Dual role of proliferating cell nuclear antigen monoubiquitination in facilitating Fanconi anemia-mediated interstrand crosslink repair.

The Fanconi anemia (FA) repair pathway governs repair of highly genotoxic DNA interstrand crosslinks (ICLs) and relies on translesion synthesis (TLS). TLS is facilitated by REV1 or site-specific monoubiquitination of proliferating cell nuclear antigen (PCNA) (PCNA-Ub) at lysine 164 (K164). A PcnaK164R/K164R but not Rev1-/- mutation renders mammals hypersensitive to ICLs. Besides the FA pathway, alternative pathways have been associated with ICL repair (1, 2), though the decision making between those remains elusive. To study the dependence and relevance of PCNA-Ub in FA repair, we intercrossed PcnaK164R/+; Fancg-/+ mice. A combined mutation (PcnaK164R/K164R; Fancg-/- ) was found embryonically lethal. RNA-seq of primary double-mutant (DM) mouse embryonic fibroblasts (MEFs) revealed elevated levels of replication stress-induced checkpoints. To exclude stress-induced confounders, we utilized a Trp53 knock-down to obtain a model to study ICL repair in depth. Regarding ICL-induced cell toxicity, cell cycle arrest, and replication fork progression, single-mutant and DM MEFs were found equally sensitive, establishing PCNA-Ub to be critical for FA-ICL repair. Immunoprecipitation and spectrometry-based analysis revealed an unknown role of PCNA-Ub in excluding mismatch recognition complex MSH2/MSH6 from being recruited to ICLs. In conclusion, our results uncovered a dual function of PCNA-Ub in ICL repair, i.e. exclude MSH2/MSH6 recruitment to channel the ICL toward canonical FA repair, in addition to its established role in coordinating TLS opposite the unhooked ICL.

Histone methyltransferase DOT1L controls state-specific identity during B cell differentiation.

Differentiation of naïve peripheral B cells into terminally differentiated plasma cells is characterized by epigenetic alterations, yet the epigenetic mechanisms that control B-cell fate remain unclear. Here, we identified a role for the histone H3K79 methyltransferase DOT1L in controlling B-cell differentiation. Mouse B cells lacking Dot1L failed to establish germinal centers (GC) and normal humoral immune responses in vivo. In vitro, activated B cells in which Dot1L was deleted showed aberrant differentiation and prematurely acquired plasma cell characteristics. Similar results were obtained when DOT1L was chemically inhibited in mature B cells in vitro. Mechanistically, combined epigenomics and transcriptomics analysis revealed that DOT1L promotes expression of a pro-proliferative, pro-GC program. In addition, DOT1L indirectly supports the repression of an anti-proliferative plasma cell differentiation program by maintaining the repression of Polycomb Repressor Complex 2 (PRC2) targets. Our findings show that DOT1L is a key modulator of the core transcriptional and epigenetic landscape in B cells, establishing an epigenetic barrier that warrants B-cell naivety and GC B-cell differentiation.

The Ig heavy chain protein but not its message controls early B cell development.

Development of progenitor B cells (ProB cells) into precursor B cells (PreB cells) is dictated by immunoglobulin heavy chain checkpoint (IgHCC), where the IgHC encoded by a productively rearranged Igh allele assembles into a PreB cell receptor complex (PreBCR) to generate signals to initiate this transition and suppressing antigen receptor gene recombination, ensuring that only one productive Igh allele is expressed, a phenomenon known as Igh allelic exclusion. In contrast to a productively rearranged Igh allele, the Igh messenger RNA (mRNA) (IgHR) from a nonproductively rearranged Igh allele is degraded by nonsense-mediated decay (NMD). This fact prohibited firm conclusions regarding the contribution of stable IgHR to the molecular and developmental changes associated with the IgHCC. This point was addressed by generating the IghTer5H∆TM mouse model from IghTer5H mice having a premature termination codon at position +5 in leader exon of IghTer5H allele. This prohibited NMD, and the lack of a transmembrane region (∆TM) prevented the formation of any signaling-competent PreBCR complexes that may arise as a result of read-through translation across premature Ter5 stop codon. A highly sensitive sandwich Western blot revealed read-through translation of IghTer5H message, indicating that previous conclusions regarding a role of IgHR in establishing allelic exclusion requires further exploration. As determined by RNA sequencing (RNA-Seq), this low amount of IgHC sufficed to initiate PreB cell markers normally associated with PreBCR signaling. In contrast, the IghTer5H∆TM knock-in allele, which generated stable IgHR but no detectable IgHC, failed to induce PreB development. Our data indicate that the IgHCC is controlled at the level of IgHC and not IgHR expression.

The histone methyltransferase DOT1L prevents antigen-independent differentiation and safeguards epigenetic identity of CD8+ T cells.

Cytotoxic T cell differentiation is guided by epigenome adaptations, but how epigenetic mechanisms control lymphocyte development has not been well defined. Here we show that the histone methyltransferase DOT1L, which marks the nucleosome core on active genes, safeguards normal differentiation of CD8+ T cells. T cell-specific ablation of Dot1L resulted in loss of naïve CD8+ T cells and premature differentiation toward a memory-like state, independent of antigen exposure and in a cell-intrinsic manner. Mechanistically, DOT1L controlled CD8+ T cell differentiation by ensuring normal T cell receptor density and signaling. DOT1L also maintained epigenetic identity, in part by indirectly supporting the repression of developmentally regulated genes. Finally, deletion of Dot1L in T cells resulted in an impaired immune response. Through our study, DOT1L is emerging as a central player in physiology of CD8+ T cells, acting as a barrier to prevent premature differentiation and controlling epigenetic integrity.

Influence of single-nucleotide polymorphisms in the IFNG towards susceptibility to tuberculosis in a Pakistani population.

Tuberculosis (TB) is a global issue as one-third of the population worldwide is considered to be infected. TB has become a critical public health problem as a result of increasing drug resistance, which poses a challenge to current control strategies. Similar to environmental factors, genetic makeup of the host equally contributes to disease onset. We performed genotypic analysis to examine the relationship between IFNG and TB onset and drug resistance in a Pakistani population comprising 689 subjects. Notable differences were observed in the IFNG polymorphism (+874T/A) between the case and control groups. The frequency of the wild-type genotype (TT) in the controls (43.2%) was significantly higher than in the cases (25.3%) (odds ratio [OR] = 0.77, p < 0.0001), while the mutant genotype frequency (AA) (38.57%) in the cases was significantly higher than in the controls (22.6%) (OR = 1.46, p < 0.0001). The heterozygous genotype frequency (TA) did not significantly differ between the control and case groups. Compared with the controls, the variant allele (A) was approximately twice as frequent in the cases. Females and older people have a higher chance of disease development. Finally, the IFNG (+874T/A) polymorphism was not associated with drug sensitivity or resistance. However, a genotypic polymorphism of IFNG (+874T/A) was significantly associated with susceptibility to TB, and the T allele conferred protection against TB. Additional studies involving larger cohorts are needed to further explore this relationship between genetics and disease vulnerability.

Genetic polymorphism in association with susceptibility to tuberculosis: a study in a Pakistani population.

Tuberculosis is becoming a global issue with raising occurrences; particularly in developing countries, the situation is alarming. Besides environmental factors, host genetic factors are vital in disease development. A demographical and genotypic analysis in relation to tuberculosis commencement is conducted in a Pakistani population, and genotypic frequency of EBI3 (rs4740) was analyzed. Allelic frequencies of EBI3 (rs4740) were significantly associated with disease susceptibility in the reviewed population. Analysis for EBI3 (rs4740) genotyping showed a significant association of "GG" with reduced risk for disease. Moreover, females and older age found to be more perilous to develop TB while smoking and a family history of TB are additional risk factors for disease development. Further work with a larger population is necessary to identify the true causative variants of tuberculosis.

Towards an understanding of C9orf82 protein/CAAP1 function.

C9orf82 protein, or conserved anti-apoptotic protein 1 or caspase activity and apoptosis inhibitor 1 (CAAP1) has been implicated as a negative regulator of the intrinsic apoptosis pathway by modulating caspase expression and activity. In contrast, an independent genome wide screen for factors capable of driving drug resistance to the topoisomerase II (Topo II) poisons doxorubicin and etoposide, implicated a role for the nuclear protein C9orf82 in delaying DSBs repair downstream of Topo II, hereby sensitizing cells to DSB induced apoptosis. To determine its function in a genetically defined setting in vivo and ex vivo, we here employed CRISPR/Cas9 technology in zygotes to generate a C9orf82 knockout mouse model. C9orf82ko/ko mice were born at a Mendelian ratio and did not display any overt macroscopic or histological abnormalities. DSBs repair dependent processes like lymphocyte development and class switch recombination (CSR) appeared normal, arguing against a link between the C9orf82 encoded protein and V(D)J recombination or CSR. Most relevant, primary pre-B cell cultures and Tp53 transformed mouse embryo fibroblasts (MEFs) derived from C9orf82ko/ko E14.5 and wild type embryos displayed comparable sensitivity to a number of DNA lesions, including DSBs breaks induced by the topoisomerase II inhibitors, etoposide and doxorubicin. Likewise, the kinetics of γH2AX formation and resolution in response to etoposide of C9orf82 protein proficient, deficient and overexpressing MEFs were indistinguishable. These data argue against a direct role of C9orf82 protein in delaying repair of Topo II generated DSBs and regulating apoptosis. The genetically defined systems generated in this study will be of value to determine the actual function of C9orf82 protein.

Precision cancer therapy: profiting from tumor specific defects in the DNA damage tolerance system.

DNA damage tolerance (DDT) enables replication to continue in the presence of a damaged template and constitutes a key step in DNA interstrand crosslink repair. In this way DDT minimizes replication stress inflicted by a wide range of endogenous and exogenous agents, and provides a critical first line defense against alkylating and platinating chemotherapeutics. Effective DDT strongly depends on damage-induced, site-specific PCNA-ubiquitination at Lysine (K) 164 by the E2/E3 complex (RAD6/18). A survey of The Cancer Genome Atlas (TCGA) revealed a high frequency of tumors presents RAD6/RAD18 bi-allelic inactivating deletions. For instance, 11% of renal cell carcinoma and 5% of pancreatic tumors have inactivating RAD18-deletions and 7% of malignant peripheral nerve sheath tumors lack RAD6B. To determine the potential benefit for tumor-specific DDT defects, we followed a genetic approach by establishing unique sets of DDT-proficient PcnaK164 and -defective PcnaK164R lymphoma and breast cancer cell lines. In the absence of exogenous DNA damage, PcnaK164R tumors grew comparably to their PcnaK164 controls in vitro and in vivo. However, DDT-defective lymphomas and breast cancers were compared to their DDT-proficient controls hypersensitive to the chemotherapeutic drug cisplatin (CsPt), both in vitro and in vivo. CsPt strongly inhibited tumor growth and the overall survival of tumor bearing mice greatly improved in the DDT-defective condition. These insights open new therapeutic possibilities for precision cancer medicine with DNA damaging chemotherapeutics and optimize Next-Generation-Sequencing (NGS)-based cancer-diagnostics, -therapeutics, and -prognosis.

Association of Polymorphisms (rs 1799782, rs25489 and rs25487) in XRCC1 and (rs 13181) XPD genes with Acute Coronary Artery Syndrome in Subjects from Multan, Pakistan.

Acute coronary artery syndrome (ACS) is the major cause of mortality in Pakistan with genetic and environmental influence on the incidence of the disease. This case-control study was designed to find out if a correlation is existing between ACS and single nucleotide polymorphisms (SNPs) in DNA repair genes XPD [at codon 751, rs 13181 (Lys to Gln)] and XRCC1 [at codon 399, rs25487 (Arg to Gln); 280, rs25489 (Arg to His) and 194, rs 1799782 (Arg to Trp)] either individually or in various combination with each other (haplotype analysis). The objective of this study was to find out the association of various studied risk factors and serum lipid profile of the subjects with the disease, if any. PCR-RFLP method was used to determine genotype at specific codon in 221 subjects (115 ACS patients and 106 healthy controls) from Southern Punjab population. Genotypic and allelic frequency distribution among the cases and controls revealed that all the studied SNPs were not individually associated with the ACS. Haplotype analysis revealed that subjects having wild type combination of all three XRCC1 SNPs had greater susceptibility to ACS than any other studied genotypic combinations. Analysis of risk factors revealed that hypertension (P<0.001), age (P=0.05), education (P<0.001), gender (P<0.001), family history (P=0.005), smoking habit (P=0.002) and diabetes (P<0.001) were significantly associated with the incidence of ACS. Serum lipid profile analysis indicated that cholesterol level was significantly higher (P=0.048) in patients (161.5mg/dL) than controls (142.1mg/dL) while triglyceride remained unaffected (P=0.87) when compared between the two treatments.

A PCR-RFLP based protocol for the detection of hepatitis B virus variants in some lamivudine-untreated chronic hepatitis B virus carriers in Pakistan.

Hepatitis B virus (HBV) affects more than 350 million people worldwide and is a leading cause of morbidity and mortality in developing countries like Pakistan. Lamivudine has potential to inhibit hepatitis B virus (HBV) replication but long term lamivudine treatment results in mutations in YMDD region of HBV, making this therapy ineffective. In this study, we have optimized a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) based protocol to detect two mutations in HBV DNA polymerase gene (at codon 528 and 552) in chronic hepatitis patients, without any prior lamivudine treatment. HBV genome was extracted and tested by PCR-RFLP for detection of mutations in polymerase gene. Variations in HBV genome were not detected in enrolled patients confirming that lamivudine can be used to treat chronic Hepatitis B in these patients. Several studies have reported the natural occurrence of mutation in YMDD motif of polymerase gene in chronic hepatitis B patients, not treated with lamivudine, but these mutants were not detected in Pakistani lamivudine-untreated chronic hepatitis B patients.

Combined effect of menopause age and genotype on occurrence of breast cancer risk in Pakistani population.

UNLABELLED: Cancer incidences and mortality rates are rapidly increasing and breast cancer is among the most frequent malignancy experienced in women worldwide. The occurrence of breast cancer could be associated with various social, cultural, environmental, life-style, hormonal and genetic factors. OBJECTIVE: To establish if PvuII and XbaI polymorphisms of estrogen receptor alpha would make Pakistani women more susceptible to breast cancer. Furthermore, association between breast cancer and various factors was also explored to establish the contributing factors in breast cancer in Pakistani population. SUBJECTS AND METHODS: Two hundred samples, aged 15-65 years, consisting of 100 breast cancer patients and 100 control samples were ascertained for this case-control study in order to evaluate the factors related to disease incidence. 5-7 ml of blood sample of each participating women in the study was collected and analyzed for polymorphisms of PvuII and XbaI using PCR-RFLP method. RESULTS: The menopause had strong influence on incidences of cancer with ca 18-fold increase in risk of breast cancer in women with menopause compared with non-menopaused. Furthermore significant impact of menopause age (P<0.0001) was observed on the incidence of cancer, as high rate of cancer incidence was observed in patients with age between 36 and 45 years (P<0.0001). Similarly, the genotype XbaI had significant influence on the incidence of the disease with heterozygous genotype of XbaI was 45% higher than wild type in cancerous cases. The menopausal women having heterozygous and homozygous mutants of PvuII or XbaI genotypes were strongly correlated with breast cancer (P<0.01). CONCLUSION: The polymorphism of genes involving estrogen-metabolizing pathway and estrogen receptor pathway may play an important role in the etiology of breast cancer in Pakistani women.