CUX1 transcription factor is required for optimal ATM/ATR-mediated responses to DNA damage

The p110 Cut homeobox 1 (CUX1) transcription factor regulates genes involved in DNA replication and chromosome segregation. Using a genome-wide-approach, we now demonstrate that CUX1 also modulates the constitutive expression of DNA damage response genes, including ones encoding ATM and ATR, as well as proteins involved in DNA damage-induced activation of, and signaling through, these kinases. Consistently, RNAi knockdown or genetic inactivation of CUX1 reduced ATM/ATR expression and negatively impacted hallmark protective responses mediated by ATM and ATR following exposure to ionizing radiation (IR) and UV, respectively. Specifically, abrogation of CUX1 strongly reduced ATM autophosphorylation after IR, in turn causing substantial decreases in (i) levels of phospho-Chk2 and p53, (ii) γ-H2AX and Rad51 DNA damage foci and (iii) the efficiency of DNA strand break repair. Similarly remarkable reductions in ATR-dependent responses, including phosphorylation of Chk1 and H2AX, were observed post-UV. Finally, multiple cell cycle checkpoints and clonogenic survival were compromised in CUX1 knockdown cells. Our results indicate that CUX1 regulates a transcriptional program that is necessary to mount an efficient response to mutagenic insult. Thus, CUX1 ensures not only the proper duplication and segregation of the genetic material, but also the preservation of its integrity

Differential requirement of a distal regulatory region for pre-initiation complex formation at globin gene promoters

Although distal regulatory regions are frequent throughout the genome, the molecular mechanisms by which they act in a promoter-specific manner remain to be elucidated. The human β-globin locus constitutes an extremely well-established multigenic model to investigate this issue. In erythroid cells, the β-globin locus control region (LCR) exerts distal regulatory function by influencing local chromatin organization and inducing high-level expression of individual β-like globin genes. Moreover, in transgenic mice expressing the entire human β-globin locus, deletion of LCR-hypersensitive site 2 (HS2) can alter β-like globin gene expression. Here, we show that abnormal expression of human β-like globin genes in the absence of HS2 is associated with decreased efficacy of pre-initiation complex formation at the human ɛ- and γ-promoters, but not at the β-promoter. This promoter-specific phenomenon is associated with reduced long-range interactions between the HS2-deleted LCR and human γ-promoters. We also find that HS2 is dispensable for high-level human β-gene transcription, whereas deletion of this hypersensitive site can alter locus chromatin organization; therefore the functions exerted by HS2 in transcriptional enhancement and locus chromatin organization are distinct. Overall, our data delineate one mechanism whereby a distal regulatory region provides promoter-specific transcriptional enhancement

PI 3 Kinase Related Kinases-Independent Proteolysis of BRCA1 Regulates Rad51 Recruitment during Genotoxic Stress in Human Cells

The function of BRCA1 in response to ionizing radiation, which directly generates DNA double strand breaks, has been extensively characterized. However previous investigations have produced conflicting data on mutagens that initially induce other classes of DNA adducts. Because of the fundamental and clinical importance of understanding BRCA1 function, we sought to rigorously evaluate the role of this tumor suppressor in response to diverse forms of genotoxic stress.We investigated BRCA1 stability and localization in various human cells treated with model mutagens that trigger different DNA damage signaling pathways. We established that, unlike ionizing radiation, either UVC or methylmethanesulfonate (MMS) (generating bulky DNA adducts or alkylated bases respectively) induces a transient downregulation of BRCA1 protein which is neither prevented nor enhanced by inhibition of PIKKs. Moreover, we found that the proteasome mediates early degradation of BRCA1, BARD1, BACH1, and Rad52 implying that critical components of the homologous recombination machinery need to be functionally abrogated as part of the early response to UV or MMS. Significantly, we found that inhibition of BRCA1/BARD1 downregulation is accompanied by the unscheduled recruitment of both proteins to chromatin along with Rad51. Consistently, treatment of cells with MMS engendered complete disassembly of Rad51 from pre-formed ionizing radiation-induced foci. Following the initial phase of BRCA1/BARD1 downregulation, we found that the recovery of these proteins in foci coincides with the formation of RPA and Rad51 foci. This indicates that homologous recombination is reactivated at later stage of the cellular response to MMS, most likely to repair DSBs generated by replication blocks.Taken together our results demonstrate that (i) the stabilities of BRCA1/BARD1 complexes are regulated in a mutagen-specific manner, and (ii) indicate the existence of mechanisms that may be required to prevent the simultaneous recruitment of conflicting signaling pathways to sites of DNA damage

The Matrix Revolution: Matricellular Proteins and Restructuring of the Cancer Microenvironment

The extracellular matrix (ECM) surrounding cells is indispensable for regulating their behavior. The dynamics of ECM signaling are tightly controlled throughout growth and development. During tissue remodeling, matricellular proteins (MCP) are secreted into the ECM. These factors do not serve classical structural roles, but rather regulate matrix proteins and cell-matrix interactions to influence normal cellular functions. In the tumor microenvironment, it is becoming increasingly clear that aberrantly expressed MCPs can support multiple hallmarks of carcinogenesis by interacting with various cellular components that are coupled to an array of downstream signals. Moreover, MCPs also reorganize the biomechanical properties of the ECM to accommodate metastasis and tumor colonization. This realization is stimulating new research on MCPs as reliable and accessible biomarkers in cancer, as well as effective and selective therapeutic targets

A Majority of Human Melanoma Cell Lines Exhibits an S Phase-Specific Defect in Excision of UV-Induced DNA Photoproducts

<div><p>It is well established that efficient removal of highly-promutagenic UV-induced dipyrimidine photoproducts <i>via</i> nucleotide excision repair (NER) is required for protection against sunlight-associated malignant melanoma. Nonetheless, the extent to which reduced NER capacity might contribute to individual melanoma susceptibility in the general population remains unclear. Here we show that among a panel of 14 human melanoma strains, 11 exhibit significant inhibition of DNA photoproduct removal during S phase relative to G0/G1 or G2/M. Evidence is presented that this cell cycle-specific NER defect correlates with enhanced apoptosis and reduced clonogenic survival following UV irradiation. In addition, melanoma strains deficient in S phase-specific DNA photoproduct removal manifest significantly lower levels of phosphorylated histone H2AX at 1 h post-UV, suggesting diminished activation of ataxia telangiectasia and Rad 3-related (ATR) kinase, i.e., a primary orchestrator of the cellular response to UV-induced DNA replication stress. Consistently, in the case of DNA photoproduct excision-proficient melanoma cells, siRNA-mediated depletion of ATR (but not of its immediate downstream effector kinase Chk1) engenders deficient NER specifically during S. On the other hand simultaneous siRNA-mediated depletion of ataxia telangiectasia mutated kinase (ATM) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) exerts no significant effect on either phosphorylation of H2AX at 1 h post-UV or the efficiency of DNA photoproduct removal. Our data suggest that defective NER exclusively during S phase, possibly associated with decreased ATR signaling, may constitute an heretofore unrecognized determinant in melanoma pathogenesis.</p></div

Cell cycle-specific CPD excision in melanoma cell lines.

<p>Cells were irradiated with 10/m² UVC and then labeled with BrdU for 1 h (except for the 0 h time point, where BrdU labeling was performed for 30 min prior to UV). At the indicated times post-UV, cells were stained with anti-CPD (FITC), PI, and anti-BrdU (Alexa-647) and analyzed by flow cytometry. <b>A</b>) Bivariate plots of WM35 showing the distribution of BrdU-positive cells at different times post-UV. The S' population indicated with an arrow at 24 h post-UV have traversed mitosis after increasing their DNA content and are excluded from the analysis. The extent of CPD removal is compared for cells in S phase at the time of irradiation (designated S), vs. cells in G1 at the time of irradiation which includes a minor proportion of cells that were initially in G2 and have migrated into the G1 compartment during the post-UV incubation period (designated G1/G2) (see text for details). <b>B</b>) Graphical representation of CPD excision in WM35 (top), WM3248 (middle), and XPA control skin fibroblasts (bottom). * and **, two-tailed paired t-test comparing CPD excision during S-phase vs G1/G2; p<0.02 and 0.001 at 12 and 24 h, respectively. For all panels in this figure, values represent the mean ± SEM of three independent experiments.</p

Cell cycle-specific 6–4PP excision in normal melanocytes and melanoma cell lines.

<p><b>A</b>) Graphical representation of 6–4PP removal as a function of cell cycle at 6 h post-UVC for 3 primary melanocyte strains and XPA-deficient skin fibroblasts. <b>B</b>) Same as A, but for 14 melanoma strains. In the case of all SPR-deficient strains, excision during S phase is significantly slower relative to other phases (p<0.01; two-tailed paired t-test). <b>C</b>) Representative bivariate dot plots showing WM35 and WM3248 (SPR-proficient and -deficient, respectively) either UV- or mock-irradiated as indicated, and stained with PI and anti-6-4PP antibody. Cells were gated in each phase of the cell cycle as shown for WM35 (no UV). <b>D</b>) Graphical representation of 6–4PP excision as a function of cell cycle at 6 h post-UVB (290–320-nm; 300 J/m²) in WM35 and WM3248. For all panels in this figure, values represent the mean ± SEM of three independent experiments.</p

Downregulation of ATR but not Chk1 engenders defective SPR in melanoma cells.

<p><b>A</b>) Each of the three SPR-proficient strains in our collection were incubated with siRNA pools against ATR or Chk1, or with non-targeting control siRNA, and treated with UVC or mock-irradiated. Protein levels for ATR, Chk1, and p-Chk1(S345) were determined by western blotting. YY1 and actin were used as loading control for ATR and Chk1, respectively. <b>B</b>) SPR-proficient strains were incubated with combined siRNA pools targeting both ATM and DNA-PKcs (A+D) or with non-targeting control siRNA, and treated with 6 Gy of IR or mock-irradiated. Protein levels for ATM, DNA-PKcs, and γH2AX were determined by western blotting. YY1 and GAPDH were used as loading controls for ATM/DNA-PKcs and γH2AX, respectively. <b>C</b>) γH2AX induction as a function of cell cycle at 1 h post-UV for siATR knockdown and ATM/DNA-PKcs double knockdown vs. non-targeting siRNA control. * p<0.01, two-tailed unpaired t-test comparing γH2AX induction in S-phase for siATR knockdown cells vs. non-targeting siRNA control. <b>D</b>) Cell cycle-specific excision of 6–4PP at 6 h post-UV in ATR, Chk1 and ATM/DNA-PK siRNA knockdown cells vs. non-targeting siRNA control. * p<0.005, two-tailed paired t-test comparing the extent of 6–4PP removal in G1 vs S. For all panels in this figure, values represent the mean ± SEM of three independent experiments.</p