DNA cytosine methylation at the lexA promoter of Escherichia coli is stationary phase specific

The bacterial DNA damage response pathway (SOS response) is composed of a network of genes regulated by a single transcriptional repressor, LexA. The lexA promoter, itself, contains two LexA operators, enabling negative feedback. In Escherichia coli, the downstream operator contains a conserved DNA cytosine methyltransferase (Dcm) site that is predicted to be methylated to 5-methylcytosine (5mC) specifically during stationary phase growth, suggesting a regulatory role for DNA methylation in the SOS response. To test this, we quantified 5mC at the lexA locus, and then examined the effect of LexA on Dcm activity, as well as the impact of this 5mC mark on LexA binding, lexA transcription, and SOS response induction. We found that 5mC at the lexA promoter is specific to stationary phase growth, but that it does not affect lexA expression. Our data support a model where LexA binding at the promoter inhibits Dcm activity without an effect on the SOS regulon.

RTT109 AND Fun30 proteins mediate epigenetic regulation of the DNA damage response pathway in C. albicans

Fun30, an ATP-dependent chromatin remodeller, from S. cerevisiae mediates both regulation of gene expression as well as DNA damage response/repair. In this paper, we have characterized the biochemical and physiological function of Fun30 from the opportunistic fungi, C. albicans. Biochemically, the protein shows DNA-stimulated ATPase activity. Physiologically, the protein co-regulates transcription of RTT109, TEL1, MEC1, and SNF2-genes that encode for proteins involved in DNA damage response and repair pathway. The expression of FUN30, in turn, is regulated by histone H3 acetylation catalysed by Rtt109 encoded by RTT109. The RTT109Hz/FUN30Hz mutant strain shows sensitivity to oxidative stress and resistance to MMS as compared to the wild type strain. Quantitative PCR showed that the sensitivity to oxidative stress results from downregulation of MEC1, RAD9, MRC1 and RAD5 expression; ChIP experiments showed Fun30 but not H3ac regulates the expression of these genes in response to oxidative stress. In contrast, on treatment with MMS, the expression of RAD9 is upregulated and this upregulation is co-regulated by both Fun30 and H3 acetylation catalysed by Rtt109. Thus, Fun30 and H3 acetylation mediate the response of the fungal cell to genotoxic agents in C. albicans by regulating the expression of DNA damage response and repair pathway genes.

CDX2 inducible microRNAs sustain colon cancer by targeting multiple DNA damage response pathway factors

Meta-analysis of transcripts in colon adenocarcinoma patient tissues led to the identification of a DNA damage responsive miR signature called DNA damage sensitive miRs (DDSMs). DDSMs were experimentally validated in the cancerous colon tissues obtained from an independent cohort of colon cancer patients and in multiple cellular systems with high levels of endogenous DNA damage. All the tested DDSMs were transcriptionally upregulated by a common intestine-specific transcription factor, CDX2. Reciprocally, DDSMs were repressed via the recruitment of HDAC1/2 containing complexes onto the CDX2 promoter. These miRs downregulated multiple key targets in the DNA damage response (DDR) pathway, namely BRCA1, ATM, Chk1 and RNF8. CDX2 directly regulated the DDSMs which led to increased tumor volume and metastasis in multiple preclinical models. In colon cancer patient tissues the DDSMs negatively correlated with BRCA1 levels, were associated with decreased probability of survival, and thereby could be used as a prognostic biomarker.

New familial cases of karyomegalic interstitial nephritis with mutations in the FAN1 gene

Background Karyomegalic interstitial nephritis (KIN) is a rare disease entity first described by Burry in 1974. The term KIN was introduced by Mihatsch et al. in 1979. KIN is characterized by chronic tubulointerstitial nephritis associated with enlarged tubular epithelial cell nuclei, which leads to a progressive decline of renal function. The prevalence of this disease is less than 1% of all biopsies, and its pathogenesis is unclear. KIN results from mutations in FAN1 (FANCD2/FANCI-Associated Nuclease 1), a gene involved in the DNA damage response pathway, particularly in the kidney. In this study, we report two Tunisian consanguineous families with KIN caused by mutations in the FAN1 gene. Methods Direct sequencing of the coding regions and flanking intronic sequences of the FAN1 gene was performed in three affected members. Three prediction programs (Polyphen-2 software, SIFT, and MutationTaster) were used to predict the functional effect of the detected variations. Results Two causative frameshift variants in the FAN1 gene were identified in each family: The previously described frameshift mutation c.2616delA (p.Asp873ThrfsTer17) and a novel mutation c.2603delT (p.Leu868ArgfsTer22) classified as "pathogenic" according to the American College of Medical Genetics and Genomics (ACMG) guidelines. Conclusion To our best knowledge, this is the first Tunisian study involving familial cases of KIN with mutations in the FAN1 gene. We hypothesize that these findings can expand the mutational spectrum of KIN and provide valuable information on the genetic cause of KIN.

Inhibition of DNA damage response pathway using combination of DDR pathway inhibitors and radiation in treatment of acute lymphoblastic leukemia cells

An alarming increase in acute lymphoblastic leukemia cases among children and adults has attracted the attention of researchers to discover new therapeutic strategies with a better prognosis. In cancer cells, the DNA damage response (DDR) pathway elements have been recognized to protect tumor cells from various stresses and cause tumor progression; targeting these DDR members is an attractive strategy for treatment of cancers. The inhibition of the DDR pathway in cancer cells for the treatment of cancers has recently been introduced. Hence, effective treatment strategies are needed for this purpose. Chemotherapy in combination with radiotherapy is considered a potential therapeutic strategy for acute leukemia. This review aims to assess the synergistic effects of these inhibitors with irradiation for the treatment of leukemia.

Arabidopsis Casein Kinase 2 triggers Stem Cell Exhaustion under Al Toxicity and Phosphate Deficiency Through activation of the DNA Damage Response pathway

Aluminum (Al) toxicity and inorganic phosphate (Pi) limitation are widespread chronic abiotic and mutually enhancing stresses that profoundly affect crop yield. Both stresses cause a strong inhibition of root growth, resulting from a progressive exhaustion of the stem cell niche. Here, we report on a casein kinase 2 (CK2) inhibitor identified by its capability to maintain a functional root stem cell niche under Al toxic conditions. CK2 operates through phosphorylation of the cell cycle checkpoint activator SUPPRESSOR OF GAMMA RADIATION1 (SOG1), priming its activity under DNA-damaging conditions. In addition to yielding Al tolerance, CK2 and SOG1 inactivation prevents meristem exhaustion under Pi starvation, revealing the existence of a low Pi-induced cell cycle checkpoint that depends on the DNA damage activator ATAXIA-TELANGIECTASIA MUTATED. Overall, our data reveal an important physiological role for the plant DNA damage response pathway under agriculturally limiting growth conditions, opening new avenues to cope with Pi limitation.

NUSAP1 Promotes Cell Proliferation Via the DNA Damage Response Pathway in Diffuse Large B-Cell Lymphoma

Introduction: Nucleolar spindle-associated protein 1 (NUSAP1), a microtubule binding protein with molecular weight of 55KD, plays an important role to ensure the normal regulation of cell cycle in chromosome separation, spindle assembly and DNA repair. NUSAP1 has been shown to be highly expressed in a variety of tumors, involved in tumor occurrence, invasion, migration, and drug resistance. Moreover, it is associated with poor prognosis. Whereas, no research has been reported regarding the role of NUSAP1 in diffuse large B-cell lymphoma (DLBCL). Methods: Peripheral blood samples from de novo DLBCL patients and healthy volunteers were collected with informed consents at the Department of Hematology in Shandong Provincial Hospital Affiliated to Shandong University (SPHASU). Microarray datasets GSE83632 and GSE32918 were obtained from Gene Expression Omnibus. Kaplan-Meier survival curves with log-rank test of overall survival (OS) were analyzed. Immunohistochemistry staining (IHC) was performed to assess NUSAP1 expression in specimens. Expression levels of NUSAP1 mRNA and protein were detected by quantitative RT-PCR and western blotting. The DLBCL cells were transfected by lentiviral shRNA and vectors to stably silence and up-regulate NUSAP1. Effects of doxorubicin on cell viabilities were assessed by cell counting kit-8. Besides, apoptosis and cell cycle were respectively detected by annexin V-PE/7AAD and PI/RNase staining via flow cytometry. Invasion ability was analyzed by transwell assay. ShNUSAP1 cells and Scramble cells were subcutaneously injected to SCID-Beige mice to establish xenograft models. Animal experiments were performed in accordance with the principles of the Institutional Animal Care. Results: According to clinical specimens and bioinformatics analysis, the expression level of NUSAP1 gene in samples of DLBCL patients was significantly increased than that of healthy donors (P<0.05) (Figure A, B). Besides, patients with high expression of NUSAP1 was related to shorter overall survival (HR=0.456, P<0.01), indicating poor prognosis (Figure C). Stronger positivity of NUSAP1 was markedly observed in DLBCL lymph nodes than reactive hyperplasia group, which was associated with Ann Arbor stage of DLBCL patients (Figure D). Compared with normal B cells, both the mRNA and protein level of NUSAP1 were up-regulated in DLBCL cell lines (Figure E, F). After transfected with lentivirus, the proliferation rate of NUSAP1 knockdown group was lower than that of the control group, while the overexpression group was faster than control (Figure G). Through flow cytometry, silencing of NUSAP1 led to increased apoptotic rates of LY1 and LY3 cells (Figure H). Along with the recovery of NUSAP1 expression level, apoptosis rates were improved again. With the addition of doxorubicin at 100nM, interference of NUSAP1 could increase the sensitivity of cells to doxorubicin (Figure I). NUSAP1 intervention induced obvious cell cycle arrest in G1 phase of LY1 and LY3 cell lines, with concomitant reduction of cell proportion in S phase (Figure J). Moreover, known as EMT biomarkers, the expression of ZEB1 and VIMENTIN were decreased with reduction of NUSAP1. Likewise, we also investigated NUSAP1 overexpression has promoted EMT-like process of LY1 and LY3 cells (Figure K). Surppression of NUSAP1 inhibited the action of DNA damage repair pathway (Figure L). Moreover, we also found that NUSAP1 knockdown induced striking reduction in invasion cells (Figure M). We found that tumor growth was significantly suppressed by inhibiting NUSAP1 in xenograft models (Figure N, O). IHC for NUSAP1 and Ki67 on tumor sections showed that tumors derived from shNUSAP1 cells exhibited significantly lower levels of Ki67 compared to control group (Figure P). Conclusions: This study first identified that the high expression of NUSAP1 in DLBCL patients is associated with poor prognosis through database analysis and in vitro experiments. Interference of NUSAP1 expression led to a slower DLBCL cell proliferation and a higher apoptosis rate, meanwhile induced the G1 phase arrest and promoted EMT-like process. Collectively, our study demonstrated that NUSAP1 plays a role in promoting tumor growth both in vivo and vitro through DNA damage response pathway, which providing a new direction for prognosis assessment and targeted therapy of DLBCL. Figure Disclosures No relevant conflicts of interest to declare.

New Familial Cases of Karyomegalic Interstitial Nephritis With Mutations in the FAN1 Gene

Karyomegalic interstitial nephritis (KIN) is a rare disease entity that was first described by Burry in 1974 and given this term by Mihatsch 1979 and al five years later. KIN is characterized by chronic tubulo-interstitial nephritis associated with enlarged tubular epithelial cell nuclei, which leads to progressive decline of renal function. The prevalence of this disease is less than 1% and its pathogenesis is unclear. KIN results from the mutation in the FAN 1 (FANCD2/FANCI-Associated Nuclease 1) gene, a gene involved in the DNA damage response pathway, particularly in the kidney. KIN seems to be rather multifactorial involving an environmental factor Ochratoxin A (OTA) but especially a genetic predisposition. In this article, we report 6 additional cases of KIN with histological evidence in 3 patients. Family clustering and differences in susceptibility to develop the nephropathy in spite of a high OTA contamination in all subjects show that there is a genetic susceptibility. So we completed our explorations by a genetic study. This study allowed us to identify novel mutations in the FAN1 gene in the affected members. To our best knowledge, this is the first Tunisian study involving familial cases of KIN with mutations on the FAN1 gene.