h2av

UNLABELLED: When replication forks encounter damaged DNA, cells utilize DNA damage tolerance mechanisms to allow replication to proceed. These include translesion synthesis at the fork, postreplication gap filling, and template switching via fork reversal or homologous recombination. The extent to which these different damage tolerance mechanisms are utilized depends on cell, tissue, and developmental context-specific cues, the last two of which are poorly understood. To address this gap, we have investigated damage tolerance responses following alkylation damage in Drosophila melanogaster ...

PARP-1 participates in various cellular processes, including gene regulation. In Drosophila , PARP-1 mutants undergo developmental arrest during larval-to-pupal transition. In this study, we investigated PARP-1 binding and its transcriptional regulatory role at this stage. Our findings revealed that PARP-1 binds and represses active metabolic genes, including glycolytic genes, whereas activating low-expression developmental genes, including a subset of "bivalent" genes in third-instar larvae. These bivalent promoters, characterized by dual enrichment of low H3K4me3 and high H3K27me3, a unimodal H3K4me1 enrichment at the transcription start site (conserved in C...

DNA damage response (DDR) pathways in keto-enol genotoxicity have not been characterized, and few studies have reported genotoxic effects in non-target organisms. The present study shows that concentrations of 11.2, 22.4, 37.3 mg/L of Movento® 240SC and 12.3, 24.6, 41.1 mg/L of Envidor® 240SC for 72 h oral exposure induced DSBs by significantly increasing the percentage of γH2AV expression in regions 2b and 3 from the germarium of wild type females of Drosophila melanogaster Oregon R, compared to the control group (0...

Nuclear markers for live imaging are useful for counting and tracking cells, visualizing cell division, and examining the regulation of proteins that are controlled via entry or exit from the nucleus. Near-infrared fluorescent proteins have advantages over shorter wavelength fluorescent proteins, including reduced phototoxicity, less light scattering, and enabling multicolor live imaging. We have constructed and tested transgenic Drosophila expressing Histone H2Av iRFP fusion proteins, and confirmed that they can be used to label nuclei in both fixed and live tissue at multiple stages of development...

Chromatin insulators are responsible for orchestrating long-range interactions between enhancers and promoters throughout the genome and align with the boundaries of Topologically Associating Domains (TADs). Here, we demonstrate an association between gypsy insulator proteins and the phosphorylated histone variant H2Av (γH2Av), normally a marker of DNA double strand breaks. Gypsy insulator components colocalize with γH2Av throughout the genome, in polytene chromosomes and in diploid cells in which Chromatin IP data shows it is enriched at TAD boundaries...

Programmed Cell Death (PCD) or apoptosis is a highly conserved biological process and plays essential roles both in the development and stress context. In Drosophila, expression of pro-apoptotic genes, including reaper (rpr), head involution defective (hid), grim, and sickle (skl), is sufficient to induce cell death. Here, we demonstrate that the chromatin remodeler Dmp18, the homolog of mammalian Znhit1, plays a crucial role in regulating apoptosis in eye and wing development. We showed that loss of Dmp18 disrupted eye and wing development, up-regulated transcription of pro-apoptotic genes, and induced apoptosis...

Insects depend on the innate immune response for defense against a wide array of pathogens. Central to Drosophila immunity are antimicrobial peptides (AMPs), released into circulation when pathogens trigger either of the two widely studied signal pathways, Toll or IMD. The Toll pathway responds to infection by Gram-positive bacteria and fungi while the IMD pathway is activated by Gram-negative bacteria. During activation of the IMD pathway, the NF-κB-like transcription factor Relish is phosphorylated and then cleaved, which is crucial for IMD-dependent AMP gene induction...

Because both dearth and overabundance of histones result in cellular defects, histone synthesis and demand are typically tightly coupled. In Drosophila embryos, histones H2B, H2A and H2Av accumulate on lipid droplets (LDs), which are cytoplasmic fat storage organelles. Without LD binding, maternally provided H2B, H2A and H2Av are absent; however, how LDs ensure histone storage is unclear. Using quantitative imaging, we uncover when during oogenesis these histones accumulate, and which step of accumulation is LD dependent...

Because both dearth and overabundance of histones result in cellular defects, histone synthesis and demand are typically tightly coupled. In Drosophila embryos, histones H2B/H2A/H2Av accumulate on lipid droplets (LDs), cytoplasmic fat storage organelles. Without LD-binding, maternally provided H2B/H2A/H2Av are absent, but how LDs ensure histone storage is unclear. Using quantitative imaging, we uncover when during oogenesis these histones accumulate, and which step of accumulation is LD-dependent. LDs originate in nurse cells (NCs) and are transported to the oocyte...

Estrogen exposure has already been considered to be associated with tumorigenesis and breast cancer progression. To study the epigenetic regulation mechanism in MCF-7 cells under estrogen exposure, which normally results in cell proliferation and malignancy, a stable isotope labeling of amino acid (SILAC) based quantitative proteomics strategy was used to analyse histone post-translational modifications (PTMs) and protein differential expressions. In total, we have unambiguously identified 49 histone variants and quantified 42 of them, in which two differentially expressed proteins were found to be associated with breast cancers...

Several oncogenes induce untimely entry into S phase and alter replication timing and progression, thereby generating replicative stress, a well-known source of genomic instability and a hallmark of cancer. Using an epithelial model in Drosophila, we show that the RAS oncogene, which triggers G1/S transition, induces DNA damage and, at the same time, silences the DNA damage response pathway. RAS compromises ATR-mediated phosphorylation of the histone variant H2Av and ATR-mediated cell-cycle arrest in G2 and blocks, through ERK, Dp53-dependent induction of cell death...

Aurora-A is a conserved mitotic kinase overexpressed in many types of cancer. Growing evidence shows that Aurora-A plays a crucial role in DNA damage response (DDR) although this aspect has been less characterized. We isolated a new aur-A mutation, named aur-A949 , in Drosophila, and we showed that it causes chromosome aberrations (CABs). In addition, aur-A949 mutants were sensitive to X-ray treatment and showed impaired γ-H2Av foci dissolution kinetics. To identify the pathway in which Aur-A works, we conducted an epistasis analysis by evaluating CAB frequencies in double mutants carrying aur-A949 mutation combined to mutations in genes related to DNA damage response (DDR)...

Regulating nuclear histone balance is essential for survival, yet in early Drosophila melanogaster embryos many regulatory strategies employed in somatic cells are unavailable. Previous work had suggested that lipid droplets (LDs) buffer nuclear accumulation of the histone variant H2Av. Here, we elucidate the buffering mechanism and demonstrate that it is developmentally controlled. Using live imaging, we find that H2Av continuously exchanges between LDs. Our data suggest that the major driving force for H2Av accumulation in nuclei is H2Av abundance in the cytoplasm and that LD binding slows nuclear import kinetically, by limiting this cytoplasmic pool...

A model has been proposed in which JIL-1 kinase-mediated H3S10 and H2Av phosphorylation is required for transcriptional elongation and heat shock-induced chromatin decondensation. However, here we show that although H3S10 phosphorylation is indeed compromised in the H2Av null mutant, chromatin decondensation at heat shock loci is unaffected in the absence of JIL-1 as well as of H2Av and that there is no discernable decrease in the elongating form of RNA polymerase II in either mutant. Furthermore, mRNA for the major heat shock protein Hsp70 is transcribed at robust levels in both H2Av and JIL-1 null mutants...

In Drosophila melanogaster, DNA double-strand breaks (DSBs) created by exposure to gamma or X-ray radiation can be quantified by immunofluorescent detection of phosphorylated histone H2Av (γ-H2Av) foci in imaginal disc tissues. This technique has been less useful for studying DSBs in imaginal discs exposed to DSB-inducing chemicals, since standard protocols require raising larvae in food treated with liquid chemical suspensions. These protocols typically take 3-4 days to complete and result in heterogeneous responses that do not provide information about the kinetics of DSB formation and repair...

Replication-independent histone variants can replace the canonical replication-dependent histones. Vertebrates have multiple H2A variant histones, including H2AZ and H2AX that are present in most eukaryotes. H2AZ regulates transcriptional activation as well as the maintenance of gene silencing, while H2AX is important in DNA damage repair. The fruit fly Drosophila melanogaster has only one histone H2A variant (H2AV), which is a chimera of H2AZ and H2AX. In this study we found that lack of H2AV led to the formation of black melanotic masses in Drosophila third instar larvae...

Piwi-interacting RNAs (piRNAs) are 26-30-nucleotide germ line-specific small non-coding RNAs that have evolutionarily conserved function in mobile genetic element (transposons) silencing and maintenance of genome integrity. Drosophila Hsp70/90-organizing protein homolog (Hop), a co-chaperone, interacts with piRNA-binding protein Piwi and mediates silencing of phenotypic variations. However, it is not known whether Hop has a direct role in piRNA biogenesis and transposon silencing. Here, we show that knockdown of Hop in the germ line nurse cells (GLKD) of Drosophila ovaries leads to activation of transposons...

The protein phosphatase 2A (PP2A) is a conserved heterotrimeric enzyme that regulates several cellular processes including the DNA damage response and mitosis. Consistent with these functions, PP2A is mutated in many types of cancer and acts as a tumor suppressor. In mammalian cells, PP2A inhibition results in DNA double strand breaks (DSBs) and chromosome aberrations (CABs). However, the mechanisms through which PP2A prevents DNA damage are still unclear. Here, we focus on the role of the Drosophila twins ( tws ) gene in the maintenance of chromosome integrity; tws encodes the B regulatory subunit (B/B55) of PP2A...

Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) has been instrumental in inferring the roles of histone post-translational modifications in the regulation of transcription, chromatin compaction and other cellular processes that require modulation of chromatin structure. However, analysis of ChIP-seq data is challenging when the manipulation of a chromatin-modifying enzyme significantly affects global levels of histone post-translational modifications. For example, small molecule inhibition of the methyltransferase EZH2 reduces global levels of histone H3 lysine 27 trimethylation (H3K27me3)...

The histone locus body (HLB) assembles at replication-dependent histone genes and concentrates factors required for histone messenger RNA (mRNA) biosynthesis. FLASH (Flice-associated huge protein) and U7 small nuclear RNP (snRNP) are HLB components that participate in 3' processing of the nonpolyadenylated histone mRNAs by recruiting the endonuclease CPSF-73 to histone pre-mRNA. Using transgenes to complement a FLASH mutant, we show that distinct domains of FLASH involved in U7 snRNP binding, histone pre-mRNA cleavage, and HLB localization are all required for proper FLASH function in vivo...