UV-exposure, endogenous DNA damage, and DNA replication errors shape the spectra of genome changes in human skin.

Human skin is continuously exposed to environmental DNA damage leading to the accumulation of somatic mutations over the lifetime of an individual. Mutagenesis in human skin cells can be also caused by endogenous DNA damage and by DNA replication errors. The contributions of these processes to the somatic mutation load in the skin of healthy humans has so far not been accurately assessed because the low numbers of mutations from current sequencing methodologies preclude the distinction between sequencing errors and true somatic genome changes. In this work, we sequenced genomes of single cell-derived clonal lineages obtained from primary skin cells of a large cohort of healthy individuals across a wide range of ages. We report here the range of mutation load and a comprehensive view of the various somatic genome changes that accumulate in skin cells. We demonstrate that UV-induced base substitutions, insertions and deletions are prominent even in sun-shielded skin. In addition, we detect accumulation of mutations due to spontaneous deamination of methylated cytosines as well as insertions and deletions characteristic of DNA replication errors in these cells. The endogenously induced somatic mutations and indels also demonstrate a linear increase with age, while UV-induced mutation load is age-independent. Finally, we show that DNA replication stalling at common fragile sites are potent sources of gross chromosomal rearrangements in human cells. Thus, somatic mutations in skin of healthy individuals reflect the interplay of environmental and endogenous factors in facilitating genome instability and carcinogenesis.

DNA damage responses in murine Pre-B cells with genetic deficiencies in damage response genes.

DNA damage can be generated in multiple ways from genotoxic and physiologic sources. Genotoxic damage is known to disrupt cellular functions and is lethal if not repaired properly. We compare the transcriptional programs activated in response to genotoxic DNA damage induced by ionizing radiation (IR) in abl pre-B cells from mice deficient in DNA damage response (DDR) genes Atm, Mre11, Mdc1, H2ax, 53bp1, and DNA-PKcs. We identified a core IR-specific transcriptional response that occurs in abl pre-B cells from WT mice and compared the response of the other genotypes to the WT response. We also identified genotype specific responses and compared those to each other. The WT response includes many processes involved in lymphocyte development and immune response, as well as responses associated with the molecular mechanisms of cancer, such as TP53 signaling. As expected, there is a range of similarity in transcriptional profiles in comparison to WT cells, with Atm-/- cells being the most different from the core WT DDR and Mre11 hypomorph (Mre11A/A) cells also very dissimilar to WT and other genotypes. For example, NF-kB-related signaling and CD40 signaling are deficient in both Atm-/- and Mre11A/A cells, but present in all other genotypes. In contrast, IR-induced TP53 signaling is seen in the Mre11A/A cells, while these responses are not seen in the Atm-/- cells. By examining the similarities and differences in the signaling pathways in response to IR when specific genes are absent, our results further illustrate the contribution of each gene to the DDR. The microarray gene expression data discussed in this paper have been deposited in NCBI's Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) and are accessible under accession number GSE116388.

p53-responsive TLR8 SNP enhances human innate immune response to respiratory syncytial virus.

The Toll-like receptor 8 (TLR8) has an important role in innate immune responses to RNA viral infections, including respiratory syncytial virus (RSV). We previously reported that TLR8 expression was increased directly by the tumor suppressor and transcription factor p53 via a single nucleotide polymorphism (SNP) (rs3761624) in the TLR8 promoter, thereby placing TLR8 in the p53/immune axis. Because this SNP is in linkage disequilibrium with other SNPs associated with several infectious diseases, we addressed the combined influence of p53 and the SNP on downstream inflammatory signaling in response to a TLR8 cognate ssRNA ligand. Using human primary lymphocytes, p53 induction by chemotherapeutic agents such as ionizing radiation caused SNP-dependent synergistic increases in IL-6 following incubation with an ssRNA ligand, as well as TLR8 RNA and protein expression along with p53 binding at the TLR-p53 SNP site. Because TLR8 is X-linked, the increases were generally reduced in heterozygous females. We found a corresponding association of the p53-responsive allele with RSV disease severity in infants hospitalized with RSV infection. We conclude that p53 can strongly influence TLR8-mediated immune responses and that knowledge of the p53-responsive SNP can inform diagnosis and prognosis of RSV disease and other diseases that might have a TLR8 component, including cancer.

Crohn's disease IRGM risk alleles are associated with altered gene expression in human tissues.

Crohn's disease (CD) is a chronic inflammatory gastrointestinal disorder. Genetic association studies have implicated dysregulated autophagy in CD. Among risk loci identified are a promoter single nucleotide polymorphism (SNP)( rs13361189 ) and two intragenic SNPs ( rs9637876 , rs10065172 ) in immunity-related GTPase family M ( IRGM) a gene that encodes a protein of the autophagy initiation complex. All three SNPs have been proposed to modify IRGM expression, but reports have been divergent and largely derived from cell lines. Here, analyzing RNA-Sequencing data of human tissues from the Genotype-Tissue Expression Project, we found that rs13361189 minor allele carriers had reduced IRGM expression in whole blood and terminal ileum, and upregulation in ileum of ZNF300P1, a locus adjacent to IRGM on chromosome 5q33.1 that encodes a long noncoding RNA. Whole blood and ileum from minor allele carriers had altered expression of multiple additional genes that have previously been linked to colitis and/or autophagy. Notable among these was an increase in ileum of LTF (lactoferrin), an established fecal inflammatory biomarker of CD, and in whole blood of TNF, a key cytokine in CD pathogenesis. Last, we confirmed that risk alleles at all three loci associated with increased risk for CD but not ulcerative colitis in a case-control study. Taken together, our findings suggest that genetically encoded IRGM deficiency may predispose to CD through dysregulation of inflammatory gene networks. Gene expression profiling of disease target tissues in genetically susceptible populations is a promising strategy for revealing new leads for the study of molecular pathogenesis and, potentially, for precision medicine. NEW & NOTEWORTHY Single nucleotide polymorphisms in immunity-related GTPase family M ( IRGM), a gene that encodes an autophagy initiation protein, have been linked epidemiologically to increased risk for Crohn's disease (CD). Here, we show for the first time that subjects with risk alleles at two such loci, rs13361189 and rs10065172 , have reduced IRGM expression in whole blood and terminal ileum, as well as dysregulated expression of a wide array of additional genes that regulate inflammation and autophagy.

Toll-like Receptor 4 Pathway Polymorphisms Interact with Pollution to Influence Asthma Diagnosis and Severity.

Asthma is a common chronic lung disease, the incidence and severity of which may be influenced by gene-environment interactions. Our objective was to examine associations between single nucleotide polymorphisms (SNPs) and combinations of SNPs in the toll-like receptor 4 (TLR4) pathway, residential distance to roadway as a proxy for traffic-related air pollution exposure, and asthma diagnosis and exacerbations. We obtained individual-level data on genotype, residential address, and asthma diagnosis and exacerbations from the Environmental Polymorphisms Registry. Subjects (n = 2,704) were divided into three groups (hyper-responders, hypo-responders, and neither) based on SNP combinations in genes along the TLR4 pathway. We geocoded subjects and calculated distance, classified as <250 m or ≥250 m, between residence and nearest major road. Relationships between genotype, distance to road, and odds of asthma diagnosis and exacerbations were examined using logistic regression. Odds of an asthma diagnosis among hyper-responders <250 m from a major road was 2.37(0.97, 6.01) compared to the reference group (p < 0.10). Hypo-responders ≥250 m from the nearest road had lower odds of activity limitations (0.46 [0.21, 0.95]) and sleeplessness (0.36 [0.12, 0.91]) compared to neither-responders (p < 0.05). Specific genotype combinations when combined with an individual's proximity to roadways, possibly due to traffic-related air pollution exposure, may affect the likelihood of asthma diagnosis and exacerbations.

A type I IFN-dependent DNA damage response regulates the genetic program and inflammasome activation in macrophages.

Macrophages produce genotoxic agents, such as reactive oxygen and nitrogen species, that kill invading pathogens. Here we show that these agents activate the DNA damage response (DDR) kinases ATM and DNA-PKcs through the generation of double stranded breaks (DSBs) in murine macrophage genomic DNA. In contrast to other cell types, initiation of this DDR depends on signaling from the type I interferon receptor. Once activated, ATM and DNA-PKcs regulate a genetic program with diverse immune functions and promote inflammasome activation and the production of IL-1ß and IL-18. Indeed, following infection with Listeria monocytogenes, DNA-PKcs-deficient murine macrophages produce reduced levels of IL-18 and are unable to optimally stimulate IFN-γ production by NK cells. Thus, genomic DNA DSBs act as signaling intermediates in murine macrophages, regulating innate immune responses through the initiation of a type I IFN-dependent DDR.

Depletion of ATR selectively sensitizes ATM-deficient human mammary epithelial cells to ionizing radiation and DNA-damaging agents.

DNA damage response (DDR) to double strand breaks is coordinated by 3 phosphatidylinositol 3-kinase-related kinase (PIKK) family members: the ataxia-telangiectasia mutated kinase (ATM), the ATM and Rad3-related (ATR) kinase and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). ATM and ATR are central players in activating cell cycle checkpoints and function as an active barrier against genome instability and tumorigenesis in replicating cells. Loss of ATM function is frequently reported in various types of tumors, thus placing more reliance on ATR for checkpoint arrest and cell survival following DNA damage. To investigate the role of ATR in the G2/M checkpoint regulation in response to ionizing radiation (IR), particularly when ATM is deficient, cell lines deficient of ATM, ATR, or both were generated using a doxycycline-inducible lentiviral system. Our data suggests that while depletion of ATR or ATM alone in wild-type human mammary epithelial cell cultures (HME-CCs) has little effect on radiosensitivity or IR-induced G2/M checkpoint arrest, depletion of ATR in ATM-deficient cells causes synthetic lethality following IR, which correlates with severe G2/M checkpoint attenuation. ATR depletion also inhibits IR-induced autophagy, regardless of the ATM status, and enhances IR-induced apoptosis particularly when ATM is deficient. Collectively, our results clearly demonstrate that ATR function is required for the IR-induced G2/M checkpoint activation and subsequent survival of cells with ATM deficiency. The synthetic lethal interaction between ATM and ATR in response to IR supports ATR as a therapeutic target for improved anti-cancer regimens, especially in tumors with a dysfunctional ATM pathway.

Gene expression signatures but not cell cycle checkpoint functions distinguish AT carriers from normal individuals.

Ataxia telangiectasia (AT) is a rare autosomal recessive disease caused by mutations in the ataxia telangiectasia-mutated gene (ATM). AT carriers with one mutant ATM allele are usually not severely affected although they carry an increased risk of developing cancer. There has not been an easy and reliable diagnostic method to identify AT carriers. Cell cycle checkpoint functions upon ionizing radiation (IR)-induced DNA damage and gene expression signatures were analyzed in the current study to test for differential responses in human lymphoblastoid cell lines with different ATM genotypes. While both dose- and time-dependent G1 and G2 checkpoint functions were highly attenuated in ATM-/- cell lines, these functions were preserved in ATM+/- cell lines equivalent to ATM+/+ cell lines. However, gene expression signatures at both baseline (consisting of 203 probes) and post-IR treatment (consisting of 126 probes) were able to distinguish ATM+/- cell lines from ATM+/+ and ATM-/- cell lines. Gene ontology (GO) and pathway analysis of the genes in the baseline signature indicate that ATM function-related categories, DNA metabolism, cell cycle, cell death control, and the p53 signaling pathway, were overrepresented. The same analyses of the genes in the IR-responsive signature revealed that biological categories including response to DNA damage stimulus, p53 signaling, and cell cycle pathways were overrepresented, which again confirmed involvement of ATM functions. The results indicate that AT carriers who have unaffected G1 and G2 checkpoint functions can be distinguished from normal individuals and AT patients by expression signatures of genes related to ATM functions.

Genome-wide small RNA sequencing and gene expression analysis reveals a microRNA profile of cancer susceptibility in ATM-deficient human mammary epithelial cells.

Deficiencies in the ATM gene are the underlying cause for ataxia telangiectasia, a syndrome characterized by neurological, motor and immunological defects, and a predisposition to cancer. MicroRNAs (miRNAs) are useful tools for cancer profiling and prediction of therapeutic responses to clinical regimens. We investigated the consequences of ATM deficiency on miRNA expression and associated gene expression in normal human mammary epithelial cells (HME-CCs). We identified 81 significantly differentially expressed miRNAs in ATM-deficient HME-CCs using small RNA sequencing. Many of these have been implicated in tumorigenesis and proliferation and include down-regulated tumor suppressor miRNAs, such as hsa-miR-29c and hsa-miR-16, as well as over-expressed pro-oncogenic miRNAs, such as hsa-miR-93 and hsa-miR-221. MicroRNA changes were integrated with genome wide gene expression profiles to investigate possible miRNA targets. Predicted mRNA targets of the miRNAs significantly regulated after ATM depletion included many genes associated with cancer formation and progression, such as SOCS1 and the proto-oncogene MAF. While a number of miRNAs have been reported as altered in cancerous cells, there is little understanding as to how these small RNAs might be driving cancer formation or how they might be used as biomarkers for cancer susceptibility. This study provides preliminary data for defining miRNA profiles that may be used as prognostic or predictive biomarkers for breast cancer. Our integrated analysis of miRNA and mRNA expression allows us to gain a better understanding of the signaling involved in breast cancer predisposition and suggests a mechanism for the breast cancer-prone phenotype seen in ATM-deficient patients.

Dissecting cellular responses to irradiation via targeted disruptions of the ATM-CHK1-PP2A circuit.

Exposure of proliferating cells to genotoxic stresses activates a cascade of signaling events termed the DNA damage response (DDR). The DDR preserves genetic stability by detecting DNA lesions, activating cell cycle checkpoints and promoting DNA damage repair. The phosphoinositide 3-kinase-related kinases (PIKKs) ataxia telangiectasia-mutated (ATM), ATM and Rad 3-related kinase (ATR) and DNA-dependent protein kinase (DNA-PK) are crucial for sensing lesions and signal transduction. The checkpoint kinase 1 (CHK1) is a traditional ATR target involved in DDR and normal cell cycle progression and represents a pharmacological target for anticancer regimens. This study employed cell lines stably depleted for CHK1, ATM or both for dissecting cross-talk and compensatory effects on G(2)/M checkpoint in response to ionizing radiation (IR). We show that a 90% depletion of CHK1 renders cells radiosensitive without abrogating their IR-mediated G(2)/M checkpoint arrest. ATM phosphorylation is enhanced in CHK1-deficient cells compared with their wild-type counterparts. This correlates with lower nuclear abundance of the PP2A catalytic subunit in CHK1-depleted cells. Stable depletion of CHK1 in an ATM-deficient background showed only a 50% reduction from wild-type CHK1 protein expression levels and resulted in an additive attenuation of the G(2)/M checkpoint response compared with the individual knockdowns. ATM inhibition and 90% CHK1 depletion abrogated the early G(2)/M checkpoint and precluded the cells from mounting an efficient compensatory response to IR at later time points. Our data indicates that dual targeting of ATM and CHK1 functionalities disrupts the compensatory response to DNA damage and could be exploited for developing efficient anti-neoplastic treatments.

DNA damage activates a complex transcriptional response in murine lymphocytes that includes both physiological and cancer-predisposition programs.

BACKGROUND: Double strand (ds) DNA breaks are a form of DNA damage that can be generated from both genotoxic exposures and physiologic processes, can disrupt cellular functions and can be lethal if not repaired properly. Physiologic dsDNA breaks are generated in a variety of normal cellular functions, including the RAG endonuclease-mediated rearrangement of antigen receptor genes during the normal development of lymphocytes. We previously showed that physiologic breaks initiate lymphocyte development-specific transcriptional programs. Here we compare transcriptional responses to physiological DNA breaks with responses to genotoxic DNA damage induced by ionizing radiation. RESULTS: We identified a central lymphocyte-specific transcriptional response common to both physiologic and genotoxic breaks, which includes many lymphocyte developmental processes. Genotoxic damage causes robust alterations to pathways associated with B cell activation and increased proliferation, suggesting that genotoxic damage initiates not only the normal B cell maturation processes but also mimics activated B cell response to antigenic agents. Notably, changes including elevated levels of expression of Kras and mmu-miR-155 and the repression of Socs1 were observed following genotoxic damage, reflecting induction of a cancer-prone phenotype. CONCLUSIONS: Comparing these transcriptional responses provides a greater understanding of the mechanisms cells use in the differentiation between types of DNA damage and the potential consequences of different sources of damage. These results suggest genotoxic damage may induce a unique cancer-prone phenotype and processes mimicking activated B cell response to antigenic agents, as well as the normal B cell maturation processes.

DNA double-strand breaks activate a multi-functional genetic program in developing lymphocytes.

DNA double-strand breaks are generated by genotoxic agents and by cellular endonucleases as intermediates of several important physiological processes. The cellular response to genotoxic DNA breaks includes the activation of transcriptional programs known primarily to regulate cell-cycle checkpoints and cell survival. DNA double-strand breaks are generated in all developing lymphocytes during the assembly of antigen receptor genes, a process that is essential for normal lymphocyte development. Here we show that in murine lymphocytes these physiological DNA breaks activate a broad transcriptional program. This program transcends the canonical DNA double-strand break response and includes many genes that regulate diverse cellular processes important for lymphocyte development. Moreover, the expression of several of these genes is regulated similarly in response to genotoxic DNA damage. Thus, physiological DNA double-strand breaks provide cues that can regulate cell-type-specific processes not directly involved in maintaining the integrity of the genome, and genotoxic DNA breaks could disrupt normal cellular functions by corrupting these processes.

DNA protein kinase-dependent G2 checkpoint revealed following knockdown of ataxia-telangiectasia mutated in human mammary epithelial cells.

Members of the phosphatidylinositol 3-kinase-related kinase family, in particular the ataxia-telangiectasia mutated (ATM) kinase and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), regulate cellular responses to DNA double-strand breaks. Increased sensitivity to ionizing radiation (IR) in DNA-PKcs- or ATM-deficient cells emphasizes their important roles in maintaining genome stability. Furthermore, combined knockout of both kinases is synthetically lethal, suggesting functional complementarity. In the current study, using human mammary epithelial cells with ATM levels stably knocked down by >90%, we observed an IR-induced G(2) checkpoint that was only slightly attenuated. In marked contrast, this G(2) checkpoint was significantly attenuated with either DNA-PK inhibitor treatment or RNA interference knockdown of DNA-PKcs, the catalytic subunit of DNA-PK, indicating that DNA-PK contributes to the G(2) checkpoint in these cells. Furthermore, in agreement with the checkpoint attenuation, DNA-PK inhibition in ATM-knockdown cells resulted in reduced signaling of the checkpoint kinase CHK1 as evidenced by reduced CHK1 phosphorylation. Taken together, these results show a DNA-PK-dependent component to the IR-induced G(2) checkpoint, in addition to the well-defined ATM-dependent component. This may have important implications for chemotherapeutic strategies for breast cancers.

ATM requirement in gene expression responses to ionizing radiation in human lymphoblasts and fibroblasts.

The heritable disorder ataxia telangiectasia (AT) is caused by mutations in the AT-mutated (ATM) gene with manifestations that include predisposition to lymphoproliferative cancers and hypersensitivity to ionizing radiation (IR). We investigated gene expression changes in response to IR in human lymphoblasts and fibroblasts from seven normal and seven AT-affected individuals. Both cell types displayed ATM-dependent gene expression changes after IR, with some responses shared and some responses varying with cell type and dose. Interestingly, after 5 Gy IR, lymphoblasts displayed ATM-independent responses not seen in the fibroblasts at this dose, which likely reflect signaling through ATM-related kinases, e.g., ATR, in the absence of ATM function.

ATM-dependent and -independent gene expression changes in response to oxidative stress, gamma irradiation, and UV irradiation.

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by progressive cerebellar degeneration, immunodeficiencies, telangiectasias, sensitivity to ionizing radiation, and high predisposition for malignancies. The ataxia telangiectasia mutated (ATM) gene encodes a protein (ATM) with serine/threonine kinase activity. DNA-double strand breaks are known to increase its kinase activity. While cells from individuals with AT are attenuated in their G(1)-, S- and G(2)-phase cell cycle checkpoint functions in response to gamma irradiation and oxidative stress, their response to UV irradiation appears to be equivalent to that of wild-type cells. In this study, we investigated changes in gene expression in response to gamma irradiation, oxidative stress, and UV irradiation, focusing on the dependence on ATM. Doses for all three treatments were selected that resulted in roughly an equivalent induction of a G(1) checkpoint response and inhibition of progression through S phase. To investigate gene expression changes, logarithmically growing wild-type and AT dermal diploid fibroblasts were exposed to either gamma radiation (5 Gy), oxidative stress (75 micro M t-butyl-hydroperoxide), or UV radiation (7.5 J/m(2)), and RNA was harvested 6 h after treatment. Gene expression analysis was performed using the NIEHS Human ToxChip 2.0 with approximately 1900 cDNA clones representing known genes and ESTs. All three treatments resulted in distinct patterns of gene expression changes, as shown previously. ATM-dependent and ATM-independent components were detected within these patterns, as were novel indications of involvement of ATM in regulation of transcription factors such as SP1, AP1 and MTF1.

Identification of distinct and common gene expression changes after oxidative stress and gamma and ultraviolet radiation.

The human genome is exposed to many different kinds of DNA-damaging agents. While most damage is detected and repaired through complex damage recognition and repair machineries, some damage has the potential to escape these mechanisms. Unrepaired DNA damage can give rise to alterations and mutations in the genome in an individual cell, which can result in malignant transformation, especially when critical genes are deregulated. In this study, we investigated gene expression changes in response to oxidative stress, gamma (gamma) radiation, and ultraviolet (UV) radiation and their potential implications in cancer development. Doses were selected for each of the three treatments, based on their ability to cause a similar G(1) checkpoint induction and slow down in early S-phase progression, as reflected by a comparable reduction in cyclin E-associated kinase activity of at least 75% in logarithmically growing human dermal diploid fibroblasts. To investigate gene expression changes, logarithmically growing dermal diploid fibroblasts were exposed to either gamma radiation (5 Gy), oxidative stress (75 microM of tert-butyl hydroperoxide (t-butyl-OOH)), or UV radiation (UVC) (7.5 J/m(2)) and RNA was harvested 6 h after treatment. Gene expression was analyzed using the NIEHS Human ToxChip 2.0 with approximately 1901 cDNA clones representing known genes and expressed sequence tags (ESTs). We were able to identify common and distinct responses in dermal diploid fibroblasts to the three different stimuli used. Within our analysis, gene expression profiles in response to gamma radiation and oxidative stress appeared to be more similar than profiles expressed after UV radiation. Interestingly, equivalent cyclin E-associated kinase activity reduction with all the three treatments was associated with greater transcriptional changes after UV radiation than after gamma radiation and oxidative stress. While samples treated with UV radiation displayed modulations of their mitogen activated protein kinase (MAPK) pathway, gamma radiation had its major influence on cell-cycle progression in S-phase and mitosis. In addition, cell cultures from different individuals displayed significant differences in their gene expression responses to DNA damage.