Regulation Of DNA Repair Genes Research Articles

NtcA, LexA and heptamer repeats involved in the multifaceted regulation of DNA repair genes recF, recO and recR in the cyanobacterium Nostoc PCC7120

Regulation of DNA repair genes in cyanobacteria is an unexplored field despite some of them exhibiting high radio-resistance. With RecF pathway speculated to be the major double strand break repair pathway in Nostoc sp. strain PCC7120, regulation of recF, recO and recR genes was investigated. Bioinformatic approach-based identification of promoter and regulatory elements was validated using qRT-PCR analysis, reporter gene and DNA binding assays. Different deletion constructs of the upstream regulatory regions of these genes were analysed in host Nostoc as well as heterologous system Escherichia coli. Studies revealed: (1) Positive regulation of all three genes by NtcA, (2) Negative regulation by LexA, (3) Involvement of contiguous heptamer repeats with/without its yet to be identified interacting partner in regulating (i) binding of NtcA and LexA to recO promoter and its translation, (ii) transcription or translation of recF, (4) Translational regulation of recF and recO through non-canonical and distant S.D. sequence and of recR through a rare initiation codon. Presence of NtcA either precludes binding of LexA to AnLexA-Box or negates its repressive action resulting in higher expression of these genes under nitrogen-fixing conditions in Nostoc. Thus, in Nostoc, expression of recF, recO and recR genes is intricately regulated through multiple regulatory elements/proteins. Contiguous heptamer repeats present across the Nostoc genome in the vicinity of start codon or promoter is likely to have a global regulatory role. This is the first report detailing regulation of DSB repair genes in any algae.

Coexistence of SOS-Dependent and SOS-Independent Regulation of DNA Repair Genes in Radiation-Resistant Deinococcus Bacteria.

Deinococcus bacteria are extremely resistant to radiation and able to repair a shattered genome in an essentially error-free manner after exposure to high doses of radiation or prolonged desiccation. An efficient, SOS-independent response mechanism to induce various DNA repair genes such as recA is essential for radiation resistance. This pathway, called radiation/desiccation response, is controlled by metallopeptidase IrrE and repressor DdrO that are highly conserved in Deinococcus. Among various Deinococcus species, Deinococcus radiodurans has been studied most extensively. Its genome encodes classical DNA repair proteins for error-free repair but no error-prone translesion DNA polymerases, which may suggest that absence of mutagenic lesion bypass is crucial for error-free repair of massive DNA damage. However, many other radiation-resistant Deinococcus species do possess translesion polymerases, and radiation-induced mutagenesis has been demonstrated. At least dozens of Deinococcus species contain a mutagenesis cassette, and some even two cassettes, encoding error-prone translesion polymerase DnaE2 and two other proteins, ImuY and ImuB-C, that are probable accessory factors required for DnaE2 activity. Expression of this mutagenesis cassette is under control of the SOS regulators RecA and LexA. In this paper, we review both the RecA/LexA-controlled mutagenesis and the IrrE/DdrO-controlled radiation/desiccation response in Deinococcus.

WNT inhibition creates a BRCA‐like state in Wnt‐addicted cancer

Wnt signaling maintains diverse adult stem cell compartments and is implicated in chemotherapy resistance in cancer. PORCN inhibitors that block Wnt secretion have proven effective in Wnt‐addicted preclinical cancer models and are in clinical trials. In a survey for potential combination therapies, we found that Wnt inhibition synergizes with the PARP inhibitor olaparib in Wnt‐addicted cancers. Mechanistically, we find that multiple genes in the homologous recombination and Fanconi anemia repair pathways, including BRCA1, FANCD2, and RAD51, are dependent on Wnt/β‐catenin signaling in Wnt‐high cancers, and treatment with a PORCN inhibitor creates a BRCA‐like state. This coherent regulation of DNA repair genes occurs in part via a Wnt/β‐catenin/MYBL2 axis. Importantly, this pathway also functions in intestinal crypts, where high expression of BRCA and Fanconi anemia genes is seen in intestinal stem cells, with further upregulation in Wnt‐high APCmin mutant polyps. Our findings suggest a general paradigm that Wnt/β‐catenin signaling enhances DNA repair in stem cells and cancers to maintain genomic integrity. Conversely, interventions that block Wnt signaling may sensitize cancers to radiation and other DNA damaging agents.

Chaga mushroom (Inonotus obliquus) polysaccharides exhibit genoprotective effects in UVB-exposed embryonic zebrafish (Danio rerio) through coordinated expression of DNA repair genes

Chaga mushroom is one of the promising beneficial mushrooms thriving in the colder parts of Northern hemisphere. Chaga polysaccharides (IOP) have been reported to enhance immune response and alleviate oxidative stress during development. However, the effects of IOP on the genotoxicity in model organisms are yet to be clarified. In this study, IOP was extracted using hot water extraction method, followed by GC analysis. Zebrafish embryos (12 h post fertilization, hpf) were exposed to transient UVB (12 J/m2/s, 305–310nm) for 10 s using a UV hybridisation chamber, followed by IOP treatment (2.5 mg/mL) at 24 hpf for up to 7 days post fertilization (dpf). The genotoxic effects were assessed using acridine orange staining, alkaline comet assay, and qRT-PCR for screening DNA repair genes. Significant reduction in DNA damage and amelioration of the deformed structures in the IOP-treated zebrafish exposed to UVB (p < 0.05) was observed at 5 dpf and thereafter. The relative mRNA expressions of XRCC-5, XRCC-6, RAD51, and GADD45 were significantly upregulated, whereas p53 and BAX were downregulated in IOP-treated UVB-exposed zebrafish compared to UVB-exposed zebrafish. ELISA analysis revealed significantly decreased expression of XRCC5 and RAD51 in UVB-exposed compared to IOP-treated UVB-exposed and control zebrafish (7 dpf). However, p53 and BAX levels were high in UVB-exposed zebrafish, indicating higher apoptosis. Pathway analysis demonstrated coordinated regulation of DNA repair genes; p53 playing a pivotal role in regulating the expression of BAX, thereby promoting apoptosis in UVB-exposed zebrafish. Overall, IOP treatment ameliorated the genotoxic effects in UVB-exposed zebrafish by enhanced expression of DNA repair genes, which assisted in normal development. The study delineated the efficacy of IOP in mitigating UV-induced DNA damage in zebrafish.

PF230 THE RNA HELICASE DDX17 PROTECTS ACUTE MYELOID LEUKEMIA CELLS AGAINST DNA DAMAGE

Background:Alternative splicing (AS) is essentially regulated by RNA‐binding proteins (RBPs) and is one of many deregulated cellular processes in Acute Myeloid Leukemia (AML). The alternative usage of exons substantially increases the coding potential of mRNAs and frequently leads to the expression of tumor‐promoting mRNA variants in cancer modulating apoptosis, proliferation, invasion and migration of cancer cells. This underlines the great plasticity of AS in cancer and justifies the need to elucidate the function of nuclear RBPs in specific cancer types. We found that the RNA helicase DDX17 is significantly more highly expressed in primary human AML specimens with high versus low Leukemia Stem Cell (LSC) frequency.Aims:In this study, we aimed to understand the functional role of DDX17 in normal hematopoietic stem and progenitor cells (HSPCs) and in AML cells. In particular, we sought to determine whether DDX17 exerts its function in the hematopoietic system primarily via AS or AS‐unrelated mechanisms.Methods:We performed knockdown (KD) experiments using small hairpin (sh) RNAs to determine whether DDX17 was essential for primary human cord blood CD34+ HSPCs and leukemia cells. Furthermore, mRFP tagged DDX17 was overexpressed through lentiviral transduction to track protein localization by immunofluorescence (IF) imaging. Lentivirally transduced HSPCs and leukemia cells were analyzed in vitro for proliferative output, viability, immunophenotype, and colony formation potential. RNA sequencing (RNA‐Seq) was used to determine how DDX17 KD affected gene expression and alternative exon usage. Pull down experiments combined with mass spectrometry revealed binding partners in presence and absence of chemotherapeutic agents. Finally, we performed homologous recombination (HR) and drug sensitivity assays to determine the role of DDX17 in DNA damage.Results:Knockdown of DDX17 induced DNA damage and severely impaired the function of healthy HSPCs and TP53 wild type AML cells. TP53 null cells maintained their proliferative capacity during short term in vitro expansion in absence of DDX17, but were significantly sensitized towards DNA damaging agents. In line, overexpression of DDX17 protected the cells against double strand break (DSB) inducing drugs. IF imaging revealed direct recruitment of DDX17 to DNA damage sites, while RNA‐Seq showed suppression of multiple DNA repair genes.Summary/Conclusion:RNA‐Seq, IF imaging, proteomics, and HR assays suggest an essential role of DDX17 in DNA repair via different mechanisms including direct recruitment to DNA damage sites, alternative splicing, and regulation of DNA repair genes on the transcriptional level.

Investigating the relationship between BRCA1 and BRCA2 genes methylation profile and sperm DNA fragmentation in infertile men.

The purpose of the study was to investigate whether the promoter methylation status of BRCA1 and BRCA2 DNA repair genes is associated with sperm DNA fragmentation (sDF) in infertile men with oligoasthenoteratozoospermia (OAT) which emerges due to various reasons and is effective in male infertility. Seventy-three infertile men with OAT and 20 normozoospermic volunteers participated in the study. To investigate sDF and methylation patterns of BRCA1 and BRCA2 gene promoters, TUNEL assay and methylation-specific PCR (MS-PCR) were used. The mean sDF ratio for the patients was calculated as 22.50%. The calculated cut-off value for sDF ratio was 17.0% in ROC curve analysis. Regarding sDF, a significant difference between the normozoospermic group and the OAT group with abnormal semen parameters (p<0.001) was found. sDF demonstrated a significant effect on the semen parameters and negative correlations on sDF ratios and sperm motility, concentration and morphology. There was no statistically significant association between sDF and the methylation status of the promoter of either BRCA1 or BRCA2 genes. In routine clinical practice, sperm DNA integrity should be investigated before applying assisted reproductive techniques. To understand better the relationship between epigenetic regulation of DNA repair genes and male infertility, additional studies are required.

MicroRNAs and DNA-Damaging Drugs in Breast Cancer: Strength in Numbers.

MicroRNAs are a class of small non-coding regulatory RNAs playing key roles in cancer. Breast cancer is the most common female malignancy worldwide and is categorized into four molecular subtypes: luminal A and B, HER2+ and triple-negative breast cancer (TNBC). Despite the development of multiple targeted therapies for luminal and HER2+ breast tumors, TNBC lacks specific therapeutic approaches, thus they are treated mainly with radio- and chemotherapy. The effectiveness of these therapeutic regimens is based on their ability to induce DNA damage, which is differentially resolved and repaired by normal vs. cancer cells. Recently, drugs directly targeting DNA repair mechanisms, such as PARP inhibitors, have emerged as attractive candidates for the future molecular targeted-therapy in breast cancer. These compounds prevent cancer cells to appropriate repair DNA double strand breaks and induce a phenomenon called synthetic lethality, that results from the concurrent inhibition of PARP and the absence of functional BRCA genes which prompt cell death. MicroRNAs are relevant players in most of the biological processes including DNA damage repair mechanisms. Consistently, the downregulation of DNA repair genes by miRNAs have been probe to improve the therapeutic effect of genotoxic drugs. In this review, we discuss how microRNAs can sensitize cancer cells to DNA-damaging drugs, through the regulation of DNA repair genes, and examine the most recent findings on their possible use as a therapeutic tools of treatment response in breast cancer.

Epigenetic regulation of DNA repair genes and implications for tumor therapy

DNA repair represents the first barrier against genotoxic stress causing metabolic changes, inflammation and cancer. Besides its role in preventing cancer, DNA repair needs also to be considered during cancer treatment with radiation and DNA damaging drugs as it impacts therapy outcome. The DNA repair capacity is mainly governed by the expression level of repair genes. Alterations in the expression of repair genes can occur due to mutations in their coding or promoter region, changes in the expression of transcription factors activating or repressing these genes, and/or epigenetic factors changing histone modifications and CpG promoter methylation or demethylation levels. In this review we provide an overview on the epigenetic regulation of DNA repair genes. We summarize the mechanisms underlying CpG methylation and demethylation, with de novo methyltransferases and DNA repair involved in gain and loss of CpG methylation, respectively. We discuss the role of components of the DNA damage response, p53, PARP-1 and GADD45a on the regulation of the DNA (cytosine-5)-methyltransferase DNMT1, the key enzyme responsible for gene silencing. We stress the relevance of epigenetic silencing of DNA repair genes for tumor formation and tumor therapy. A paradigmatic example is provided by the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT), which is silenced in up to 40% of various cancers through CpG promoter methylation. The CpG methylation status of the MGMT promoter strongly correlates with clinical outcome and, therefore, is used as prognostic marker during glioblastoma therapy. Mismatch repair genes are also subject of epigenetic silencing, which was shown to correlate with colorectal cancer formation. For many other repair genes shown to be epigenetically regulated the clinical outcome is not yet clear. We also address the question of whether genotoxic stress itself can lead to epigenetic alterations of genes encoding proteins involved in the defense against genotoxic stress.

Abstract 1431: Therapeutic exploitation of mutant BRAF in childhood glioma

Abstract Low grade gliomas (LGG) are the most common tumors of the central nervous system in children, accounting for about 50% of all brain tumors. They represent a heterogeneous group of grade I and II tumors according to the WHO classification. Pediatric LGG, is associated with activation of BRAF through a tandem duplication that results in the KIAA1549-BRAF fusion or through an activating point mutation of BRAF (predominantly V600E). More recent findings suggest that the KIAA1549-BRAF fusion is restricted to Grade 1 tumors (70-90%) whereas BRAF(V600E) occurs more frequently in Grade 2-4 tumors (~23%). Findings for BRAF mutation, similar to other tumors with activated BRAF (e.g. melanoma), and the phase I activity of MEK inhibitor in the PBTC-029 protocol, suggest that activated BRAF may provide a validated drug target. Previous studies from our lab indicate that, in the context of mutant BRAF, inhibition of MEK inhibits TORC1 signaling and may induce a ‘BRCA-like’ phenotype, through depletion of FANCD2. Potentially suppression of TORC1 could have effects on other DNA damage response pathways that could compensate for loss of FANCD2. To understand the consequences of MEK inhibition in the context of BRAF abreation in LGG we surveyed DNA repair genes that may be regulated via the MEK/TORC1 pathway in BRAF mutant cells. The objective of this particular study was to examine the mechanism and significance of MEK inhibition to the repair of DNA damage by the homologous recombination (HR) pathway. For this study, we have used glioma cell lines having BRAF(V600E) mutation; the BT40 cell line was developed from patient-derived astrocytoma xenograft (PDX) model in mice in our lab and AM38c1 and DBTRG-05MG cells were generously provided by T. Nicolades. Annexin binding assay demonstrated that MEKi treatment significantly increased the percentage of apoptosis and necrosis of glioma cells. Human DNA repair PCR Array analysis identified MEKi induced down regulation of genes involved in Base Excision Repair (BER), Nucleotide Excision Repair (NER), Mismatch Repair (MMR) and Double-Strand Break (DSB) repair pathways. DSB repair genes were further validated by real time qPCR analysis. Immunoblot analysis of glioma cell lines indicate that MEKi treatment enhanced the gamma-H2AX levels. To elucidate the mechanism of DSB repair pathway in presence of MEKi, clonogenic assay, nuclear foci formation assay, and GFP reporter assay for homologous recombination (HR) and non-homologous end-joining (NHEJ) are ongoing. Collectively, these findings demonstrate for the first time a previously unknown role for MEKi in treatment of glioma cells that involves inhibition of DNA double-strand break repair pathways. Further, we will examine the mechanism and significance of MEK/ERK and TORC1 signaling axis in regulation of DNA repair genes and its effect on LGG. Understanding how MEK plays a role in DNA repair pathways will be useful in maximizing treatment opportunities for childhood glioma. Citation Format: Sudipa Saha Roy, Terry Shackleford, Peter Houghton. Therapeutic exploitation of mutant BRAF in childhood glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1431. doi:10.1158/1538-7445.AM2017-1431

MP99-05 ELL2 REGULATES DNA DOUBLE-STRAND BREAK REPAIR IN PROSTATE CANCER CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology IV1 Apr 2017MP99-05 ELL2 REGULATES DNA DOUBLE-STRAND BREAK REPAIR IN PROSTATE CANCER CELLS Yachen zang, Yibin Zhou, Leizhen Wei, Joel B. Nelson, Lan LI, Boxin xue, Yuxi Shan, and Zhou Wang Yachen zangYachen zang More articles by this author , Yibin ZhouYibin Zhou More articles by this author , Leizhen WeiLeizhen Wei More articles by this author , Joel B. NelsonJoel B. Nelson More articles by this author , Lan LILan LI More articles by this author , Boxin xueBoxin xue More articles by this author , Yuxi ShanYuxi Shan More articles by this author , and Zhou WangZhou Wang More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.3091AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Androgens are known to protect prostate cancer cells from DNA damage. Recent studies showed regulation of DNA repair genes by androgen receptor (AR) signaling in prostate cancers. We recently reported that androgen-regulated protein and potential tumor suppressor ELL2-associated factor 2 (EAF2) can enhance DNA repair through Ku70/Ku80 in the prostate. ELL2 (elongation factor, RNA polymerase II, 2), a component of the super elongation complex (SEC), is an important factor for RNA Pol II transiting from promoter-proximal paused state into elongation state. ELL2 is also regulated by androgens and frequently down-regulated in prostate cancer. These observations led to our hypothesis that ELL2 can regulate DNA repair through Ku70/Ku80 in prostate cancer cells. METHODS Prostate cancer cells, in the presence or absence of siRNA of ELL2, were treated with ?-irradiation or doxorubicin and then collected for detecting the level of DNA damage marker ?H2ax or neutral comet assay. Nonhomologous end-joining (NHEJ) and homologous recombination (HR) assays were used to test the role of ELL2 in these two double-strand break (DSB) repair pathways. Co-immunoprecipitation was used to determine the interaction between ELL2 and NHEJ pathway proteins Ku70 and Ku80. We examined the effect of ELL2 knockdown on Ku70 and Ku80 recruitment in response to laser microirradiation. RESULTS Knockdown of ELL2 sensitized prostate cancer cells to DNA damage and overexpression of ELL2 protected prostate cancer cells from DNA damage. Knockdown of ELL2 impaired NHEJ repair but not HR. Transfected ELL2 co-immunoprecipitated with both Ku70 and Ku80 proteins. ELL2 could binds to and co-accumulated with Ku70/Ku80 proteins at sites of DNA damage. Knockdown of ELL2 dramatically/significantl inhibited Ku70 and Ku80 accumulation and retention at DSB sites in prostate cancer cells, and also impaired recruitment of Ku70/Ku80 to DSB sites. The impaired recruitment of Ku70 and Ku80 proteins to DNA damage sites upon ELL2 knockdown was rescued by re-expression of an ELL2 transgene insensitive to siELL2. CONCLUSIONS This study suggests that ELL2 is an important factor mediating androgen protection of DNA damage via Ku70/Ku80 in prostate cancer cells. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1321-e1322 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Yachen zang More articles by this author Yibin Zhou More articles by this author Leizhen Wei More articles by this author Joel B. Nelson More articles by this author Lan LI More articles by this author Boxin xue More articles by this author Yuxi Shan More articles by this author Zhou Wang More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

EAF2 regulates DNA repair through Ku70/Ku80 in the prostate

Androgens are known to protect prostate cancer cells from DNA damage. Recent studies showed regulation of DNA repair genes by androgen receptor signaling in prostate cancers. ELL-associated factor 2 (EAF2) is an androgen-regulated tumor suppressor and its intracellular localization can be modulated by ultraviolet light, suggesting a potential role for EAF2 in androgen regulation of DNA repair in prostate cancer cells. Here we show that knockdown of EAF2 or its homolog EAF1 sensitized prostate cancer cells to DNA damage and the sensitization did not require p53. EAF2 knockout mouse prostate was also sensitized to γ-irradiation. Furthermore, EAF2 knockdown blocked androgen repression of LNCaP or C4-2 cells from doxorubicin induction of γH2ax, a DNA damage marker. In human prostate cancer specimens, EAF2 expression was inversely correlated with the level of γH2ax. Further analysis showed that EAF2 and EAF1 are required for the recruitment and retention of Ku70/Ku80 to DNA damage sites and play a functional role in nonhomologous end-joining DNA repair. These findings provide evidence for EAF2 as a key factor mediating androgen protection of DNA damage via Ku70/Ku80 in prostate cancer cells.

ΔNp63 activates the Fanconi anemia DNA repair pathway and limits the efficacy of cisplatin treatment in squamous cell carcinoma.

TP63, a member of the p53 gene family gene, encodes the ΔNp63 protein and is one of the most frequently amplified genes in squamous cell carcinomas (SCC) of the head and neck (HNSCC) and lungs (LUSC). Using an epiallelic series of siRNAs with intrinsically different knockdown abilities, we show that the complete loss of ΔNp63 strongly impaired cell proliferation, whereas partial ΔNp63 depletion rendered cells hypersensitive to cisplatin accompanied by an accumulation of DNA damage. Expression profiling revealed wide-spread transcriptional regulation of DNA repair genes and in particular Fanconi anemia (FA) pathway components such as FANCD2 and RAD18 - known to be crucial for the repair of cisplatin-induced interstrand crosslinks. In SCC patients ΔNp63 levels significantly correlate with FANCD2 and RAD18 expression confirming ΔNp63 as a key activator of the FA pathway in vivo. Mechanistically, ΔNp63 bound an upstream enhancer of FANCD2 inactive in primary keratinocytes but aberrantly activated by ΔNp63 in SCC. Consistently, depletion of FANCD2 sensitized to cisplatin similar to depletion of ΔNp63. Together, our results demonstrate that ΔNp63 directly activates the FA pathway in SCC and limits the efficacy of cisplatin treatment. Targeting ΔNp63 therefore would not only inhibit SCC proliferation but also sensitize tumors to chemotherapy.

Specific histone modifications were associated with the PAH-induced DNA damage response in coke oven workers.

To investigate whether polycyclic aromatic hydrocarbon (PAH) exposure is associated with specific histone modifications and whether DNA damage triggers epigenetic alterations, we recruited 190 male workers with occupational exposure to PAHs and 100 male control workers from Benxi Steel Plant, Liaoning province, China. Urinary 1-hydroxypyrene (1-OHP), DNA damage, specific histone modification levels and the expression of selected DNA damage response (DDR) genes were measured in peripheral blood lymphocytes (PBLCs) of the subjects. The results showed that trimethylated Lys 27 of histone H3 (H3K27me3) and trimethylated Lys 36 of histone H3 (H3K36me3) were elevated in the PAH-exposed group (both P < 0.001), while trimethylated Lys H3 of histone H3 (H3K4me3) was decreased compared to the unexposed group (P < 0.001). Notably, H3K36me3 was positively associated with the level of internal exposure marker 1-OHP (β = 0.197; P < 0.001) and the degree of DNA damage (β = 0.175; P < 0.001) in all subjects, indicating that the PAH-induced DNA damage response might be mediated by H3K36me3 and/or H3K4me3 modifications. Particularly, the ChIP-qPCR assay revealed that the modifications of H3K36me3 were enriched in the gene body of DDR genes, MGMT and MLH1. The up-regulation of MGMT and MLH1 was correlated with the elevated H3K36me3 in the PAH-exposed workers (P < 0.001). Collectively, we revealed that H3K36me3 could be an indicator of PAH exposure and might be involved in the transcriptional regulation of DNA repair genes in response to DNA damage.

CREB Increases Chemotherapy Resistance through Regulation of the DNA Damage Repair Pathway in AML Cells

CREB (cAMP Response Element Binding Protein) is a nuclear transcription factor that plays a critical role in regulating myeloid cell proliferation and differentiation. CREB is overexpressed in Acute Myeloid Leukemia (AML) cells from the majority of AML patients at diagnosis, and CREB overexpression is associated with a poor prognosis.Transgenic mice overexpressing CREB in myeloid cells develop myelodysplasia/myeloproliferative neoplasms. CREB also cooperates with other oncogenes, such as Sox4, to induce transformation to AML. Knockdown of CREB inhibits AML proliferation but does not affect normal hematopoietic stem cell activity, establishing the crucial role of CREB in AML cell growth and survival.In vitro, CREB overexpression leads to increased resistance to apoptosis in AML cells. Thus, we hypothesized that increased CREB expression confers chemoresistance, as this may represent one reason that patients with high CREB levels have worse prognoses and relapse following therapy. Previous studies have demonstrated that chemotherapy resistance can result from increased DNA damage repair activity, but CREB has never been implicated in these DNA damage repair processes, nor has CREB even been described as an important transcriptional regulator of DNA damage repair genes. The goal of this study was to characterize whether CREB expression confers chemoresistance through regulation of DNA repair genes in AML cells. Firstly, we established that CREB expression levels correlate with chemoresistance by treating KG-1 cells engineered to express lower and higher levels of CREB with etoposide and doxorubicin, both chemotherapy drugs used to treat AML. Cells with CREB overexpression had increased viability compared to CREB knockdown cells after treatment with both chemotherapies at a range of concentrations. To investigate the underlying mechanism, we performed CREB chromatin immunoprecipitation and RNA-seq following small molecule CREB inhibition to identify the sets of genes that are regulated by CREB in AML cells and whose expression levels are sensitive to CREB inhibition. Out of 88 DNA damage repair genes found to be CREB-bound, 41 exhibited at least a 2-fold change in expression after CREB inhibition. qPCR was performed to determine whether the expression of DNA damage repair genes were proportional to CREB levels. Transcription of ATM, ATR, RAD54L, and RAD51, genes important in sensing and repairing DNA damage, were coordinately regulated with CREB expression. ATM, ATR, RAD54L, and RAD51 were reduced by approximately 42.0%±0.1%, 44.8%±0.1%, 40.2%±0.1%, and 27.9%±0.1% respectively in CREB knockdown cells (p≤0.05). Reduced expression of these genes also had a functional consequence. CREB knockdown cells initiated a lesser DNA damage repair response in response to etoposide treatment, as determined by measured phospho-H2AX levels, compared to wild-type CREB-expressing cells. Conversely, cells with CREB overexpression exhibited the strongest DNA damage repair response following etoposide treatment.Taken together, these data demonstrate that CREB overexpression has a protective effect against DNA damage and confers chemoresistance, likely through upregulation of DNA damage repair genes. Future studies will seek to determine if small molecule inhibition of CREB can reduce the transcription of DNA damage repair genes and thus sensitize AML cells to chemotherapeutic agents. DisclosuresNo relevant conflicts of interest to declare.

Investigation of microRNA expression and DNA repair gene transcripts in human oocytes and blastocysts.

The aim of the study is to investigate the regulation of DNA repair genes by microRNAs (miRNAs). miRNAs are short non-coding RNAs that regulate transcriptional and post-transcriptional gene silencing. Several miRNAs that are expressed during preimplantation embryo development have been shown or are predicted to target genes that regulate cell cycle checkpoints and DNA repair in response to DNA damage. This study compares the expression level of 20 miRNAs and 9 target transcripts involved in DNA repair. The statistical significance of differential miRNA expression between oocytes and blastocysts was determined by t test analysis using the GraphPad Prism v6 software. The possible regulatory roles of miRNAs on their target messenger RNAs (mRNAs) were analysed using a Pearson correlation test. This study shows for the first time that several miRNAs are expressed in human oocytes and blastocysts that target key genes involved in DNA repair and cell cycle checkpoints. Blastocysts exhibited statistically significant lower expression levels for the majority of miRNAs compared to oocytes (p < 0.05). Correlation analyses showed that there was both inverse and direct association between miRNAs and their target mRNAs. miRNAs target many mRNAs including ones involved in DNA repair mechanisms. This study suggests that miRNAs and their target mRNAs involved in DNA repair are expressed in preimplantation embryos. Similar to the miRNAs expressed in adult tissues, these miRNAs seem to have regulatory roles on their target DNA repair mRNAs during preimplantation embryo development.

A functional screen identifies miRNAs that inhibit DNA repair and sensitize prostate cancer cells to ionizing radiation.

MicroRNAs (miRNAs) have been implicated in DNA repair pathways through transcriptional responses to DNA damaging agents or through predicted miRNA regulation of DNA repair genes. We hypothesized that additional DNA damage regulating miRNAs could be identified by screening a library of 810 miRNA mimetics for the ability to alter cellular sensitivity to ionizing radiation (IR). A prostate cancer Metridia luciferase cell model was applied to examine the effects of individual miRNAs on IR sensitivity. A large percentage of miRNA mimetics were found to increase cellular sensitivity to IR, while a smaller percentage were protective. Two of the most potent IR sensitizing miRNAs, miR-890 and miR-744–3p, significantly delayed IR induced DNA damage repair. Both miRNAs inhibited the expression of multiple components of DNA damage response and DNA repair. miR-890 directly targeted MAD2L2, as well as WEE1 and XPC, where miR-744–3p directly targeted RAD23B. Knock-down of individual miR-890 targets by siRNA was not sufficient to ablate miR-890 radiosensitization, signifying that miR-890 functions by regulating multiple DNA repair genes. Intratumoral delivery of miR-890 mimetics prior to IR therapy significantly enhanced IR therapeutic efficacy. These results reveal novel miRNA regulation of DNA repair and identify miR-890 as a potent IR sensitizing agent.

CD133+ cells contribute to radioresistance via altered regulation of DNA repair genes in human lung cancer cells

BackgroundRadioresistance in human tumors has been linked in part to a subset of cells termed cancer stem cells (CSCs). The prominin 1 (CD133) cell surface protein is proposed to be a marker enriching for CSCs. We explore the importance of DNA repair in contributing to radioresistance in CD133+ lung cancer cells. Materials and methodsA549 and H1299 lung cancer cell lines were used. Sorted CD133+ cells were exposed to either single 4Gy or 8Gy doses and clonogenic survival measured. ϒ-H2AX immunofluorescence and quantitative real time PCR was performed on sorted CD133+ cells both in the absence of IR and after two single 4Gy doses. Lentiviral shRNA was used to silence repair genes. ResultsA549 but not H1299 cells expand their CD133+ population after single 4Gy exposure, and isolated A549 CD133+ cells demonstrate IR resistance. This resistance corresponded with enhanced repair of DNA double strand breaks (DSBs) and upregulated expression of DSB repair genes in A549 cells. Prior IR exposure of two single 4Gy doses resulted in acquired DNA repair upregulation and improved repair proficiency in both A549 and H1299. Finally Exo1 and Rad51 silencing in A549 cells abrogated the CD133+ IR expansion phenotype and induced IR sensitivity in sorted CD133+ cells. ConclusionsCD133 identifies a population of cells within specific tumor types containing altered expression of DNA repair genes that are inducible upon exposure to chemotherapy. This altered gene expression contributes to enhanced DSB resolution and the radioresistance phenotype of these cells. We also identify DNA repair genes which may serve as promising therapeutic targets to confer radiosensitivity to CSCs.

Transcriptional regulation of human DNA repair genes following genotoxic stress: trigger mechanisms, inducible responses and genotoxic adaptation

DNA repair is the first barrier in the defense against genotoxic stress. In recent years, mechanisms that recognize DNA damage and activate DNA repair functions through transcriptional upregulation and post-translational modification were the focus of intensive research. Most DNA repair pathways are complex, involving many proteins working in discrete consecutive steps. Therefore, their balanced expression is important for avoiding erroneous repair that might result from excessive base removal and DNA cleavage. Amelioration of DNA repair requires both a fine-tuned system of lesion recognition and transcription factors that regulate repair genes in a balanced way. Transcriptional upregulation of DNA repair genes by genotoxic stress is counteracted by DNA damage that blocks transcription. Therefore, induction of DNA repair resulting in an adaptive response is only visible through a narrow window of dose. Here, we review transcriptional regulation of DNA repair genes in normal and cancer cells and describe mechanisms of promoter activation following genotoxic exposures through environmental carcinogens and anticancer drugs. The data available to date indicate that 25 DNA repair genes are subject to regulation following genotoxic stress in rodent and human cells, but for only a few of them, the data are solid as to the mechanism, homeostatic regulation and involvement in an adaptive response to genotoxic stress.

Protein Arginine Methyltransferase 7 Regulates Cellular Response to DNA Damage by Methylating Promoter Histones H2A and H4 of the Polymerase δ Catalytic Subunit Gene, POLD1

Covalent modification of histones by protein arginine methyltransferases (PRMTs) impacts genome organization and gene expression. In this report, we show that PRMT7 interacts with the BRG1-based hSWI/SNF chromatin remodeling complex and specifically methylates histone H2A Arg-3 (H2AR3) and histone H4 Arg-3 (H4R3). To elucidate the biological function of PRMT7, we knocked down its expression in NIH 3T3 cells and analyzed global gene expression. Our findings show that PRMT7 negatively regulates expression of genes involved in DNA repair, including ALKBH5, APEX2, POLD1, and POLD2. Chromatin immunoprecipitation (ChIP) revealed that PRMT7 and dimethylated H2AR3 and H4R3 are enriched at target DNA repair genes in parental cells, whereas PRMT7 knockdown caused a significant decrease in PRMT7 recruitment and H2AR3/H4R3 methylation. Decreased PRMT7 expression also resulted in derepression of target DNA repair genes and enhanced cell resistance to DNA-damaging agents. Furthermore, we show that BRG1 co-localizes with PRMT7 on target promoters and that expression of a catalytically inactive form of BRG1 results in derepression of PRMT7 target DNA repair genes. Remarkably, reducing expression of individual PRMT7 target DNA repair genes showed that only the catalytic subunit of DNA polymerase, POLD1, was able to resensitize PRMT7 knock-down cells to DNA-damaging agents. These results provide evidence for the important role played by PRMT7 in epigenetic regulation of DNA repair genes and cellular response to DNA damage.

Global Analysis of the Regulon of the Transcriptional Repressor LexA, a Key Component of SOS Response in Mycobacterium tuberculosis

The DNA damage response is crucial for bacterial survival. The transcriptional repressor LexA is a key component of the SOS response, the main mechanism for the regulation of DNA repair genes in many bacteria. In contrast, in mycobacteria gene induction by DNA damage is carried out by two mechanisms; a relatively small number of genes are thought to be regulated by LexA, and a larger number by an alternate, independent mechanism. In this study we have used ChIP-seq analysis to identify 25 in vivo LexA-binding sites, including nine regulating genes not previously known to be part of this regulon. Some of these binding sites were found to be internal to the predicted open reading frame of the gene they are thought to regulate; experimental analysis has confirmed that these LexA-binding sites regulate the expression of the expected genes, and transcriptional start site analysis has found that their apparent relative location is due to misannotation of these genes. We have also identified novel binding sites for LexA in the promoters of genes that show no apparent DNA damage induction, show positive regulation by LexA, and those encoding small RNAs.