Chromosome Research最新文献

ATM and DNA-PKcs coordinate the DNA damage response at multiple levels following the exposure to chemotherapy. The Topoisomerase II poison etoposide (ETO) is an effective chemotherapeutic agent that induces DNA double-strand breaks (DSB), but it is responsible from the chromosomal rearrangements frequently found in therapy-related secondary tumors. Targeted inhibition of DNA-PKcs in ATM-defective tumors combined with radio- or chemotherapy has been proposed as relevant therapies. Here, we explored the DNA repair mechanisms and the genetic consequences of targeting the non-oncogenic addiction to DNA-PKcs of ATM-defective tumor cells after exposure to ETO. We demonstrated that chemical inhibition of DNA-PKcs followed by treatment with ETO resulted in the accumulation of chromatid breaks and decreased mitotic index in both A-T cells and ATM-knocked-down (ATM^kd) tumor cells. The HR repair process in DNA-PKcs-inhibited ATM^kd cells amplified the RAD51 foci number, with no correlated increase in sister chromatid exchanges. The analysis of post-mitotic DNA lesions presented an augmented number of persistent unresolved DSB, without alterations in the cell cycle progression. Long-term examination of chromosome aberrations revealed a strikingly high number of chromatid and chromosome exchanges. By using genetic and pharmacological abrogation of PARP-1, we demonstrated that alternative end-joining (alt-EJ) repair pathway is responsible for those chromosome abnormalities generated by limiting c-NHEJ activities during directed inhibition of DNA-PKcs in ATM-deficient cells. Targeting the non-oncogenic addiction to DNA-PKcs of ATM-defective tumors stimulates the DSB repair by alt-EJ, which is liable for the origin of cells carrying stable chromosome aberrations that may eventually restrict the therapeutic strategy.

Homomorphic sex chromosomes and their turnover are common in teleosts. We investigated the evolution of nascent sex chromosomes in several populations of two sister species of African annual killifishes, Nothobranchius furzeri and N. kadleci, focusing on their under-studied repetitive landscape. We combined bioinformatic analyses of the repeatome with molecular cytogenetic techniques, including comparative genomic hybridization, fluorescence in situ hybridization with satellite sequences, ribosomal RNA genes (rDNA) and bacterial artificial chromosomes (BACs), and immunostaining of SYCP3 and MLH1 proteins to mark lateral elements of synaptonemal complexes and recombination sites, respectively. Both species share the same heteromorphic XY sex chromosome system, which thus evolved prior to their divergence. This was corroborated by sequence analysis of a putative master sex determining (MSD) gene gdf6Y in both species. Based on their divergence, differentiation of the XY sex chromosome pair started approximately 2 million years ago. In all populations, the gdf6Y gene mapped within a region rich in satellite DNA on the Y chromosome long arms. Despite their heteromorphism, X and Y chromosomes mostly pair regularly in meiosis, implying synaptic adjustment. In N. kadleci, Y-linked paracentric inversions like those previously reported in N. furzeri were detected. An inversion involving the MSD gene may suppress occasional recombination in the region, which we otherwise evidenced in the N. furzeri population MZCS-121 of the Limpopo clade lacking this inversion. Y chromosome centromeric repeats were reduced compared with the X chromosome and autosomes, which points to a role of relaxed meiotic drive in shaping the Y chromosome repeat landscape. We speculate that the recombination rate between sex chromosomes was reduced due to heterochiasmy. The observed differences between the repeat accumulations on the X and Y chromosomes probably result from high repeat turnover and may not relate closely to the divergence inferred from earlier SNP analyses.

Histones H1 and H3 are highly phosphorylated in mitotic HeLa cells but are rapidly dephosphorylated by endogenous protein phosphatases during the isolation of metaphase chromosomes. We show that this dephosphorylation can be prevented by including the sulfhydryl reagent 5,5'-dithiobis-(2-nitrobenzoate) (Ellman's reagent, or DTNB) in the isolation buffer. The minimal amount of DTNB required is approximately stoichiometric with the number of sulfhydryl groups in the lysate. Inhibition of the protein phosphatases can subsequently be reversed by treatment with dithiothreitol or 2-mercaptoethanol. DTNB is compatible with the isolation of either metaphase chromosome clusters or individual metaphase chromosomes. It should be useful in investigations of the structure and biochemistry of chromatin and chromosomes and in the study of possible functions for mitotic histone phosphorylation.

The human genome is divided into functional units that replicate at specific times during S-phase. This temporal program is known as replication timing (RT) and is coordinated with the spatial organization of the genome and transcriptional activity. RT is also cell type-specific, dynamically regulated during development, and alterations in RT are observed in multiple diseases. Thus, the precise measure of RT is critical to understand the role of RT in gene function regulation. Distinct methods for assaying the RT program exist; however, conventional methods require thousands of cells as input, prohibiting its applicability to samples with limited cell numbers such as those from disease patients or from early developing embryos. Although single-cell RT analyses have been developed, these methods are low throughput, require generation of numerous libraries, increased sequencing costs, and produce low resolution data. Here, we developed an improved method to measure RT genome-wide that enables high-resolution analysis of low input samples. This method incorporates direct cell sorting into lysis buffer, as well as DNA fragmentation and library preparation in a single tube, resulting in higher yields, increased quality, and reproducibility with decreased costs. We also performed a systematic data processing analysis to provide standardized parameters for RT measurement. This optimized method facilitates RT analysis and will enable its application to a broad range of studies investigating the role of RT in gene expression, nuclear architecture, and disease.

Double-strand break repair during meiosis is normally achieved using the homologous chromosome as a repair template. Heteromorphic sex chromosomes share little sequence homology, presenting unique challenges to the repair of double-strand breaks. Our understanding of how heteromorphic sex chromosomes behave during meiosis has been focused on ancient sex chromosomes, where the X and Y differ markedly in overall structure and gene content. It remains unclear how more recently evolved sex chromosomes that share considerably more sequence homology with one another pair and form double-strand breaks. One possibility is barriers to pairing evolve rapidly. Alternatively, recently evolved sex chromosomes may exhibit pairing and double-strand break repair that more closely resembles that of their autosomal ancestors. Here, we use the recently evolved X and Y chromosomes of the threespine stickleback fish (Gasterosteus aculeatus) to study patterns of pairing and double-stranded break formation using molecular cytogenetics. We found that the sex chromosomes of threespine stickleback fish did not pair exclusively in the pseudoautosomal region. Instead, the chromosomes fully paired in a non-homologous fashion. To achieve this, the X chromosome underwent synaptic adjustment during pachytene to match the axis length of the Y chromosome. Double-strand break formation and repair rate also matched that of the autosomes. Our results highlight that recently evolved sex chromosomes exhibit meiotic behavior that is reminiscent of autosomes and argues for further work to identify the homologous templates that are used to repair double-strand breaks on the X and Y chromosomes.

The tribe Phaseoleae includes several legume crops with assembled genomes. Comparative genomic studies have evidenced the preservation of large genomic blocks among legumes, although chromosome dynamics during Phaseoleae evolution has not been investigated. We conducted a comparative genomic analysis to define an informative genomic block (GB) system and to reconstruct the ancestral Phaseoleae karyotype (APK). We identified GBs based on the orthologous genes between Phaseolus vulgaris and Vigna unguiculata and searched for GBs in different genomes of the Phaseolinae (P. lunatus) and Glycininae (Amphicarpaea edgeworthii) subtribes and Spatholobus suberectus (sister to Phaseolinae and Glycininae), using Medicago truncatula as the outgroup. We also used oligo-FISH probes of two P. vulgaris chromosomes to paint the orthologous chromosomes of two non-sequenced Phaseolinae species. We inferred the APK as having n = 11 and 19 GBs (A to S), hypothesizing five chromosome fusions that reduced the ancestral legume karyotype to n = 11. We identified the rearrangements among the APK and the subtribes and species, with extensive centromere repositioning in Phaseolus. We also reconstructed the chromosome number reduction in S. suberectus. The development of the GB system and the proposed APK provide useful approaches for future comparative genomic analyses of legume species.

Most plant species have three or more SPO11/TOPOVIA homologs and two TOPOVIB homologs, which associate to trigger meiotic double-strand break (DSB) formation and subsequent meiotic recombination. In Zea mays L. (maize), ZmSPO11-1 and ZmMTOPVIB have been reported to be indispensable for the initiation of meiotic recombination, yet the function of ZmSPO11-2 remains unclear. In this study, we characterized meiotic functions of ZmSPO11-2 during male meiosis in maize. Two independent Zmspo11-1 knock-out mutants exhibited normal vegetative growth but both male and female sterility. The formation of meiotic DSBs of DNA molecules was fully abolished in the Zmspo11-2 plants, leading to the defective homologous chromosome paring, synapsis, recombination, and segregation. However, the bipolar spindle assembly was not noticeably affected in Zmspo11-2 meiocytes. Overall, our results demonstrate that as its partner ZmSPO11-1 and ZmMTOPVIB, ZmSPO11-2 plays essential roles in DSB formation and homologous recombination in maize meiosis.

Imprinted X chromosome inactivation (iXCI) balances the expression of X-linked genes in preimplantation embryos and extraembryonic tissues in rodents. Long noncoding Xist RNA drives iXCI, silencing genes and recruiting Xist-dependent chromatin repressors. Some domains on the inactive X chromosome include repressive modifications specific to constitutive heterochromatin, which show no direct link to Xist RNA. We explored the relationship between Xist RNA and chromatin silencing during iXCI in vole Microtus levis. We performed locus-specific activation of Xist transcription on the only active X chromosome using the dCas9-SAM system in XO vole trophoblast stem cells (TSCs), which allow modeling iXCI events to some extent. The artificially activated endogenous vole Xist transcript is truncated and restricted ~ 6.6 kb of the exon 1. Ectopic Xist RNA accumulates on the X chromosome and recruits Xist-dependent modifications during TSC differentiation, yet is incapable by itself repressing X-linked genes. Transcriptional silencing occurs upon ectopic Xist upregulation only when repressive marks spread from the massive telomeric constitutive heterochromatin to the X chromosome region containing genes. We hypothesize that the Xist RNA-induced propagation of repressive marks from the constitutive heterochromatin could be a mechanism involved in X chromosome inactivation.

Unisexual reproduction, which generates clonal offspring, is an alternative strategy to sexual breeding and occurs even in vertebrates. A wide range of non-sexual reproductive modes have been described, and one of the least understood questions is how such pathways emerged and how they mechanistically proceed. The Amazon molly, Poecilia formosa, needs sperm from males of related species to trigger the parthenogenetic development of diploid eggs. However, the mechanism, of how the unreduced female gametes are produced, remains unclear. Cytological analyses revealed that the chromosomes of primary oocytes initiate pachytene but do not proceed to bivalent formation and meiotic crossovers. Comparing ovary transcriptomes of P. formosa and its sexual parental species revealed expression levels of meiosis-specific genes deviating from P. mexicana but not from P. latipinna. Furthermore, several meiosis genes show biased expression towards one of the two alleles from the parental genomes. We infer from our data that in the Amazon molly diploid oocytes are generated by apomixis due to a failure in the synapsis of homologous chromosomes. The fact that this failure is not reflected in the differential expression of known meiosis genes suggests the underlying molecular mechanism may be dysregulation on the protein level or misexpression of a so far unknown meiosis gene, and/or hybrid dysgenesis because of compromised interaction of proteins from diverged genomes.