeat sequences and thus belongs to a highly conserved subfamily of proteins with such domains. This retinoblastoma-binding protein plays an important role in nucleosome assembly and histone modification, which influences gene transcription and regulates cell cycle and proliferation. Artemia sinica (brine shrimp) undergoes an unusual diapause process under stress conditions of high salinity and low temperature. However, the role of RBBP4 in diapause termination of embryo development in A. sinica remains unknown. Here, the full-length cDNA of the As-rbbp4 gene was obtained from A. sinica and found to contain 1411 nucleotides, including a 1281 bp open reading frame (ORF), 63 bp 5′-untranslated region (UTR) and a 67-bp 3′-UTR, which encodes a 427 amino acid (48 kDa) protein. Bioinformatic analysis indicated As-RBBP4 to be mainly located in the nucleus, with a theoretical isoelectric point of 4.79. Protein sequence domain analysis showed that As-RBBP4 is a conserved protein, especially in the WD40 domain. No specificity in expression of this gene was observed in tissues or organs by in situ hybridization. Real-time quantitative PCR and Western blot analyses of As-RBBP4 gene and protein expression, respectively, showed notably high levels at 10 h and a subsequent downward trend. Obvious trends in upregulation of As-RBBP4 were observed under conditions of low temperature and high salinity stress. As-E2F1 and As-CyclinE also presented similar trends as that of As-RBBP4 in Western blots. Analysis of the RBBP4 expression in early embryonic development of A. sinica indicated that this protein plays an important role in diapause termination and cell cycle regulation.

Nucleosome Assembly Factors CAF-1 and HIR Modulate Epigenetic Switching Frequencies in an H3K56 Acetylation-Associated Manner in Candida albicans

CAF-1 and HIR are highly conserved histone chaperone protein complexes that function in the assembly of nucleosomes onto chromatin. CAF-1 is characterized as having replication-coupled nucleosome activity, whereas the HIR complex can assemble nucleosomes independent of replication. Histone H3K56 acetylation, controlled by the acetyltransferase Rtt109 and deacetylase Hst3, also plays a significant role in nucleosome assembly. In this study, we generated a set of deletion mutants to genetically characterize pathway-specific and overlapping functions of CAF-1 and HIR in C. albicans. Their roles in epigenetic maintenance of cell type were examined by using the white-opaque switching system in C. albicans. We show that CAF-1 and HIR play conserved roles in UV radiation recovery, repression of histone gene expression, correct chromosome segregation, and stress responses. Unique to C. albicans, the cac2Δ/Δ mutant shows increased sensitivity to the Hst3 inhibitor nicotinamide, while the rtt109Δ/Δ cac2Δ/Δ and hir1Δ/Δ cac2Δ/Δ mutants are resistant to nicotinamide. CAF-1 plays a major role in maintaining cell types, as the cac2Δ/Δ mutant exhibited increased switching frequencies in both directions and switched at a high frequency to opaque in response to nicotinamide. Like the rtt109Δ/Δ mutant, the hir1Δ/Δ cac2Δ/Δ double mutant is defective in maintaining the opaque cell fate and blocks nicotinamide-induced opaque formation, and the defects are suppressed by ectopic expression of the master white-opaque regulator Wor1. Our data suggest an overlapping function of CAF-1 and HIR in epigenetic regulation of cell fate determination in an H3K56 acetylation-associated manner.

Scm3 Is a Centromeric Nucleosome Assembly Factor

The Cse4 nucleosome at each budding yeast centromere must be faithfully assembled each cell cycle to specify the site of kinetochore assembly and microtubule attachment for chromosome segregation. Although Scm3 is required for the localization of the centromeric H3 histone variant Cse4 to centromeres, its role in nucleosome assembly has not been tested. We demonstrate that Scm3 is able to mediate the assembly of Cse4 nucleosomes in vitro, but not H3 nucleosomes, as measured by a supercoiling assay. Localization of Cse4 to centromeres and the assembly activity depend on an evolutionarily conserved core motif in Scm3, but localization of the CBF3 subunit Ndc10 to centromeres does not depend on this motif. The centromere targeting domain of Cse4 is sufficient for Scm3 nucleosome assembly activity. Assembly does not depend on centromeric sequence. We propose that Scm3 plays an active role in centromeric nucleosome assembly.

Unique Gene Expression Patterns in Hematopoietic Stem Cells from Various Sources

Hematopoietic stem cells are involved in the production of blood cells. These cells are derived from various origins. In this study, we examined, using microarray software GeneSpring GX, whether HSCs derived from four different origins i.e., cord blood, liver, bone marrow and peripheral blood, exhibit unique gene expression pattern. A total of 23 individual normal hematopoietic cell expression profiles were identified in NCBI's GEO database -10 normal CD34+ bone marrow sample, 4 normal CD34+ liver sample, 5 normal CD34+ cord blood sample and 4 normal CD34+ peripheral blood sample. These studies were performed using U133A array platform. The data analysis was done by GeneSpring GX Microarray software 7.3 version. Our results indicated that HSCs derived from various origins exhibited unique gene expression patterns. We also determined the specific biological function of HSCs using upregulated genes that are uniquely expressed in each origin. We observed the following results - cord blood HSCs play an important role in development of embryos; Liver HSCs play an important role in apoptosis, angiogenesis and neural transdifferentiation; Bone marrow HSCs play an important role in cell proliferation, cell migration and cell retention and peripheral blood HSCs play an important role in nucleosome assembly. These HSCs genes present in peripheral blood, when altered, get involved in Systemic Lupus Erythematosus Pathway.

Codanin-1, the Product of the Gene Mutated In Congenital Dyserythropoietic Anemia Type I (CDA I), Binds to Histone Chaperone Asf1a and Inhibits Its Nucleosome Assembly Activity

AbstractAbstract 1004Congenital dyserythropoietic anemia type I (CDA1) is an inherited recessive macrocytic anemia associated with ineffective erythropoiesis. The disorder is characterized by the accumulation of erythroid precursors containing spongy heterochromatin and internuclear chromatin bridges. The mutated gene (CDAN1) encodes an ubiquitously expressed protein (codanin-1) of unknown function. We have previously shown that codanin-1 is a direct transcriptional target of the E2F1 transcription factor and that the levels of codanin-1 increase during S-phase and decrease during mitosis. In an attempt to further define the role of codanin-1, we conducted a yeast two-hybrid screen using a human bone marrow library and found that codanin-1 binds to Asf1a. Asf1 (anti silencing function) is a H3/H4 histone chaperone involved in the chromatin structure dynamics by its role in nucleosome assembly and disassembly. Using coimmunoprecipitation experiments we confirmed that histone chaperone Asf1a is a direct binding partner of codanin-1. Minimal 100 amino acids domain of codanin-1, involved in binding Asf1, was identified and defined as the Asf1-binding domain. We found that codanin-1 binds to the conserved N-terminal core of Asf1a, where histones and other histone chaperones also bind. FLAG-tagged codanin-1 or its Asf1-binding domain immunoprecipitated from transfected Hela cells and subsequently coimmunoprecipitated histone H3 and Asf1a simultaneously. A pull-down assay of purified Asf1-binding domain in the presence of core histones showed, however, no direct binding of this domain of codanin-1 to H3/H4 histones. By using the replication-independent nucleosome formation assay we noticed that the nucleosome assembly activity of GST-Asf1a was severely decreased by the addition of the purified Asf1-binding domain of codanin-1, a phenotype similar to the one observed in Asf1 depletion. One possible explanation is that binding of codanin-1 inhibits dissociation of histones from Asf1a, which therefore cannot be deposited onto DNA. It will be of interest to determine if codanin-1 is involved in modulating Asf1a activity in vivo and also in response to DNA replication or damage and to determine its role in erythroid heterochromatin formation. Disclosures:No relevant conflicts of interest to declare.

Identification of a structural and functional domain in xNAP1 involved in protein–protein interactions

xNAP1 (Xenopus nucleosome assembly protein) belongs to the family of nucleosome assembly proteins (NAPs) and shares 92% identity with human and mouse NAP1. NAPs have been reported to have a role in nucleosome assembly, cell cycle regulation, cell proliferation and transcriptional control, although the precise function of NAP1 is still not clear. Here we report the identification of a putative domain of xNAP1 by limited proteolysis. This domain has been mapped in the xNAP1 protein sequence to residues 38–282 and thus lacks the acidic sequences at the N- and C-termini. We have studied this domain and related fragments in vitro and by a functional assay involving over-expression of the protein in Xenopus laevis embryos. Analytical ultracentrifugation shows that removal of the acidic N- and C-terminal regions does not prevent the formation of larger multimers, which are predominantly hexadecamers. Injection of mRNA encoding the full-length xNAP1 or the putative domain and other related constructs into Xenopus embryos gave identical phenotypes. These results are discussed in relation to protein–protein interactions between NAP1 octamers and a possible ‘squelching’ mechanism.

Sperm decondensation in Xenopus egg cytoplasm is mediated by nucleoplasmin

At fertilization, sperm chromatin decondenses in two stages, which can be mimicked in extracts of Xenopus eggs. Rapid, limited decondensation is followed by slower, membrane-dependent decondensation and swelling. Nucleoplasmin, an acidic nuclear protein, occurs at high concentration in Xenopus eggs and has a histone-binding role in nucleosome assembly. Immunodepleting nucleoplasmin from egg extracts inhibits the initial rapid stage of sperm decondensation, and also the decondensation of myeloma nuclei, relative to controls of mock depletion and TFIIIA depletion. Readdition of purified nucleoplasmin recues depleted extracts. A physiological concentration of purified nucleoplasmin alone decondenses both sperm and myeloma nuclei. We conclude that nucleoplasmin is both necessary and sufficient for the first stage of sperm decondensation in Xenopus eggs.