A Transcriptional Analysis of the Human c-Ha-ras1 Oncogene

The pattern of transcription initiation for the human c-Ha-rasl gene has been investigated using several experimental methods. These analyses demonstrated multiple clusters of transcription initiation sites distributed over an approximately 200 bp non-coding, upstream exon (termed exon -1), which is separated from the ATG codon by an 1040 bp intron. Mutational analysis of the promoter region identified a short positive regulatory element, located between positions -243 to -196, relative to the donor splice site of exon -1. This element contains known regulatory sequence motifs. Furthermore, a putative negative regulatory element, with an unusual DNA sequence, was identified between positions -103 to -34, relative to the same donor splice site. It has also been demonstrated that the human c-Ha-rasl promoter region can function bidirectionally. The sequence directing the "reverse-orientation" promoter activity was located to between positions, -392 to -196, relative to the exon -1 donor splice site of the c-Ha-rasl gene. Transcription of the c-Ha-rasl gene was shown to be increased approximately 20 fold when covalently linked to the SV40 enhancer element. This result is the first direct demonstration that the SV40 enhancer can increase transcription of "housekeeping-type" genes and this result also has important implications for the possible methods of oncogenic activation of this gene. The 1040 bp intron located between exon -1 and the first coding exon (exon 1) was found to contain sequences within its 5' end, which were moderately repetitive within the human and mouse genomes, and homologous to abundantly transcribed, non-polyadenylated RNAs from various human cell lines. However, the functional significance, if any, of this result is unclear

BRCA1 is required for maintenance of phospho-Chk1 and G₂/M arrest during DNA cross-link repair in DT40 cells

The Fanconi anemia DNA repair pathway is pivotal for the efficient repair of DNA interstrand cross-links. Here, we show that FA-defective (Fancc(-)) DT40 cells arrest in G(2) phase following cross-link damage and trigger apoptosis. Strikingly, cell death was reduced in Fancc(-) cells by additional deletion of the BRCA1 tumor suppressor, resulting in elevated clonogenic survival. Increased resistance to cross-link damage was not due to loss of toxic BRCA1-mediated homologous recombination but rather through the loss of a G(2) checkpoint. This proapoptotic role also required the BRCA1-A complex member ABRAXAS (FAM175A). Finally, we show that BRCA1 promotes G(2) arrest and cell death by prolonging phosphorylation of Chk1 on serine 345 after DNA damage to sustain arrest. Our data imply that DNA-induced cross-link death in cells defective in the FA pathway is dependent on the ability of BRCA1 to prolong cell cycle arrest in G(2) phase

Regulation of the DNA Damage Response and Gene Expression by the Dot1L Histone Methyltransferase and the 53Bp1 Tumour Suppressor

Dot1L, a histone methyltransferase that targets histone H3 lysine 79 (H3K79), has been implicated in gene regulation and the DNA damage response although its functions in these processes remain poorly defined.Using the chicken DT40 model system, we generated cells in which the Dot1L gene is disrupted to examine the function and focal recruitment of the 53Bp1 DNA damage response protein. Detailed kinetic and dose response assays demonstrate that, despite the absence of H3K79 methylation demonstrated by mass spectrometry, 53Bp1 focal recruitment is not compromised in these cells. We also describe, for the first time, the phenotypes of a cell line lacking both Dot1L and 53Bp1. Dot1L⁻/⁻ and wild type cells are equally resistant to ionising radiation, whereas 53Bp1⁻/⁻/Dot1L⁻/⁻ cells display a striking DNA damage resistance phenotype. Dot1L and 53Bp1 also affect the expression of many genes. Loss of Dot1L activity dramatically alters the mRNA levels of over 1200 genes involved in diverse biological functions. These results, combined with the previously reported list of differentially expressed genes in mouse ES cells knocked down for Dot1L, demonstrates surprising cell type and species conservation of Dot1L-dependent gene expression. In 53Bp1⁻/⁻ cells, over 300 genes, many with functions in immune responses and apoptosis, were differentially expressed. To date, this is the first global analysis of gene expression in a 53Bp1-deficient cell line.Taken together, our results uncover a negative role for Dot1L and H3K79 methylation in the DNA damage response in the absence of 53Bp1. They also enlighten the roles of Dot1L and 53Bp1 in gene expression and the control of DNA double-strand repair pathways in the context of chromatin

Genetic Evidence for Single-Strand Lesions Initiating Nbs1-Dependent Homologous Recombination in Diversification of Ig V in Chicken B Lymphocytes

Homologous recombination (HR) is initiated by DNA double-strand breaks (DSB). However, it remains unclear whether single-strand lesions also initiate HR in genomic DNA. Chicken B lymphocytes diversify their Immunoglobulin (Ig) V genes through HR (Ig gene conversion) and non-templated hypermutation. Both types of Ig V diversification are initiated by AID-dependent abasic-site formation. Abasic sites stall replication, resulting in the formation of single-stranded gaps. These gaps can be filled by error-prone DNA polymerases, resulting in hypermutation. However, it is unclear whether these single-strand gaps can also initiate Ig gene conversion without being first converted to DSBs. The Mre11-Rad50-Nbs1 (MRN) complex, which produces 3′ single-strand overhangs, promotes the initiation of DSB-induced HR in yeast. We show that a DT40 line expressing only a truncated form of Nbs1 (Nbs1p70) exhibits defective HR-dependent DSB repair, and a significant reduction in the rate—though not the fidelity—of Ig gene conversion. Interestingly, this defective gene conversion was restored to wild type levels by overproduction of Escherichia coli SbcB, a 3′ to 5′ single-strand–specific exonuclease, without affecting DSB repair. Conversely, overexpression of chicken Exo1 increased the efficiency of DSB-induced gene-targeting more than 10-fold, with no effect on Ig gene conversion. These results suggest that Ig gene conversion may be initiated by single-strand gaps rather than by DSBs, and, like SbcB, the MRN complex in DT40 may convert AID-induced lesions into single-strand gaps suitable for triggering HR. In summary, Ig gene conversion and hypermutation may share a common substrate—single-stranded gaps. Genetic analysis of the two types of Ig V diversification in DT40 provides a unique opportunity to gain insight into the molecular mechanisms underlying the filling of gaps that arise as a consequence of replication blocks at abasic sites, by HR and error-prone polymerases

RNAi Screening Implicates a SKN-1-Dependent Transcriptional Response in Stress Resistance and Longevity Deriving from Translation Inhibition

Caenorhabditis elegans SKN-1 (ortholog of mammalian Nrf1/2/3) is critical for oxidative stress resistance and promotes longevity under reduced insulin/IGF-1-like signaling (IIS), dietary restriction (DR), and normal conditions. SKN-1 inducibly activates genes involved in detoxification, protein homeostasis, and other functions in response to stress. Here we used genome-scale RNA interference (RNAi) screening to identify mechanisms that prevent inappropriate SKN-1 target gene expression under non-stressed conditions. We identified 41 genes for which knockdown leads to activation of a SKN-1 target gene (gcs-1) through skn-1-dependent or other mechanisms. These genes correspond to multiple cellular processes, including mRNA translation. Inhibition of translation is known to increase longevity and stress resistance and may be important for DR-induced lifespan extension. One model postulates that these effects derive from reduced energy needs, but various observations suggest that specific longevity pathways are involved. Here we show that translation initiation factor RNAi robustly induces SKN-1 target gene transcription and confers skn-1-dependent oxidative stress resistance. The accompanying increases in longevity are mediated largely through the activities of SKN-1 and the transcription factor DAF-16 (FOXO), which is required for longevity that derives from reduced IIS. Our results indicate that the SKN-1 detoxification gene network monitors various metabolic and regulatory processes. Interference with one of these processes, translation initiation, leads to a transcriptional response whereby SKN-1 promotes stress resistance and functions together with DAF-16 to extend lifespan. This stress response may be beneficial for coping with situations that are associated with reduced protein synthesis

Correction: The RSF1 Histone-Remodelling Factor Facilitates DNA Double Strand Break Repair by Recruiting Centromeric and Fanconi Anaemia Proteins.

[This corrects the article DOI: 10.1371/journal.pbio.1001856.]

Hypoxia enhances the radioresistance of mouse mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) are radioresistant bone marrow progenitors that support hematopoiesis and its reconstitution following total body irradiation. MSCs reside in hypoxic niches within the bone marrow and tumor microenvironments. The DNA damage response (DDR) represents a network of signaling pathways that enable cells to activate biological responses to DNA damaging agents. Hypoxia-mediated alterations in the DDR contribute to the increased radioresistance of hypoxic cancer cells, limiting therapeutic efficacy. The DDR is important in mediating mouse MSC radioresistance. However, the effects of hypoxia on MSC radioresistance are currently unknown. In this report, hypoxia was found to (a) increase MSC proliferation rate and colony size; (b) increase long-term survival post-irradiation (IR), and (c) improve MSC recovery from IR-induced cell cycle arrest. DNA double-strand break (DSB) repair in MSCs was upregulated in hypoxia, accelerating the resolution of highly genotoxic IR-induced DNA DSBs. In addition, HIF-1 alpha was found to contribute to this enhanced DSB repair by regulating (a) the expression of DNA ligase IV and DNA-PKcs and (b) Rad51 foci formation in response to DNA DSBs in hypoxic MSCs. We have demonstrated, for the first time, that hypoxia enhances mouse MSC radioresistance in vitro. These findings have important implications for our understanding of MSC functions in supporting allogeneic bone marrow transplantation and in tumorigenesis