Chromatin profiling using targeted DNA adenine methyltransferase

Chromatin is the highly complex structure consisting of DNA and hundreds of associated proteins. Most chromatin proteins exert their regulatory and structural functions by binding to specific chromosomal loci. Knowledge of the identity of these in vivo target loci is essential for the understanding of the functions and mechanisms of action of chromatin proteins. We report here large-scale mapping of in vivo binding sites of chromatin proteins, using a novel approach based on a combination of targeted DNA methylation and microarray technology. We show that three distinct chromatin proteins in Drosophila melanogaster cells each associate with specific sets of genes. HP1 binds predominantly to pericentric genes and transposable elements. GAGA factor associates with euchromatic genes that are enriched in (GA)n motifs. A Drosophila homolog of Saccharomyces cerevisiae Sir2p is associated with several active genes and is excluded from heterochromatin. High-resolution, genome-wide maps of target loci of chromatin proteins ('chromatin profiles') provide new insights into chromatin structure and gene regulation.     

Analysis of expressed sequence tags indicates 35,000 human genes

The number of protein-coding genes in an organism provides a useful first measure of its molecular complexity. Single-celled prokaryotes and eukaryotes typically have a few thousand genes; for example, Escherichia coli has 4,300 and Saccharomyces cerevisiae has 6,000. Evolution of multicellularity appears to have been accompanied by a several-fold increase in gene number, the invertebrates Caenorhabditis elegans and Drosophila melanogaster having 19,000 and 13,600 genes, respectively. Here we estimate the number of human genes by comparing a set of human expressed sequence tag (EST) contigs with human chromosome 22 and with a non-redundant set of mRNA sequences. The two comparisons give mutually consistent estimates of approximately 35,000 genes, substantially lower than most previous estimates. Evolution of the increased physiological complexity of vertebrates may therefore have depended more on the combinatorial diversification of regulatory networks or alternative splicing than on a substantial increase in gene number.     

Mitotic replication initiation proteins are not required for pre-meiotic S phase

Initiation of mitotic DNA replication in eukaryotes requires conserved factors, including Cdc18/CDC6 and minichromosome maintenance (MCM) proteins. We show here that these proteins are not essential for meiotic DNA replication or subsequent meiotic divisions in fission yeast. In addition, vegetative replication checkpoint genes are not required for the arrest of meiotic divisions in response to pre-meiotic S-phase delays. Genes essential for other aspects of vegetative DNA replication, however, including polymerases and DNA ligase, are also required for pre-meiotic DNA synthesis. Our results indicate that the process of replication initiation and checkpoint control may be fundamentally different in mitotic and meiotic cells.     

Meta-analyses identify 13 loci associated with age at menopause and highlight DNA repair and immune pathways

To newly identify loci for age at natural menopause, we carried out a meta-analysis of 22 genome-wide association studies (GWAS) in 38,968 women of European descent, with replication in up to 14,435 women. In addition to four known loci, we identified 13 loci newly associated with age at natural menopause (at P < 5 × 10(-8)). Candidate genes located at these newly associated loci include genes implicated in DNA repair (EXO1, HELQ, UIMC1, FAM175A, FANCI, TLK1, POLG and PRIM1) and immune function (IL11, NLRP11 and PRRC2A (also known as BAT2)). Gene-set enrichment pathway analyses using the full GWAS data set identified exoDNase, NF-κB signaling and mitochondrial dysfunction as biological processes related to timing of menopause.     

Efficient and accurate replication in the presence of 7,8-dihydro-8-oxoguanine by DNA polymerase eta

Oxidative damage to DNA has been proposed to have a role in cancer and ageing. Oxygen-free radicals formed during normal aerobic cellular metabolism attack bases in DNA, and 7, 8-dihydro-8-oxoguanine (8-oxoG) is one of the adducts formed. Eukaryotic replicative DNA polymerases replicate DNA containing 8-oxoG by inserting an adenine opposite the lesion; consequently, 8-oxoG is highly mutagenic and causes G:C to T:A transversions. Genetic studies in yeast have indicated a role for mismatch repair in minimizing the incidence of these mutations. In Saccharomyces cerevisiae, deletion of OGG1, encoding a DNA glycosylase that functions in the removal of 8-oxoG when paired with C, causes an increase in the rate of G:C to T:A transversions. The ogg1Delta msh2Delta double mutant displays a higher rate of CAN1S to can1r forward mutations than the ogg1Delta or msh2Delta single mutants, and this enhanced mutagenesis is primarily due to G:C to T:A transversions. The gene RAD30 of S. cerevisiae encodes a DNA polymerase, Poleta, that efficiently replicates DNA containing a cis-syn thymine-thymine (T-T) dimer by inserting two adenines across from the dimer. In humans, mutations in the yeast RAD30 counterpart, POLH, cause the variant form of xeroderma pigmentosum (XP-V), and XP-V individuals suffer from a high incidence of sunlight-induced skin cancers. Here we show that yeast and human POLeta replicate DNA containing 8-oxoG efficiently and accurately by inserting a cytosine across from the lesion and by proficiently extending from this base pair. Consistent with these biochemical studies, a synergistic increase in the rate of spontaneous mutations occurs in the absence of POLeta in the yeast ogg1Delta mutant. Our results suggest an additional role for Poleta in the prevention of internal cancers in humans that would otherwise result from the mutagenic replication of 8-oxoG in DNA.     

Gross chromosomal rearrangements in Saccharomyces cerevisiae replication and recombination defective mutants.

Cancer progression is often associated with the accumulation of gross chromosomal rearrangements (GCRs), such as translocations, deletion of a chromosome arm, interstitial deletions or inversions. In many instances, GCRs inactivate tumour-suppressor genes or generate novel fusion proteins that initiate carcinogenesis. The mechanism underlying GCR formation appears to involve interactions between DNA sequences of little or no homology. We previously demonstrated that mutations in the gene encoding the largest subunit of the Saccharomyces cerevisiae single-stranded DNA binding protein (RFA1) increase microhomology-mediated GCR formation. To further our understanding of GCR formation, we have developed a novel mutator assay in S. cerevisiae that allows specific detection of such events. In this assay, the rate of GCR formation was increased 600-5, 000-fold by mutations in RFA1, RAD27, MRE11, XRS2 and RAD50, but was minimally affected by mutations in RAD51, RAD54, RAD57, YKU70, YKU80, LIG4 and POL30. Genetic analysis of these mutants suggested that at least three distinct pathways can suppress GCRs: two that suppress microhomology-mediated GCRs (RFA1 and RAD27) and one that suppresses non-homology-mediated GCRs (RAD50/MRE11/XRS2).     

Regulatory element detection using correlation with expression

We present here a new computational method for discovering cis-regulatory elements that circumvents the need to cluster genes based on their expression profiles. Based on a model in which upstream motifs contribute additively to the log-expression level of a gene, this method requires a single genome-wide set of expression ratios and the upstream sequence for each gene, and outputs statistically significant motifs. Analysis of publicly available expression data for Saccharomyces cerevisiae reveals several new putative regulatory elements, some of which plausibly control the early, transient induction of genes during sporulation. Known motifs generally have high statistical significance.     

An ACF1-ISWI chromatin-remodeling complex is required for DNA replication through heterochromatin

The mechanism by which the eukaryotic DNA-replication machinery penetrates condensed chromatin structures to replicate the underlying DNA is poorly understood. Here we provide evidence that an ACF1-ISWI chromatin-remodeling complex is required for replication through heterochromatin in mammalian cells. ACF1 (ATP-utilizing chromatin assembly and remodeling factor 1) and an ISWI isoform, SNF2H (sucrose nonfermenting-2 homolog), become specifically enriched in replicating pericentromeric heterochromatin. RNAi-mediated depletion of ACF1 specifically impairs the replication of pericentromeric heterochromatin. Accordingly, depletion of ACF1 causes a delay in cell-cycle progression through the late stages of S phase. In vivo depletion of SNF2H slows the progression of DNA replication throughout S phase, indicating a functional overlap with ACF1. Decondensing the heterochromatin with 5-aza-2-deoxycytidine reverses the effects of ACF1 and SNF2H depletion. Expression of an ACF1 mutant that cannot interact with SNF2H also interferes with replication of condensed chromatin. Our data suggest that an ACF1-SNF2H complex is part of a dedicated mechanism that enables DNA replication through highly condensed regions of chromatin.     

Common variation in GPC5 is associated with acquired nephrotic syndrome

Severe proteinuria is a defining factor of nephrotic syndrome irrespective of the etiology. Investigation of congenital nephrotic syndrome has shown that dysfunction of glomerular epithelial cells (podocytes) plays a crucial role in this disease. Acquired nephrotic syndrome is also assumed to be associated with podocyte injury. Here we identify an association between variants in GPC5, encoding glypican-5, and acquired nephrotic syndrome through a genome-wide association study and replication analysis (P value under a recessive model (P(rec)) = 6.0 × 10(-11), odds ratio = 2.54). We show that GPC5 is expressed in podocytes and that the risk genotype is associated with higher expression. We further show that podocyte-specific knockdown and systemic short interfering RNA injection confers resistance to podocyte injury in mouse models of nephrosis. This study identifies GPC5 as a new susceptibility gene for nephrotic syndrome and implicates GPC5 as a promising therapeutic target for reducing podocyte vulnerability in glomerular disease.     

RPA regulates telomerase action by providing Est1p access to chromosome ends

Replication protein A (RPA) is a highly conserved single-stranded DNA-binding protein involved in DNA replication, recombination and repair. We show here that RPA is present at the telomeres of the budding yeast Saccharomyces cerevisiae, with a maximal association in S phase. A truncation of the N-terminal region of Rfa2p (associated with the rfa2Delta40 mutated allele) results in severe telomere shortening caused by a defect in the in vivo regulation of telomerase activity. Cells carrying rfa2Delta40 show impaired binding of the protein Est1p, which is required for telomerase action. In addition, normal telomere length can be restored by expressing a Cdc13-Est1p hybrid protein. These findings indicate that RPA activates telomerase by loading Est1p onto telomeres during S phase. We propose a model of in vivo telomerase action that involves synergistic action of RPA and Cdc13p at the G-rich 3' overhang of telomeric DNA.     

Robustness against mutations in genetic networks of yeast

There are two principal mechanisms that are responsible for the ability of an organism's physiological and developmental processes to compensate for mutations. In the first, genes have overlapping functions, and loss-of-function mutations in one gene will have little phenotypic effect if there are one or more additional genes with similar functions. The second mechanism has its origin in interactions between genes with unrelated functions, and has been documented in metabolic and regulatory gene networks. Here I analyse, on a genome-wide scale, which of these mechanisms of robustness against mutations is more prevalent. I used functional genomics data from the yeast Saccharomyces cerevisiae to test hypotheses related to the following: if gene duplications are mostly responsible for robustness, then a correlation is expected between the similarity of two duplicated genes and the effect of mutations in one of these genes. My results demonstrate that interactions among unrelated genes are the major cause of robustness against mutations. This type of robustness is probably an evolved response of genetic networks to stabilizing selection.