UV Damage Response Research Articles

Abstract 2029: The ER stress protein IRE1a regulates intracellular calcium, ROS, and the UV damage response through the IP3R inhibitor CIB1

Abstract UV induced DNA damage is the primary driver of human skin cancer. UV can also cause endoplasmic reticulum (ER) stress, but the role of unfolded protein response (UPR) in the cellular response to UV exposure is not clear. Inositol-requiring enzyme 1α (IRE1α) is one of the key regulators of the UPR but its function in the UV response has not been resolved. Here we show that IRE1α, is transiently dephosphorylated and its RNase activity downregulated by UVB resulting in reduced XBP1 splicing. Keratinocytes with either IRE1α knockout or knockdown have elevated cytosolic calcium and ROS resulting in increased apoptosis after UV. Pharmacological inhibition of the ER inositol triphosphate receptor (IP3R) or phospholipase C (PLC) restored normal cytosolic calcium and ROS levels to cells lacking IRE1α. While loss of IRE1α did not alter the frequency of cells with UV-induced cyclo-pyrimidine dimers (CPD), it significantly reduced the percent of γH2AX positive keratinocytes 6 hours after UVB irradiation. This percent of CPD positive cells and the level of γH2AX 6 hours after UV suggesting a defect in the DNA repair response. Pharmacological inhibition of IRE1α RNase or Xbp1 knockdown did not phenocopy IRE1α loss, but overexpression of IRE1α in IRE1α knockdown cells restored γH2AX expression and reduced keratinocyte apoptosis. IRE1α overexpression also enhanced the survival of keratinocytes deficient in p53 function after UV irradiation. Additionally, mice with an epidermal deletion of IRE1α showed more rapid loss of UV-induced CPD positive keratinocytes compared to wildtype mice, primarily through accelerated sloughing of UV damaged cells from the skin. Knockdown of Calcium and integrin binding protein 1 (CIB1), which binds and inhibits the IP3R, elevated cytosolic calcium in keratinocytes and suppressed UV-induced γH2AX. Knockdown of Apoptosis signal-regulating kinase 1 (ASK1), which can interact with IRE1α through TRAF2 as well as CIB1, restored normal levels of UV-induced γH2AX to IRE1α knockdown keratinocytes. Together these results show that IRE1α is essential for maintaining normal cellular calcium and ROS levels and the UV-induced repair response, and suggest a model where IRE1a activation state drives CIB1 binding either to the IP3R or ASK1 to regulate ER calcium efflux, ROS and DNA repair responses following UV. Citation Format: Jeongin Son, Saie Mogre, Adam Glick. The ER stress protein IRE1a regulates intracellular calcium, ROS, and the UV damage response through the IP3R inhibitor CIB1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2029.

UV Laser-Induced, Time-Resolved Transcriptome Responses of Saccharomyces cerevisiae.

We determined the effect on gene transcription of laser-mediated, long-wavelength UV-irradiation of Saccharomyces cerevisiae by RNAseq analysis at times T15, T30, and T60 min after recovery in growth medium. Laser-irradiated cells were viable, and the transcriptional response was transient, with over 400 genes differentially expressed at T15 or T30, returning to basal level transcription by T60. Identification of transcripts exhibiting enhanced differential expression that were unique to UV laser-irradiation were identified by imposing a stringent significance cut-off (P < 0.05, log2 difference >2) then filtering out genes known as environmental stress response (ESR) genes. Using these rigorous criteria, 56 genes were differentially expressed at T15; at T30 differential expression was observed for 57 genes, some of which persisted from T15. Among the highly up-regulated genes were those supporting amino acid metabolic processes sulfur amino acids, methionine, aspartate, cysteine, serine), sulfur regulation (hydrogen sulfite metabolic processes, sulfate assimilation, sulfate reduction), proteasome components, amino acid transporters, and the iron regulon. At T30, the expression profile shifted to expression of transcripts related to catabolic processes (oxidoreductase activity, peptidase activity). Transcripts common to both T15 and T30 suggested an up-regulation of catabolic events, including UV damage response genes, and protein catabolism via proteasome and peptidase activity. Specific genes encoding tRNAs were among the down-regulated genes adding to the suggestion that control of protein biosynthesis was a major response to long-wave UV laser irradiation. These transcriptional responses highlight the remarkable ability of the yeast cell to respond to a UV-induced environmental insult.

Rev1 plays central roles in mammalian DNA-damage tolerance in response to UV irradiation.

Rev1, a Y-family DNA polymerase, is involved in the tolerance of DNA damage by translesion DNA synthesis (TLS). Previous studies have shown that the C-terminal domain (CTD) and ubiquitin (Ub)-binding (UBM) domains of Rev1 play important roles in UV-damage tolerance, but how these domains contribute to the process remains unclear. In this study, we created Ub mutations in a proliferating cell nuclear antigen (PCNA)-Ub fusion that differentially affect its interaction with Rev1 and Polη and found that UV-damage tolerance depends on its interaction with Rev1 but not Polη. We also created Rev1-UBM mutations altering its interaction with a PCNA-Ub fusion and Rev1-CTD mutations affecting its interaction with Polη and the Rev7 subunit of Polζ. We thus demonstrated that elevated expression of Rev1 alone is sufficient to confer enhanced UV-damage tolerance and that this tolerance depends on its physical interaction with monoubiquitinated PCNA and Polζ but is independent of Polη. Collectively, these studies reveal central roles played by Rev1 in coordinating UV-damage response pathway choice in mammalian cells.

The Role of Dynamic m6 A RNA Methylation in Photobiology.

N6 -methyladenosine (m6 A) is the most abundant internal RNA modification among numerous post-transcriptional modifications identified in eukaryotic mRNA. m6 A modification of RNA is catalyzed by the "writer" m6 A methyltransferase enzyme complex, consisting of METTL3, METTL14, WTAP and KIAA1429. The m6 A modification is reversible and can be removed by "eraser" m6 A demethylase enzymes, namely, FTO and ALKBH5. The biological function of m6 A modification on RNA is carried out by RNA-binding effector proteins called "readers." Varied functions of the reader proteins regulate mRNA metabolism by affecting stability, translation, splicing or nuclear export. The epitranscriptomic gene regulation by m6 A RNA methylation regulates various pathways, which contribute to basic cellular processes essential for cell maintenance, development and cell fate, and affect response to external stimuli and stressors. In this review, we summarize the recent advances in the regulation and function of m6 A RNA methylation, with a focus on UV-induced DNA damage response and the circadian clock machinery. Insights into the mechanisms of m6 A RNA regulation and post-transcriptional regulatory function in these biological processes may facilitate the development of new preventive and therapeutic strategies for various diseases related to dysregulation of UV damage response and circadian rhythm.

Segmental duplications and evolutionary acquisition of UV damage response in the SPATA31 gene family of primates and humans

BackgroundSegmental duplications are an abundant source for novel gene functions and evolutionary adaptations. This mechanism of generating novelty was very active during the evolution of primates particularly in the human lineage. Here, we characterize the evolution and function of the SPATA31 gene family (former designation FAM75A), which was previously shown to be among the gene families with the strongest signal of positive selection in hominoids. The mouse homologue for this gene family is a single copy gene expressed during spermatogenesis.ResultsWe show that in primates, the SPATA31 gene duplicated into SPATA31A and SPATA31C types and broadened the expression into many tissues. Each type became further segmentally duplicated in the line towards humans with the largest number of full-length copies found for SPATA31A in humans. Copy number estimates of SPATA31A based on digital PCR show an average of 7.5 with a range of 5–11 copies per diploid genome among human individuals. The primate SPATA31 genes also acquired new protein domains that suggest an involvement in UV response and DNA repair. We generated antibodies and show that the protein is re-localized from the nucleolus to the whole nucleus upon UV-irradiation suggesting a UV damage response. We used CRISPR/Cas mediated mutagenesis to knockout copies of the gene in human primary fibroblast cells. We find that cell lines with reduced functional copies as well as naturally occurring low copy number HFF cells show enhanced sensitivity towards UV-irradiation.ConclusionThe acquisition of new SPATA31 protein functions and its broadening of expression may be related to the evolution of the diurnal life style in primates that required a higher UV tolerance. The increased segmental duplications in hominoids as well as its fast evolution suggest the acquisition of further specific functions particularly in humans.

Abstract 3023: Chromatin accessibility underlies the tumor suppressor role of BAF (mSWI/SNF) complexes in many human cancers

Abstract Recent exome sequencing studies of human cancer indicate that ∼20% of malignancies have mutations in the subunits of the BAF (mSWI/SNF) chromatin remodeling complex, implicating the complex as a major tumor suppressor. However, the mechanisms of tumor suppression remain uncertain. To understand how defective BAF subunits contribute to cancer, we analyzed the genomic states of 5,659 patient samples derived from 22 tumor types, and compared samples containing genetic defects of the BAF complex against samples containing no such defects. We show that in the majority of cancer types, tumors with BAF subunit defects have significantly higher mutation counts than cancers of the same tissue lacking BAF defects. By reconstructing the mutational processes active in each patient sample, we find that excess mutations in BAF-deficient cancers represent the signatures of defective mismatch repair, cytidine deamination, UV-damage response, DNA end joining, Pol epsilon function, and other enzymatic processes acting on DNA. In four cancer types where ChIP-seq tracks were available for the healthy cell type of origin, we tracked the locations of over 280,000 mutations, and find that excess mutations in BAF-deficient tumors are biased towards regions of elevated H3K36me3 and low H3K27me3 in their corresponding non-cancerous tissues. We hypothesized that these mutations resulted from inaccessibility for repair and maintenance factors when a BAF subunit is defective. We tested this hypothesis using topoisomerase IIa binding as model for processes that require enzymatic access to DNA, and found that rapid conditional deletion of the ATPase Brg led to an increase in genome-wide deposition of H3K27me3 at Brg-dependent topoisomerase IIa sites from an initial low state. Our data indicate that the tumor-suppressive functions of BAF complexes arise largely from the requirement for BAF complexes to maintain genomic stability by providing access to DNA for repair and maintenance mechanisms. Note: This abstract was not presented at the meeting. Citation Format: Courtney Hodges, Diana C. Hargreaves, Erik L. Miller, Gerald R. Crabtree. Chromatin accessibility underlies the tumor suppressor role of BAF (mSWI/SNF) complexes in many human cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3023. doi:10.1158/1538-7445.AM2015-3023

Abstract NG05: Chromatin accessibility underlies the tumor-suppression role of BAF (mSWI/SNF) complexes in many malignancies

Abstract The mammalian SWI/SNF (mSWI/SNF or BAF) complex is an ATP-dependent chromatin remodeler long thought to be a tumor suppressor because it is frequently inactivated in cell lines. It was definitively identified as a tumor suppressor in malignant rhabdoid tumors (MRTs), an aggressive pediatric cancer. In most of these tumors, the dedicated BAF subunit SMARCB1 (BAF47) undergoes loss-of-heterozygosity, with no other large-scale chromosomal instability, leading to the conclusion that the tumor-suppression mechanism in MRTs is “epigenetic” in nature, meaning that it leads to changes in gene expression without necessarily altering other sequences of DNA. However, these tumors account for a small fraction of all cancers containing mutations in BAF subunits, and other evidence implicates the BAF complex in DNA repair processes, making its tumor-suppression mechanism uncertain. We sought to determine the applicability of this epigenetic model of tumor suppression in the large number of other cancers bearing BAF subunit mutations. To answer this question, we analyzed exome sequencing and genomic copy-number data derived from 5,659 patient samples, together representing 22 cancer types and over 4,500,000 genetic alterations, to compare the genomic states of tumors with BAF subunit defects against similar tumors that lacked such defects. We find that BAF subunits collectively are mutated significantly more frequently than expected in 19 of the 22 cancer types examined, far more than expected. The increase in BAF subunit mutations was statistically robust, both in samples with low mutation rates (e.g., lower-grade glioma), as well as those with high rates (e.g., melanoma, tumors of the lung), indicating that mutation of the complex is under positive selection in the vast majority of cancers. We also performed an analysis of copy-number variation of BAF subunits in each cancer, and find recurrent patterns of biallelic deletions and amplifications. In many malignancies, mutation and biallelic deletion of BAF subunits occurs at rates comparable to or higher than well-known tumor suppressors, such as TP53, PTEN, and RB1. In the majority of cancer types, tumors with BAF subunit defects have higher mutational rates than cancers of the same tissue lacking such defects. By reconstructing the mutational processes active in each patient sample, we find that excess mutations in BAF-deficient cancers represent the signatures of defective mismatch repair, cytidine deamination, UV-damage response, DNA end joining, Pol epsilon function, and other enzymatic processes acting on DNA. In four cancer types where ChIP-seq tracks were available for the healthy cell type of origin, we tracked the locations of over 280,000 mutations, and find that excess mutations in BAF-deficient tumors are biased towards regions of elevated H3K36me3 and low H3K27me3 in their corresponding non-cancerous tissues. We hypothesized that these mutations resulted from inaccessibility for repair and maintenance factors when a BAF subunit is defective. We tested this hypothesis using topoisomerase II alpha binding as representative of enzymes that require access to DNA, and find that rapid conditional deletion of the BAF ATPase Brg led to an increase in genome-wide deposition of H3K27me3 at Brg-dependent topoisomerase II alpha sites from an initial low state, contributing to loss of enzyme accessibility at its target sites. Our data indicate that the tumor suppressive functions of BAF complexes arise from their contribution to maintaining genomic stability by providing access to DNA for repair and maintenance mechanisms. Consistent with this conclusion, we examined levels of P53 and phospho-P53 in mouse embryonic fibroblasts expressing a tamoxifen-inducible deletion of Brg, the ATPase of the BAF complex. Deletion of Brg causes a significant increase in the levels of P53 and phospho-P53 following DNA damage by a panel of conditions, confirming that genomic stability and DNA repair is compromised upon loss of BAF activity. Our data are consistent with a general model, wherein DNA accessibility is maintained at a steady state arising from a balance between silencing factors and factors (including the BAF complex) that maintain accessibility. Many repair and maintenance factors operate on DNA that require at least transient accessibility, making the BAF complex an upstream contributor to several activities. When BAF activity is compromised, the equilibrium is shifted towards a state that is less accessible to repair enzymes. This disruption of normal repair and maintenance function leads to accumulation of mutations. Our analysis of 5,659 patient genomes indicates that this is the mechanism of tumor suppression under positive selection in the majority of human malignancies. Citation Format: Courtney Hodges, Diana Hargreaves, Erik Miller, Gerald R. Crabtree. Chromatin accessibility underlies the tumor-suppression role of BAF (mSWI/SNF) complexes in many malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr NG05. doi:10.1158/1538-7445.AM2015-NG05

The Deubiquitinating Enzyme USP24 Is a Regulator of the UV Damage Response

Regulation of p53 by ubiquitination and deubiquitination is important for its function. In this study, we demonstrate that USP24 deubiquitinates p53 in human cells. Functional USP24 is required for p53 stabilization, and p53 destabilization in USP24-depleted cells can be corrected by the forced expression of USP24. We show that USP24 depletion renders cells resistant to apoptosis after UV irradiation, consistent with the requirement of USP24 for p53 stabilization and PUMA activation invivo. Additionally, purified USP24 protein is able to cleave ubiquitinated p53 invitro. Importantly, cells with USP24 depletion exhibited significantly elevated mutation rates at the endogenous HPRT locus, implying an important role for USP24 in maintaining genome stability. Our data reveal that the USP24 deubiquitinase regulates the DNA damage response by directly targeting the p53 tumor suppressor.

Abstract A38: A functional connection between the SWI/SNF-BAF ATPase BRG1 and the tumor suppressor BRCA1 in DNA damage response

Abstract We and others have shown that the SWI/SNF (BAF) chromatin remodeling complex plays a role in the repair of UV-induced DNA damage. It was proposed that chromatin remodeling activities are utilized to increase the accessibility of NER machinery and checkpoint factors to the damaged DNA. It was demonstrated recently that BRCA1 contributes to UV damage response by promoting photoproduct excision, triggering post-UV checkpoint activation and post replicative repair. In this study, we show that BRCA1 rapidly binds to UV damage sites when cells are undergoing DNA synthesis. In contrast, two phosphorylated forms of BRCA1 do not accumulate at sites of UV damage. Depletion of BRG1, a core subunit of the human SWI/SNF-BAF complex, impairs the recruitment of BRCA1 to the damage sites and attenuates DNA damage induced BRCA1 phosphorylation. At UV lesions-stalled replication forks, BRG1 promotes RPA phosphorylation in response to UV irradiation, since UV-induced phosphorylation of chromatin bound RPA drops significantly when BRG1 is depleted in human cells. In addition, activation of ATR/ATM is attenuated when BRG1 is depleted. We propose that BRG1 functions upstream of BRCA1 and modulates its response to UV irradiation. Citation Format: Ling Zhang, Hua Chen, Feng Gong. A functional connection between the SWI/SNF-BAF ATPase BRG1 and the tumor suppressor BRCA1 in DNA damage response. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A38.

Abstract 613: The chromatin remodeling protein BRG1 modulates BRCA1’s response to UV irradiation.

Abstract The SWI/SNF chromatin remodeling complex plays a role in the repair of UV-induced DNA damage. It was proposed that chromatin remodeling activities are utilized to increase the accessibility of NER machinery and checkpoint factors to the damaged DNA. It was shown recently that BRCA1 contributes to UV damage response by promoting photoproduct excision, triggering post-UV checkpoint activation and post replicative repair. In this study, we show that BRCA1 rapidly binds to UV damage sites when cells are undergoing DNA synthesis. In contrast, two phosphorylated forms of BRCA1 do not accumulate at sites of UV damage. Depletion of BRG1, a core subunit of the human SWI/SNF-BAF complex, impairs the recruitment of BRCA1 to the damage sites and attenuates DNA damage induced BRCA1 phosphorylation. At UV lesions-stalled replication forks, BRG1 promotes RPA phosphorylation in response to UV irradiation, since UV-induced phosphorylation of chromatin bound RPA drops significantly when BRG1 is depleted in human cells. In addition, activation of ATR is attenuated when BRG1 is depleted. We propose that BRG1 functions upstream of BRCA1 and modulates its response to UV irradiation. Citation Format: Ling Zhang, Hua Chen, Feng Gong. The chromatin remodeling protein BRG1 modulates BRCA1’s response to UV irradiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 613. doi:10.1158/1538-7445.AM2013-613 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

The chromatin remodeling protein BRG1 modulates BRCA1 response to UV irradiation by regulating ATR/ATM activation

The SWI/SNF chromatin remodeling complex plays a role in the repair of UV-induced DNA damage. It was proposed that chromatin remodeling activities are utilized to increase the accessibility of nucleotide excision repair (NER) machinery and checkpoint factors to the damaged DNA. It was shown recently that BRCA1 contributes to UV damage response by promoting photoproduct excision, triggering post-UV checkpoint activation and post-replicative repair. In this study, we show that BRCA1 rapidly binds to UV damage sites when cells are undergoing DNA synthesis. In contrast, two phosphorylated forms of BRCA1 do not accumulate at sites of UV damage. Depletion of BRG1, a core subunit of the human SWI/SNF-BAF complex, impairs the recruitment of BRCA1 to the damage sites and attenuates DNA damage induced BRCA1 phosphorylation. At UV lesions-stalled replication forks, BRG1 promotes RPA phosphorylation in response to UV irradiation, since UV-induced phosphorylation of chromatin bound RPA drops significantly when BRG1 is depleted in human cells. Importantly, activation of the ATM/ATR kinases is attenuated when BRG1 is depleted. We propose that BRG1 modulates BRCA1 response to UV irradiation by regulating ATM/ATR activation.

γH2Ax: Biomarker of Damage or Functional Participant in DNA Repair “All that Glitters Is not Gold!”

The phosphorylation of H2Ax on its S139 site, γH2Ax, is important for the assembly of repair complexes at DNA double strand breaks (DSBs). The formation and functional role of γH2Ax after other kinds of DNA damage, especially UV light, where DSBs are rare, is less clear. Following UV light in the UVB and UVC ranges, complex distributions of γH2Ax can be identified, quite unlike the discrete enumerable foci seen after ionizing radiation. Several distinct distributions of γH2Ax occur: a low level nuclear-wide distribution of γH2Ax occurs during nucleotide excision repair; irregular focal distributions occur at arrested replication forks; high intensity nuclear-wide γH2Ax occurs in association with S-phase apoptosis. The intensity and distributions of γH2Ax vary according to the activity of excision repair, bypass polymerase and apoptotic caspases. The frequency of DSBs at arrested replication forks is low but highly variable in different cell types, and probably caused by enzymatic action. Despite the prominence of S139 phosphorylation following UV damage, mutation of this site has no influence on the UV damage response indicating that γH2Ax is a biomarker but not a participant in the UV-DNA damage response.

KIAA0101 Interacts with BRCA1 and Regulates Centrosome Number

To find genes and proteins that collaborate with BRCA1 or BRCA2 in the pathogenesis of breast cancer, we used an informatics approach and found a candidate BRCA interactor, KIAA0101, to function like BRCA1 in exerting a powerful control over centrosome number. The effect of KIAA0101 on centrosomes is likely direct, as its depletion does not affect the cell cycle, KIAA0101 localizes to regions coincident with the centrosomes, and KIAA0101 binds to BRCA1. We analyzed whether KIAA0101 protein is overexpressed in breast cancer tumor samples in tissue microarrays, and we found that overexpression of KIAA0101 correlated with positive Ki67 staining, a biomarker associated with increased disease severity. Furthermore, overexpression of the KIAA0101 gene in breast tumors was found to be associated with significantly decreased survival time. This study identifies KIAA0101 as a protein important for breast tumorigenesis, and as this factor has been reported as a UV repair factor, it may link the UV damage response to centrosome control.

Involvement of Global Genome Repair, Transcription Coupled Repair, and Chromatin Remodeling in UV DNA Damage Response Changes during Development

Nucleotide Excision Repair (NER), which removes a variety of helix-distorting lesions from DNA, is initiated by two distinct DNA damage-sensing mechanisms. Transcription Coupled Repair (TCR) removes damage from the active strand of transcribed genes and depends on the SWI/SNF family protein CSB. Global Genome Repair (GGR) removes damage present elsewhere in the genome and depends on damage recognition by the XPC/RAD23/Centrin2 complex. Currently, it is not well understood to what extent both pathways contribute to genome maintenance and cell survival in a developing organism exposed to UV light. Here, we show that eukaryotic NER, initiated by two distinct subpathways, is well conserved in the nematode Caenorhabditis elegans. In C. elegans, involvement of TCR and GGR in the UV-induced DNA damage response changes during development. In germ cells and early embryos, we find that GGR is the major pathway contributing to normal development and survival after UV irradiation, whereas in later developmental stages TCR is predominantly engaged. Furthermore, we identify four ISWI/Cohesin and four SWI/SNF family chromatin remodeling factors that are implicated in the UV damage response in a developmental stage dependent manner. These in vivo studies strongly suggest that involvement of different repair pathways and chromatin remodeling proteins in UV-induced DNA repair depends on developmental stage of cells.

Abstract 3935: H3K56 is deacetylated in response to UV-irradiation and recovery of acetylation is regulated by ATR and ATM checkpoint kinases

Abstract DNA damage results in cell cycle arrest by checkpoint activation until successful completion of DNA repair. Cell cycle arrest and DNA repair processes involve changes in chromatin architecture, which help access of proteins involved in these processes. After DNA repair, the chromatin structure and cell cycle progression are restored, but the mechanism is largely unknown. Recent studies implicated a role of histone chaperones, CAF1 and ASF1 in DNA damage repair through histone modification as well as histone assembly and disassembly. CAF1 depletion can lead to a G1 and S phase checkpoint response, in an ATR-dependent manner. In yeast, ASF1 chaperone associates with Rtt109 HAT complex which is required for H3K56 acetylation and is important for checkpoint recovery. Recently, Das et. al. showed that mammalian ASF1 interacts with p300/CBP histone acetylase and acetylates H3K56, which is deposited at the damage site in a CAF1-dependent manner. In contrast, Tjeertes et. al. showed that H3K56Ac is present in undamaged cells and is reduced in response to DNA damage. Due to the contradictory nature of H3K56 acetylation from these studies, we wanted to establish a solid foundation related to the nature of this important UV damage response event in human cells. For this, cells were irradiated and the status of H3K56 acetylation was determined immediately and up to 48 hr after UV-irradiation. Our data unambiguously show that in comparison to the steady levels of control actin, H3K56 acetylation decreased gradually and was completely eliminated at 24 hr following 20 J/m2 UV dose. However, H3K56 acetylation exhibited full recovery after 48 hr at 2.5, 5 and 10 J/m2 UV doses. Thus, UV-irradiation causes H3K56 deacetylation, which is promptly restored possibly due to the successful repair and checkpoint recovery. We observed a similar effect on H3K56 deacetylation in response to UV damage using NHF cells. Since, H3K56 acetylation has been implicated in checkpoint recovery after completion of DNA repair, we tested whether H3K56 acetylation/deacetylation is regulated by ATR and ATM checkpoint kinases. Interestingly, in Seckel cells (lower ATR level) H3K56 acetylation is dramatically reduced even in the absence of UV-irradiation suggesting that H3K56 acetylation is regulated by ATR. In contrast, in A-T cells (ATM-deficient), H3K56 acetylation did not change in the absence of UV-irradiation. In A-T cells, H3K56 is deacetylated in response to UV-irradiation as in NHF cells, but the acetylation did not recover after 48 hr post-repair. These data clearly indicate a role of ATR/ATM in recovery of H3K56 acetylation. We propose that H3K56 acetylation presents a histone mark needed for ATR/ATM-mediated checkpoint recovery. (This work was supported by NIH grants CA93413, ES2388 and ES12991) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3935.

Genome-wide analysis of YY2 versus YY1 target genes

Yin Yang 1 (YY1) is a critical transcription factor controlling cell proliferation, development and DNA damage responses. Retrotranspositions have independently generated additional YY family members in multiple species. Although Drosophila YY1 [pleiohomeotic (Pho)] and its homolog [pleiohomeotic-like (Phol)] redundantly control homeotic gene expression, the regulatory contributions of YY1-homologs have not yet been examined in other species. Indeed, targets for the mammalian YY1 homolog YY2 are completely unknown. Using gene set enrichment analysis, we found that lentiviral constructs containing short hairpin loop inhibitory RNAs for human YY1 (shYY1) and its homolog YY2 (shYY2) caused significant changes in both shared and distinguishable gene sets in human cells. Ribosomal protein genes were the most significant gene set upregulated by both shYY1 and shYY2, although combined shYY1/2 knock downs were not additive. In contrast, shYY2 reversed the anti-proliferative effects of shYY1, and shYY2 particularly altered UV damage response, platelet-specific and mitochondrial function genes. We found that decreases in YY1 or YY2 caused inverse changes in UV sensitivity, and that their combined loss reversed their respective individual effects. Our studies show that human YY2 is not redundant to YY1, and YY2 is a significant regulator of genes previously identified as uniquely responding to YY1.

Role of the mammalian SWI/SNF chromatin remodeling complex in the cellular response to UV damage

Mammalian cells exhibit complex cellular responses to DNA damage, including cell cycle arrest, DNA repair and apoptosis. Defects in any one of these responses can result in carcinogenesis. Absence of the chromatin remodeling complex Swi/Snf is found in many instances of cancer, and we have investigated its role in the UV damage response. The human carcinoma cell line SW13 is deficient in Swi/Snf and is very sensitive to UV radiation. In contrast, SW13 cells with ectopic Brg1 expression regain active Swi/Snf and become significantly more resistant to UV radiation. Sensitivity to UV light correlates well with dramatic UV induced apoptosis in SW13 cells, but not in SW13 cells expressing Brg1. We show that SW13 cells synchronized at the G1/S border progress into S phase after UV irradiation, and this checkpoint deficiency is corrected after Brg1 expression is restored. Interestingly, Brg1 expression in SW13 cells restores expression of two DNA damage responsive genes, Gadd45a and p21. Furthermore, Gadd45a induction and p21 degradation were observed in the Brg1-expressing SW13 cells after UV irradiation. Our findings demonstrate that Swi/Snf protects cells against deleterious consequences of UV induced DNA damage. These results also indicate that Swi/Snf may play a role in replication checkpoint activation after UV damage via regulation of the two PCNA-binding proteins Gadd45a and p21.

Sensitive, real-time monitoring of UV-induced stress in a single, live plant cell using an optical trap

An optical trap is used to monitor bio-deleterious effects induced by ultra-violet (UV) radiation in a single, live cell. When placed in a laser optical trap, Chlamydomonas reinhardtii cells undergo rotation because of the interplay of flagellar-based forces and optical forces generated by the laser light. Such rotation is sustained by the trapped cell's flagellar action, thereby unveiling a robust and quantitative cellular assay for flagellar function. UV induces flagellar damage, leading to dose-dependent attenuation of cellular rotation. At UV doses larger than ∼9Jm−2 rotational motion ceases. The ability of the cell to rotate in an optical trap is, therefore, a measure of the damage response of the cell. By monitoring cell rotation, we can quantify UV damage with high sensitivity, at threshold doses as low as 2Jm−2. Apart from the capability of UV-damage detection, our rotation assay is also sensitive to the cellular protective responses against such damage. To illustrate this additional facet, we quantify the efficacy of ascorbic acid in combating the UV-damage response of individual cells. Upon addition of this antioxidant, there is no cessation of rotations for doses as high as 25Jm−2. This represents the first single-cell sensor that robustly quantifies a complex pleiotropic cellular response.

Nucleolar protein NPM interacts with HDM2 and protects tumor suppressor protein p53 from HDM2-mediated degradation

Nucleophosmin (NPM, B23) is an abundant nucleolar phosphoprotein involved in ribosome biogenesis, and interacts with tumor suppressor proteins p53 and Rb. Here we show that NPM is a UV damage response protein that undergoes nucleoplasmic redistribution and regulates p53 and HDM2 levels and their interaction. By utilizing RNAi approaches and analyses of endogenous and ectopically expressed proteins, we demonstrate that NPM binds HDM2 and acts as a negative regulator of p53-HDM2 interaction. Viral stress, enforced by expression of Kaposi's sarcoma virus K cyclin, causes NPM redistribution, K cyclin-NPM association, and p53 stabilization by dissociation of HDM2-p53 complexes. The results demonstrate novel associations of HDM2 and K cyclin with NPM and implicate NPM as a crucial controller of p53 through inhibition of HDM2.