Nap1 regulates proper CENP-B binding to nucleosomes

CENP-B is a widely conserved centromeric satellite DNA-binding protein, which specifically binds to a 17-bp DNA sequence known as the CENP-B box. CENP-B functions positively in the de novo assembly of centromeric nucleosomes, containing the centromere-specific histone H3 variant, CENP-A. At the same time, CENP-B also prevents undesired assembly of the CENP-A nucleosome through heterochromatin formation on satellite DNA integrated into ectopic sites. Therefore, improper CENP-B binding to chromosomes could be harmful. However, no CENP-B eviction mechanism has yet been reported. In the present study, we found that human Nap1, an acidic histone chaperone, inhibited the non-specific binding of CENP-B to nucleosomes and apparently stimulated CENP-B binding to its cognate CENP-B box DNA in nucleosomes. In human cells, the CENP-B eviction activity of Nap1 was confirmed in model experiments, in which the CENP-B binding to a human artificial chromosome or an ectopic chromosome locus bearing CENP-B boxes was significantly decreased when Nap1 was tethered near the CENP-B box sequence. In contrast, another acidic histone chaperone, sNASP, did not promote CENP-B eviction in vitro and in vivo and did not stimulate specific CENP-B binding to CENP-A nucleosomes in vitro. We therefore propose a novel mechanism of CENP-B regulation by Nap1.     

Short-term variations of mycosporine-like amino acids in the carrageenan-producing red macroalga Mazzaella laminarioides (Gigartinales,Rhodophyta) are related to nitrate availability

Journal of Applied Phycology - The effect of solar UV radiation exposure and NO3– supply on mycosporine-like amino acids (MAAs) accumulation in the carrageenan-producing red macroalga...     

Evolutionary diversification of SPANX-N sperm protein gene structure and expression

The sperm protein associated with nucleus in the X chromosome (SPANX) genes cluster at Xq27 in two subfamilies, SPANX-A/D and SPANX-N. SPANX-A/D is specific for hominoids and is fairly well characterized. The SPANX-N gave rise to SPANX-A/D in the hominoid lineage approximately 7 MYA. Given the proposed role of SPANX genes in spermatogenesis, we have extended studies to SPANX-N gene evolution, variation, regulation of expression, and intra-sperm localization. By immunofluorescence analysis, SPANX-N proteins are localized in post-meiotic spermatids exclusively, like SPANX-A/D. But in contrast to SPANX-A/D, SPANX-N are found in all ejaculated spermatozoa rather than only in a subpopulation, are localized in the acrosome rather than in the nuclear envelope, and are expressed at a low level in several nongametogenic adult tissues as well as many cancers. Presence of a binding site for CTCF and its testis-specific paralogue BORIS in the SPANX promoters suggests, by analogy to MAGE-A1 and NY-ESO-1, that their activation in spermatogenesis is mediated by the programmed replacement of CTCF by BORIS. Based on the relative density of CpG, the more extended expression of SPANX-N compared to SPANX-A/D in nongametogenic tissues is likely attributed to differences in promoter methylation. Our findings suggest that the recent duplication of SPANX genes in hominoids was accompanied by different localization of SPANX-N proteins in post-meiotic sperm and additional expression in several nongonadal tissues. This suggests a corresponding functional diversification of SPANX gene families in hominoids. SPANX proteins thus provide unique targets to investigate their roles in the function of spermatozoa, selected malignancies, and for SPANX-N, in other tissues as well.     

Stable complex formation of CENP-B with the CENP-A nucleosome

CENP-A and CENP-B are major components of centromeric chromatin. CENP-A is the histone H3 variant, which forms the centromere-specific nucleosome. CENP-B specifically binds to the CENP-B box DNA sequence on the centromere-specific repetitive DNA. In the present study, we found that the CENP-A nucleosome more stably retains human CENP-B than the H3.1 nucleosome in vitro. Specifically, CENP-B forms a stable complex with the CENP-A nucleosome, when the CENP-B box sequence is located at the proximal edge of the nucleosome. Surprisingly, the CENP-B binding was weaker when the CENP-B box sequence was located in the distal linker region of the nucleosome. This difference in CENP-B binding, depending on the CENP-B box location, was not observed with the H3.1 nucleosome. Consistently, we found that the DNA-binding domain of CENP-B specifically interacted with the CENP-A-H4 complex, but not with the H3.1-H4 complex, in vitro. These results suggested that CENP-B forms a more stable complex with the CENP-A nucleosome through specific interactions with CENP-A, if the CENP-B box is located proximal to the CENP-A nucleosome. Our in vivo assay also revealed that CENP-B binding in the vicinity of the CENP-A nucleosome substantially stabilizes the CENP-A nucleosome on alphoid DNA in human cells.     

Breaking the HAC Barrier: histone H3K9 acetyl/methyl balance regulates CENP-A assembly

The kinetochore is responsible for accurate chromosome segregation. However, the mechanism by which kinetochores assemble and are maintained remains unclear. Here we report that de novo CENP-A assembly and kinetochore formation on human centromeric alphoid DNA arrays is regulated by a histone H3K9 acetyl/methyl balance. Tethering of histone acetyltransferases (HATs) to alphoid DNA arrays breaks a cell type-specific barrier for de novo stable CENP-A assembly and induces assembly of other kinetochore proteins at the ectopic alphoid site. Similar results are obtained following tethering of CENP-A deposition factors hMis18α or HJURP. HAT tethering bypasses the need for hMis18α, but HJURP is still required for de novo kinetochore assembly. In contrast, H3K9 methylation following tethering of H3K9 tri-methylase (Suv39h1) to the array prevents de novo CENP-A assembly and kinetochore formation. CENP-A arrays assembled de novo by this mechanism can form human artificial chromosomes (HACs) that are propagated indefinitely in human cells.     

Cloning of human centromeres by transformation-associated recombination in yeast and generation of functional human artificial chromosomes

Human centromeres remain poorly characterized regions of the human genome despite their importance for the maintenance of chromosomes. In part this is due to the difficulty of cloning of highly repetitive DNA fragments and distinguishing chromosome-specific clones in a genomic library. In this work we report the highly selective isolation of human centromeric DNA using transformation-associated recombination (TAR) cloning. A TAR vector with alphoid DNA monomers as targeting sequences was used to isolate large centromeric regions of human chromosomes 2, 5, 8, 11, 15, 19, 21 and 22 from human cells as well as monochromosomal hybrid cells. The alphoid DNA array was also isolated from the 12 Mb human mini-chromosome ΔYq74 that contained the minimum amount of alphoid DNA required for proper chromosome segregation. Preliminary results of the structural analyses of different centromeres are reported in this paper. The ability of the cloned human centromeric regions to support human artificial chromosome (HAC) formation was assessed by transfection into human HT1080 cells. Centromeric clones from ΔYq74 did not support the formation of HACs, indicating that the requirements for the existence of a functional centromere on an endogenous chromosome and those for forming a de novo centromere may be distinct. A construct with an alphoid DNA array from chromosome 22 with no detectable CENP-B motifs formed mitotically stable HACs in the absence of drug selection without detectable acquisition of host DNAs. In summary, our results demonstrated that TAR cloning is a useful tool for investigating human centromere organization and the structural requirements for formation of HAC vectors that might have a potential for therapeutic applications.