The involvement of acidic nucleoplasmic DNA-binding protein (and-1) in the regulation of prereplicative complex (pre-RC) assembly in human cells

DNA replication in all eukaryotes starts with the process of loading the replicative helicase MCM2–7 onto chromatin during late mitosis of the cell cycle. MCM2–7 is a key component of the prereplicative complex (pre-RC), which is loaded onto chromatin by the concerted action of origin recognition complex, Cdc6, and Cdt1. Here, we demonstrate that And-1 is assembled onto chromatin in late mitosis and early G(1) phase before the assembly of pre-RC in human cells. And-1 forms complexes with MCM2–7 to facilitate the assembly of MCM2–7 onto chromatin at replication origins in late mitosis and G(1) phase. We also present data to show that depletion of And-1 significantly reduces the interaction between Cdt1 and MCM7 in G(1) phase cells. Thus, human And-1 facilitates loading of the MCM2–7 helicase onto chromatin during the assembly of pre-RC

Analysis of DNA replication, sex chromosomes, and checkpoints in the Caenorhabditis elegans germ line

Meiosis is essential for all sexually reproducing organisms and consists of a single round of DNA replication followed by two rounds of nuclear division and specialized cell divisions that result in the production of two distinct haploid gametes, eggs and sperm. Meiotic DNA replication is important for the subsequent chromosomal interactions that occur during meiotic prophase which can differ extensively between sexes of the same species. Additionally, meiosis has surveillance mechanisms to monitor genome integrity as it passes to the next generation and the extent to which checkpoints monitor and safeguard the genome can differ between sexes. Taking advantage of the unique features of the Caenorhabditis elegans germ line, I investigated aspects of DNA replication, meiotic prophase progression, sex chromosomes and germ-line checkpoints. I found that meiotic S phase is at least twice as long as mitotic S phase in C. elegans germ cells and different regions of the genome replicate with different timing. I discovered that meiotic prophase for oogenesis lasts 54-60 hours while meiotic prophase for spermatogenesis is completed by 20-24 hours. Examination of meiotic progression in sex determination mutants revealed that meiotic prophase timing and germ-line apoptosis, one output of checkpoint signaling, is dictated by the sex of the germ line (oogenesis vs. spermatogenesis). Surprisingly, in feminized, fem-3lf X0 animals a single pair of asynapsed autosomes elicits a checkpoint response, but a single X chromosome fails to induce checkpoint activation. Further analysis revealed that the chromatin/transcriptional state of the single X chromosome locally precludes checkpoint signaling. Investigation into the molecular basis for the lack of checkpoint-activated apoptosis in the male germ line revealed that the 9-1-1 complex, ATR (ATL-1), and activated CHK-1 are recruited to unrepaired breaks in the male germ line. Further, CEP-1(p53) is expressed and induces expression of the pro-apoptotic protein EGL-1 in both hermaphrodite and male germ lines under checkpoint activating conditions. The core apoptotic machinery is expressed in both hermaphrodites and males, but caspase is activated in hermaphrodite but not male germ lines. Although apoptosis is not induced in male germ lines when there are errors in meiosis, checkpoint components improve the fidelity of chromosome inheritance

A Single Unpaired and Transcriptionally Silenced X Chromosome Locally Precludes Checkpoint Signaling in the Caenorhabditis elegans Germ Line

In many organisms, female and male meiosis display extensive sexual dimorphism in the temporal meiotic program, the number and location of recombination events, sex chromosome segregation, and checkpoint function. We show here that both meiotic prophase timing and germ-line apoptosis, one output of checkpoint signaling, are dictated by the sex of the germ line (oogenesis vs. spermatogenesis) in Caenorhabditis elegans. During oogenesis in feminized animals (fem-3), a single pair of asynapsed autosomes elicits a checkpoint response, yet an unpaired X chromosome fails to induce checkpoint activation. The single X in males and fem-3 worms is a substrate for the meiotic recombination machinery and repair of the resulting double strand breaks appears to be delayed compared with worms carrying paired X chromosomes. Synaptonemal complex axial HORMA domain proteins, implicated in repair of meiotic double strand breaks (DSBs) and checkpoint function, are assembled and disassembled on the single X similarly to paired chromosomes, but the central region component, SYP-1, is not loaded on the X chromosome in males. In fem-3 worms some X chromosomes achieve nonhomologous self-synapsis; however, germ cells with SYP-1-positive X chromosomes are not preferentially protected from apoptosis. Analyses of chromatin and X-linked gene expression indicate that a single X, unlike asynapsed X chromosomes or autosomes, maintains repressive chromatin marks and remains transcriptionally silenced and suggests that this state locally precludes checkpoint signaling

Mapping Challenging Mutations by Whole-Genome Sequencing

Whole-genome sequencing provides a rapid and powerful method for identifying mutations on a global scale, and has spurred a renewed enthusiasm for classical genetic screens in model organisms. The most commonly characterized category of mutation consists of monogenic, recessive traits, due to their genetic tractability. Therefore, most of the mapping methods for mutation identification by whole-genome sequencing are directed toward alleles that fulfill those criteria (i.e., single-gene, homozygous variants). However, such approaches are not entirely suitable for the characterization of a variety of more challenging mutations, such as dominant and semidominant alleles or multigenic traits. Therefore, we have developed strategies for the identification of those classes of mutations, using polymorphism mapping in Caenorhabditis elegans as our model for validation. We also report an alternative approach for mutation identification from traditional recombinant crosses, and a solution to the technical challenge of sequencing sterile or terminally arrested strains where population size is limiting. The methods described herein extend the applicability of whole-genome sequencing to a broader spectrum of mutations, including classes that are difficult to map by traditional means

Meiotic Errors Activate Checkpoints that Improve Gamete Quality without Triggering Apoptosis in Male Germ Cells

SummaryBackgroundMeiotic checkpoints ensure the production of gametes with the correct complement and integrity of DNA; in metazoans, these pathways sense errors and transduce signals to trigger apoptosis to eliminate damaged germ cells. The extent to which checkpoints monitor and safeguard the genome differs between sexes and may contribute to the high frequency of human female meiotic errors. In the C. elegans female germline, DNA damage, chromosome asynapsis, and/or unrepaired meiotic double-strand breaks (DSBs) activate checkpoints that induce apoptosis; conversely, male germ cells do not undergo apoptosis.ResultsHere we show that the recombination checkpoint is in fact activated in male germ cells despite the lack of apoptosis. The 9-1-1 complex and the phosphatidylinositol 3-kinase-related protein kinase ATR, sensors of DNA damage, are recruited to chromatin in the presence of unrepaired meiotic DSBs in both female and male germlines. Furthermore, the checkpoint kinase CHK-1 is phosphorylated and the p53 ortholog CEP-1 induces expression of BH3-only proapoptotic proteins in germlines of both sexes under activating conditions. The core cell death machinery is expressed in female and male germlines; however, CED-3 caspase is not activated in the male germline. Although apoptosis is not triggered, checkpoint activation in males has functional consequences for gamete quality, because there is reduced viability of progeny sired by males with a checkpoint-activating defect in the absence of checkpoint function.ConclusionsWe propose that the recombination checkpoint functions in male germ cells to promote repair of meiotic recombination intermediates, thereby improving the fidelity of chromosome transmission in the absence of apoptosis

Acidic nucleoplasmic DNA-binding protein (And-1) controls chromosome congression by regulating the assembly of centromere protein A (CENP-A) at centromeres

Background: The incorporation of CENP-A at centromeres is important for chromosome segregation during mitosis. Results: And-1 together with HJURP regulates the assembly of new CENP-A onto centromeres. Conclusion: And-1 facilitates the recruitment of CENP-A to centromeres. Significance: These studies reveal a novel role of And-1 in the regulation of chromosome congression during mitosis

And-1 is required for the stability of histone acetyltransferase Gcn5.

Histone acetyltransferases (HATs) have a central role in the modification of chromatin as well as in the pathogenesis of a broad set of diseases including cancers. Gcn5 is the first identified transcription-related HAT that has been implicated in the regulation of diverse cellular functions. However, how Gcn5 proteins are regulated remains largely unknown. Here we show that acidic nucleoplasmic DNA-binding protein (And-1, a high mobility group domain-containing protein) has remarkable capability to regulate the stability of Gcn5 proteins and thereby histone H3 acetylation. We find that And-1 forms a complex with both histone H3 and Gcn5. Downregulation of And-1 results in Gcn5 degradation, leading to the reduction of H3K9 and H3K56 acetylation. And-1 overexpression stabilizes Gcn5 through protein-protein interactions in vivo. Furthermore, And-1 expression is increased in cancer cells in a manner correlating with increased Gcn5 and H3K9Ac and H3K56Ac. Thus, our data reveal not only a functional link between Gcn5 and And-1 that is essential for Gcn5 protein stability and histone H3 acetylation, but also a potential role of And-1 in cancer. © 2012 Macmillan Publishers Limited All rights reserved

Identification of Suppressors of top-2 Embryonic Lethality in Caenorhabditis elegans

Topoisomerase II is an enzyme with important roles in chromosome biology. This enzyme relieves supercoiling and DNA and RNA entanglements generated during mitosis. Recent studies have demonstrated that Topoisomerase II is also involved in the segregation of homologous chromosomes during the first meiotic division. However, the function and regulation of Topoisomerase II in meiosis has not been fully elucidated. Here, we conducted a genetic suppressor screen in Caenorhabditis elegans to identify putative genes that interact with topoisomerase II during meiosis. Using a temperature-sensitive allele of topoisomerase II, top-2(it7ts), we identified eleven suppressors of top-2-induced embryonic lethality. We used whole-genome sequencing and a combination of RNAi and CRISPR/Cas9 genome editing to identify and validate the responsible suppressor mutations. We found both recessive and dominant suppressing mutations that include one intragenic and 10 extragenic loci. The extragenic suppressors consist of a known Topoisomerase II-interacting protein and two novel interactors. We anticipate that further analysis of these suppressing mutations will provide new insights into the function of Topoisomerase II during meiosis

Acidic Nucleoplasmic DNA-binding Protein (And-1) Controls Chromosome Congression by Regulating the Assembly of Centromere Protein A (CENP-A) at Centromeres

The centromere is an epigenetically designated chromatin domain that is essential for the accurate segregation of chromosomes during mitosis. The incorporation of centromere protein A (CENP-A) into chromatin is fundamental in defining the centromeric loci. Newly synthesized CENP-A is loaded at centromeres in early G(1) phase by the CENP-A-specific histone chaperone Holliday junction recognition protein (HJURP) coupled with other chromatin assembly factors. However, it is unknown whether there are additional HJURP-interacting factor(s) involving in this process. Here we identify acidic nucleoplasmic DNA-binding protein 1 (And-1) as a new factor that is required for the assembly of CENP-A nucleosomes. And-1 interacts with both CENP-A and HJURP in a prenucleosomal complex, and the association of And-1 with CENP-A is increased during the cell cycle transition from mitosis to G(1) phase. And-1 down-regulation significantly compromises chromosome congression and the deposition of HJURP-CENP-A complexes at centromeres. Consistently, overexpression of And-1 enhances the assembly of CENP-A at centromeres. We conclude that And-1 is an important factor that functions together with HJURP to facilitate the cell cycle-specific recruitment of CENP-A to centromeres