Distinct roles for RSC and SWI/SNF chromatin remodelers in genomic excision repair.

Nucleosomes are a significant barrier to the repair of UV damage because they impede damage recognition by nucleotide excision repair (NER). The RSC and SWI/SNF chromatin remodelers function in cells to promote DNA access by moving or evicting nucleosomes, and both have been linked to NER in yeast. Here, we report genome-wide repair maps of UV-induced cyclobutane pyrimidine dimers (CPDs) in yeast cells lacking RSC or SWI/SNF activity. Our data indicate that SWI/SNF is not generally required for NER but instead promotes repair of CPD lesions at specific yeast genes. In contrast, mutation or depletion of RSC subunits causes a general defect in NER across the yeast genome. Our data indicate that RSC is required for repair not only in nucleosomal DNA but also in neighboring linker DNA and nucleosome-free regions (NFRs). Although depletion of the RSC catalytic subunit also affects base excision repair (BER) of N-methylpurine (NMP) lesions, RSC activity is less important for BER in linker DNA and NFRs. Furthermore, our data indicate that RSC plays a direct role in transcription-coupled NER (TC-NER) of transcribed DNA. These findings help to define the specific genomic and chromatin contexts in which each chromatin remodeler functions in DNA repair, and indicate that RSC plays a unique function in facilitating repair by both NER subpathways.

Evolution of Ty1 copy number control in yeast by horizontal transfer and recombination.

Transposable elements constitute a large fraction of most eukaryotic genomes. Insertion of mobile DNA sequences typically has deleterious effects on host fitness, and thus diverse mechanisms have evolved to control mobile element proliferation. Mobility of the Ty1 retrotransposon in Saccharomyces yeasts is regulated by copy number control (CNC) mediated by a self-encoded restriction factor derived from the Ty1 gag capsid gene that inhibits virus-like particle function. Here, we survey a panel of wild and human-associated strains of S. cerevisiae and S. paradoxus to investigate how genomic Ty1 content influences variation in Ty1 mobility. We observe high levels of mobility for a tester element with a gag sequence from the canonical Ty1 subfamily in permissive strains that either lack full-length Ty1 elements or only contain full-length copies of the Ty1' subfamily that have a divergent gag sequence. In contrast, low levels of canonical Ty1 mobility are observed in restrictive strains carrying full-length Ty1 elements containing a canonical gag sequence. Phylogenomic analysis of full-length Ty1 elements revealed that Ty1' is the ancestral subfamily present in wild strains of S. cerevisiae, and that canonical Ty1 in S. cerevisiae is a derived subfamily that acquired gag from S. paradoxus by horizontal transfer and recombination. Our results provide evidence that variation in the ability of S. cerevisiae and S. paradoxus strains to repress canonical Ty1 transposition via CNC is regulated by the genomic content of different Ty1 subfamilies, and that self-encoded forms of transposon control can spread across species boundaries by horizontal transfer.

The Chromatin Remodeler HELLS: A New Regulator in DNA Repair, Genome Maintenance, and Cancer.

Robust, tightly regulated DNA repair is critical to maintaining genome stability and preventing cancer. Eukaryotic DNA is packaged into chromatin, which has a profound, yet incompletely understood, regulatory influence on DNA repair and genome stability. The chromatin remodeler HELLS (helicase, lymphoid specific) has emerged as an important epigenetic regulator of DNA repair, genome stability, and multiple cancer-associated pathways. HELLS belongs to a subfamily of the conserved SNF2 ATP-dependent chromatin-remodeling complexes, which use energy from ATP hydrolysis to alter nucleosome structure and packaging of chromatin during the processes of DNA replication, transcription, and repair. The mouse homologue, LSH (lymphoid-specific helicase), plays an important role in the maintenance of heterochromatin and genome-wide DNA methylation, and is crucial in embryonic development, gametogenesis, and maturation of the immune system. Human HELLS is abundantly expressed in highly proliferating cells of the lymphoid tissue, skin, germ cells, and embryonic stem cells. Mutations in HELLS cause the human immunodeficiency syndrome ICF (Immunodeficiency, Centromeric instability, Facial anomalies). HELLS has been implicated in many types of cancer, including retinoblastoma, colorectal cancer, hepatocellular carcinoma, and glioblastoma. Here, we review and summarize accumulating evidence highlighting important roles for HELLS in DNA repair, genome maintenance, and key pathways relevant to cancer development, progression, and treatment.

Myostatin regulates pituitary development and hepatic IGF1.

Circulating myostatin-attenuating agents are being developed to treat muscle-wasting disease despite their potential to produce serious off-target effects, as myostatin/activin receptors are widely distributed among many nonmuscle tissues. Our studies suggest that the myokine not only inhibits striated muscle growth but also regulates pituitary development and growth hormone (GH) action in the liver. Using a novel myostatin-null label-retaining model (Jekyll mice), we determined that the heterogeneous pool of pituitary stem, transit-amplifying, and progenitor cells in Jekyll mice depletes more rapidly after birth than the pool in wild-type mice. This correlated with increased levels of GH, prolactin, and the cells that secrete these hormones, somatotropes and lactotropes, respectively, in Jekyll pituitaries. Recombinant myostatin also stimulated GH release and gene expression in pituitary cell cultures although inhibiting prolactin release. In primary hepatocytes, recombinant myostatin blocked GH-stimulated expression of two key mediators of growth, insulin-like growth factor (IGF)1 and the acid labile subunit and increased expression of an inhibitor, IGF-binding protein-1. The significance of these findings was demonstrated by smaller muscle fiber size in a model lacking myostatin and liver IGF1 expression (LID-o-Mighty mice) compared with that in myostatin-null (Mighty) mice. These data together suggest that myostatin may regulate pituitary development and function and that its inhibitory actions in muscle may be partly mediated by attenuating GH action in the liver. They also suggest that circulating pharmacological inhibitors of myostatin could produce unintended consequences in these and possibly other tissues.

Fyn-mediated phosphorylation of Menin disrupts telomere maintenance in stem cells.

Telomeres protect chromosome ends and determine the replication potential of dividing cells. The canonical telomere sequence TTAGGG is synthesized by telomerase holoenzyme, which maintains telomere length in proliferative stem cells. Although the core components of telomerase are well-defined, mechanisms of telomerase regulation are still under investigation. We report a novel role for the Src family kinase Fyn, which disrupts telomere maintenance in stem cells by phosphorylating the scaffold protein Menin. We found that Fyn knockdown prevented telomere erosion in human and mouse stem cells, validating the results with four telomere measurement techniques. We show that Fyn phosphorylates Menin at tyrosine 603 (Y603), which increases Menin's SUMO1 modification, C-terminal stability, and importantly, its association with the telomerase RNA component (TR). Using mass spectrometry, immunoprecipitation, and immunofluorescence experiments we found that SUMO1-Menin decreases TR's association with telomerase subunit Dyskerin, suggesting that Fyn's phosphorylation of Menin induces telomerase subunit mislocalization and may compromise telomerase function at telomeres. Importantly, we find that Fyn inhibition reduces accelerated telomere shortening in human iPSCs harboring mutations for dyskeratosis congenita.

The emerging roles of ATP-dependent chromatin remodeling enzymes in nucleotide excision repair.

DNA repair in eukaryotic cells takes place in the context of chromatin, where DNA, including damaged DNA, is tightly packed into nucleosomes and higher order chromatin structures. Chromatin intrinsically restricts accessibility of DNA repair proteins to the damaged DNA and impacts upon the overall rate of DNA repair. Chromatin is highly responsive to DNA damage and undergoes specific remodeling to facilitate DNA repair. How damaged DNA is accessed, repaired and restored to the original chromatin state, and how chromatin remodeling coordinates these processes in vivo, remains largely unknown. ATP-dependent chromatin remodelers (ACRs) are the master regulators of chromatin structure and dynamics. Conserved from yeast to humans, ACRs utilize the energy of ATP to reorganize packing of chromatin and control DNA accessibility by sliding, ejecting or restructuring nucleosomes. Several studies have demonstrated that ATP-dependent remodeling activity of ACRs plays important roles in coordination of spatio-temporal steps of different DNA repair pathways in chromatin. This review focuses on the role of ACRs in regulation of various aspects of nucleotide excision repair (NER) in the context of chromatin. We discuss current understanding of ATP-dependent chromatin remodeling by various subfamilies of remodelers and regulation of the NER pathway in vivo.

Versatile cell-based assay for measuring DNA alkylation damage and its repair.

DNA alkylation damage induced by environmental carcinogens, chemotherapy drugs, or endogenous metabolites plays a central role in mutagenesis, carcinogenesis, and cancer therapy. Base excision repair (BER) is a conserved, front line DNA repair pathway that removes alkylation damage from DNA. The capacity of BER to repair DNA alkylation varies markedly between different cell types and tissues, which correlates with cancer risk and cellular responses to alkylation chemotherapy. The ability to measure cellular rates of alkylation damage repair by the BER pathway is critically important for better understanding of the fundamental processes involved in carcinogenesis, and also to advance development of new therapeutic strategies. Methods for assessing the rates of alkylation damage and repair, especially in human cells, are limited, prone to significant variability due to the unstable nature of some of the alkyl adducts, and often rely on indirect measurements of BER activity. Here, we report a highly reproducible and quantitative, cell-based assay, named alk-BER (alkylation Base Excision Repair) for measuring rates of BER following alkylation DNA damage. The alk-BER assay involves specific detection of methyl DNA adducts (7-methyl guanine and 3-methyl adenine) directly in genomic DNA. The assay has been developed and adapted to measure the activity of BER in fungal model systems and human cell lines. Considering the specificity and conserved nature of BER enzymes, the assay can be adapted to virtually any type of cultured cells. Alk-BER offers a cost efficient and reliable method that can effectively complement existing approaches to advance integrative research on mechanisms of alkylation DNA damage and repair.

Facilitation of base excision repair by chromatin remodeling.

Base Excision Repair (BER) is a conserved, intracellular DNA repair system that recognizes and removes chemically modified bases to insure genomic integrity and prevent mutagenesis. Aberrant BER has been tightly linked with a broad spectrum of human pathologies, such as several types of cancer, neurological degeneration, developmental abnormalities, immune dysfunction and aging. In the cell, BER must recognize and remove DNA lesions from the tightly condensed, protein-coated chromatin. Because chromatin is necessarily refractory to DNA metabolic processes, like transcription and replication, the compaction of the genomic material is also inhibitory to the repair systems necessary for its upkeep. Multiple ATP-dependent chromatin remodelling (ACR) complexes play essential roles in modulating the protein-DNA interactions within chromatin, regulating transcription and promoting activities of some DNA repair systems, including double-strand break repair and nucleotide excision repair. However, it remains unclear how BER operates in the context of chromatin, and if the chromatin remodelling processes that govern transcription and replication also actively regulate the efficiency of BER. In this review we highlight the emerging role of ACR in regulation of BER.

Chromatin remodelling complex RSC promotes base excision repair in chromatin of Saccharomyces cerevisiae.

The base excision repair (BER) pathway is a conserved DNA repair system required to maintain genomic integrity and prevent mutagenesis in all eukaryotic cells. Nevertheless, how BER operates in vivo (i.e. in the context of chromatin) is poorly understood. We have investigated the role of an essential ATP-dependent chromatin remodelling (ACR) complex RSC (Remodels the Structure of Chromatin) in BER of intact yeast cells. We show that depletion of STH1, the ATPase subunit of RSC, causes enhanced sensitivity to the DNA alkylating agent methyl methanesulfonate (MMS) and results in a substantial inhibition of BER, at the GAL1 locus and in the genome overall. Consistent with this observation, the DNA in chromatin is less accessible to micrococcal nuclease digestion in the absence of RSC. Quantitative PCR results indicate that repair deficiency in STH1 depleted cells is not due to changes in the expression of BER genes. Collectively, our data indicates the RSC complex promotes efficient BER in chromatin. These results provide, for the first time, a link between ATP-dependent chromatin remodelling and BER in living cells.

Structural and functional conservation of fungal MatA and human SRY sex-determining proteins.

Sex determination in animals and fungi is regulated by specific sex-determining genes. The Aspergillus nidulans mating type gene matA and the human SRY (Sex-Determining Region Y) encode proteins containing a single HMG (high-mobility group) domain. Analysis of the amino-acid sequence of MatA and SRY transcription factors revealed significant structural similarity. The human SRY protein is able to functionally replace MatA and drives the sexual cycle in the fungus A. nidulans. Functional studies indicate that SRY drives early fruiting body development, and hybrid MatA protein carrying the SRY HMG box is fully capable of driving both early and late stages of sexual development, including gametogenesis. Our data suggest that SRY and MatA are both structurally and functionally related and conserved in regulating sexual processes. The fundamental mechanisms driving evolution of the genetic pathways underlying sex determination, sex chromosomes and sexual reproduction in eukaryotes appear similar.

Novel sexual-cycle-specific gene silencing in Aspergillus nidulans.

We report a novel sexual-cycle-specific gene-silencing system in the genetic model Aspergillus nidulans. Duplication of the mating type matA(HMG) gene in this haploid organism triggers Mat-induced silencing (MatIS) of both endogenous and transgenic matA genes, eliminates function of the encoded SRY structural ortholog, and results in formation of barren fruiting bodies. MatIS is spatiotemporally restricted to the prezygotic stage of the sexual cycle and does not interfere with vegetative growth, asexual reproduction, differentiation of early sexual tissues, or fruiting body development. MatIS is reversible upon deletion of the matA transgene. In contrast to other sex-specific silencing phenomena, MatIS silencing has nearly 100% efficiency and appears to be independent of homologous duplicated DNA segments. Remarkably, transgene-derived matA RNA might be sufficient to induce MatIS. A unique feature of MatIS is that RNA-mediated silencing is RNA interference/Argonaute-independent and is restricted to the nucleus having the duplicated gene. The silencing phenomenon is recessive and does not spread between nuclei within the common cytoplasm of a multinucleate heterokaryon. Gene silencing induced by matA gene duplication emerges as a specific feature associated with matA(HMG) regulation during sexual development.

Complex mechanisms regulate developmental expression of the matA (HMG) mating type gene in homothallic Aspergillus nidulans.

Sexual reproduction is a fundamental developmental process that allows for genetic diversity through the control of zygote formation, recombination, and gametogenesis. The correct regulation of these events is paramount. Sexual reproduction in filamentous fungi, including mating strategy (self-fertilization/homothallism or outcrossing/heterothallism), is determined by the expression of mating type genes at mat loci. Aspergillus nidulans matA encodes a critical regulator that is a fungal ortholog of the hSRY/SOX9 HMG box proteins. In contrast to well-studied outcrossing systems, the molecular basis of homothallism and role of mating type genes during a self-fertile sexual cycle remain largely unknown. In this study the genetic model organism, A. nidulans, has been used to investigate the regulation and molecular functions of the matA mating type gene in a homothallic system. Our data demonstrate that complex regulatory mechanisms underlie functional matA expression during self-fertilization and sexual reproduction in A. nidulans. matA expression is suppressed in vegetative hyphae and is progressively derepressed during the sexual cycle. Elevated levels of matA transcript are required for differentiation of fruiting bodies, karyogamy, meiosis, and efficient formation of meiotic progeny. matA expression is driven from both initiator (Inr) and novel promoter elements that are tightly developmentally regulated by position-dependent and position-independent mechanisms. Deletion of an upstream silencing element, matA SE, results in derepressed expression from wild-type (wt) promoter elements and activation of an additional promoter. These studies provide novel insights into the molecular basis of homothallism in fungi and genetic regulation of sexual reproduction in eukaryotes.

Proficient repair in chromatin remodeling defective ino80 mutants of Saccharomyces cerevisiae highlights replication defects as the main contributor to DNA damage sensitivity.

Ino80 is an evolutionarily conserved member of the SWI2/SNF2-family of ATPases in Saccharomyces cerevisiae. It resides in a multiprotein helicase/chromatin remodeling complex, and has been shown to play a key role in the stability of replication forks during replication stress. Though yeast with defects in ino80 show sensitivity to killing by a variety of DNA-damaging agents, a role for the INO80 protein complex in the repair of DNA has only been assessed for double-strand breaks, and the results are contradictory and inconclusive. We report that ino80Delta cells are hypersensitive to DNA base lesions induced by ultraviolet (UV) radiation and methyl methanesulfonate (MMS), but show little (or no) increased sensitivity to the DNA double-strand break (DSB)-inducing agents ionizing radiation and camptothecin. Importantly, ino80Delta cells display efficient removal of UV-induced cyclobutane pyrimidine dimers, and show a normal rate of removal of DNA methylation damage after MMS exposure. In addition, ino80Delta cells have an overall normal rate of repair of DSBs induced by ionizing radiation. Altogether, our data support a model of INO80 as an important suppressor of genome instability in yeast involved in DNA damage tolerance through a role in stability and recovery of broken replication forks, but not in the repair of lesions leading to such events. This conclusion is in contrast to strong evidence for the DNA repair-promoting role of the corresponding INO80 complexes in higher eukaryotes. Thus, our results provide insight into the specialized roles of the INO80 subunits and the differential needs of different species for chromatin remodeling complexes in genome maintenance.