CpG Islands: Starting Blocks for Replication and Transcription

International audienc

Preferential Localization of Human Origins of DNA Replication at the 5′-Ends of Expressed Genes and at Evolutionarily Conserved DNA Sequences

Replication of mammalian genomes requires the activation of thousands of origins which are both spatially and temporally regulated by as yet unknown mechanisms. At the most fundamental level, our knowledge about the distribution pattern of origins in each of the chromosomes, among different cell types, and whether the physiological state of the cells alters this distribution is at present very limited.We have used standard λ-exonuclease resistant nascent DNA preparations in the size range of 0.7–1.5 kb obtained from the breast cancer cell line MCF–7 hybridized to a custom tiling array containing 50–60 nt probes evenly distributed among genic and non-genic regions covering about 1% of the human genome. A similar DNA preparation was used for high-throughput DNA sequencing. Array experiments were also performed with DNA obtained from BT-474 and H520 cell lines. By determining the sites showing nascent DNA enrichment, we have localized several thousand origins of DNA replication. Our major findings are: (a) both array and DNA sequencing assay methods produced essentially the same origin distribution profile; (b) origin distribution is largely conserved (>70%) in all cell lines tested; (c) origins are enriched at the 5′ends of expressed genes and at evolutionarily conserved intergenic sequences; and (d) ChIP on chip experiments in MCF-7 showed an enrichment of H3K4Me3 and RNA Polymerase II chromatin binding sites at origins of DNA replication.Our results suggest that the program for origin activation is largely conserved among different cell types. Also, our work supports recent studies connecting transcription initiation with replication, and in addition suggests that evolutionarily conserved intergenic sequences have the potential to participate in origin selection. Overall, our observations suggest that replication origin selection is a stochastic process significantly dependent upon local accessibility to replication factors

DNA mediated chromatin pull-down for the study of chromatin replication

Chromatin replication involves duplicating DNA while maintaining epigenetic information. These processes are critical for genome stability and for preserving cell-type identity. Here we describe a simple experimental approach that allows chromatin to be captured and its content analysed after in vivo replication and labeling of DNA by cellular DNA polymerases. We show that this technique is highly specific and that proteins bound to the replicated DNA can be analyzed by both immunological techniques and large scale mass spectrometry. As proof of concept we have used this novel procedure to begin investigating the relationship between chromatin protein composition and the temporal programme of DNA replication in human cells. It is expected that this technique will become a widely used tool to address how chromatin proteins assemble onto newly replicated DNA after passage of a replication fork and how chromatin maturation is coupled to DNA synthesis

DNA Double-Strand Break Repair Genes and Oxidative Damage in Brain Metastasis of Breast Cancer

Background Breast cancer frequently metastasizes to the brain, colonizing a neuro-inflammatory microenvironment. The molecular pathways facilitating this colonization remain poorly understood. Methods Expression profiling of 23 matched sets of human resected brain metastases and primary breast tumors by two-sided paired t test was performed to identify brain metastasis–specific genes. The implicated DNA repair genes BARD1 and RAD51 were modulated in human (MDA-MB-231-BR) and murine (4T1-BR) brain-tropic breast cancer cell lines by lentiviral transduction of cDNA or short hairpin RNA (shRNA) coding sequences. Their functional contribution to brain metastasis development was evaluated in mouse xenograft models (n = 10 mice per group). Results Human brain metastases overexpressed BARD1 and RAD51 compared with either matched primary tumors (1.74-fold, P < .001; 1.46-fold, P < .001, respectively) or unlinked systemic metastases (1.49-fold, P = .01; 1.44-fold, P = .008, respectively). Overexpression of either gene in MDA-MB-231-BR cells increased brain metastases by threefold to fourfold after intracardiac injections, but not lung metastases upon tail-vein injections. In 4T1-BR cells, shRNA-mediated RAD51 knockdown reduced brain metastases by 2.5-fold without affecting lung metastasis development. In vitro, BARD1- and RAD51-overexpressing cells showed reduced genomic instability but only exhibited growth and colonization phenotypes upon DNA damage induction. Reactive oxygen species were present in tumor cells and elevated in the metastatic neuro-inflammatory microenvironment and could provide an endogenous source of genotoxic stress. Tempol, a brain-permeable oxygen radical scavenger suppressed brain metastasis promotion induced by BARD1 and RAD51 overexpression. Conclusions BARD1 and RAD51 are frequently overexpressed in brain metastases from breast cancer and may constitute a mechanism to overcome reactive oxygen species–mediated genotoxic stress in the metastatic brain

DNA Replication Fading As Proliferating Cells Advance in Their Commitment to Terminal Differentiation

Terminal differentiation is the process by which cycling cells stop proliferating to start new specific functions. It involves dramatic changes in chromatin organization as well as gene expression. In the present report we used cell flow cytometry and genome wide DNA combing to investigate DNA replication during murine erythroleukemia-induced terminal cell differentiation. The results obtained indicated that the rate of replication fork movement slows down and the inter-origin distance becomes shorter during the precommitment and commitment periods before cells stop proliferating and accumulate in G1. We propose this is a general feature caused by the progressive heterochromatinization that characterizes terminal cell differentiation

TSPYL2 Is Important for G1 Checkpoint Maintenance upon DNA Damage

Nucleosome assembly proteins play important roles in chromatin remodeling, which determines gene expression, cell proliferation and terminal differentiation. Testis specific protein, Y-encoded-like 2 (TSPYL2) is a nucleosome assembly protein expressed in neuronal precursors and mature neurons. Previous studies have shown that TSPYL2 binds cyclin B and inhibits cell proliferation in cultured cells suggesting a role in cell cycle regulation. To investigate the physiological significance of TSPYL2 in the control of cell cycle, we generated mice with targeted disruption of Tspyl2. These mutant mice appear grossly normal, have normal life span and do not exhibit increased tumor incidence. To define the role of TSPYL2 in DNA repair, checkpoint arrest and apoptosis, primary embryonic fibroblasts and thymocytes from Tspyl2 deficient mice were isolated and examined under unperturbed and stressed conditions. We show that mutant fibroblasts are impaired in G1 arrest under the situation of DNA damage induced by gamma irradiation. This is mainly attributed to the defective activation of p21 transcription despite proper p53 protein accumulation, suggesting that TSPYL2 is additionally required for p21 induction. TSPYL2 serves a biological role in maintaining the G1 checkpoint under stress condition

Predicted Functions of MdmX in Fine-Tuning the Response of p53 to DNA Damage

Tumor suppressor protein p53 is regulated by two structurally homologous proteins, Mdm2 and MdmX. In contrast to Mdm2, MdmX lacks ubiquitin ligase activity. Although the essential interactions of MdmX are known, it is not clear how they function to regulate p53. The regulation of tumor suppressor p53 by Mdm2 and MdmX in response to DNA damage was investigated by mathematical modeling of a simplified network. The simplified network model was derived from a detailed molecular interaction map (MIM) that exhibited four coherent DNA damage response pathways. The results suggest that MdmX may amplify or stabilize DNA damage-induced p53 responses via non-enzymatic interactions. Transient effects of MdmX are mediated by reservoirs of p53∶MdmX and Mdm2∶MdmX heterodimers, with MdmX buffering the concentrations of p53 and/or Mdm2. A survey of kinetic parameter space disclosed regions of switch-like behavior stemming from such reservoir-based transients. During an early response to DNA damage, MdmX positively or negatively regulated p53 activity, depending on the level of Mdm2; this led to amplification of p53 activity and switch-like response. During a late response to DNA damage, MdmX could dampen oscillations of p53 activity. A possible role of MdmX may be to dampen such oscillations that otherwise could produce erratic cell behavior. Our study suggests how MdmX may participate in the response of p53 to DNA damage either by increasing dependency of p53 on Mdm2 or by dampening oscillations of p53 activity and presents a model for experimental investigation

DNA Damage Responses in Human Induced Pluripotent Stem Cells and Embryonic Stem Cells

BACKGROUND: Induced pluripotent stem (iPS) cells have the capability to undergo self-renewal and differentiation into all somatic cell types. Since they can be produced through somatic cell reprogramming, which uses a defined set of transcription factors, iPS cells represent important sources of patient-specific cells for clinical applications. However, before these cells can be used in therapeutic designs, it is essential to understand their genetic stability.\ud \ud METHODOLOGY/PRINCIPAL FINDINGS: Here, we describe DNA damage responses in human iPS cells. We observe hypersensitivity to DNA damaging agents resulting in rapid induction of apoptosis after γ-irradiation. Expression of pluripotency factors does not appear to be diminished after irradiation in iPS cells. Following irradiation, iPS cells activate checkpoint signaling, evidenced by phosphorylation of ATM, NBS1, CHEK2, and TP53, localization of ATM to the double strand breaks (DSB), and localization of TP53 to the nucleus of NANOG-positive cells. We demonstrate that iPS cells temporary arrest cell cycle progression in the G(2) phase of the cell cycle, displaying a lack of the G(1)/S cell cycle arrest similar to human embryonic stem (ES) cells. Furthermore, both cell types remove DSB within six hours of γ-irradiation, form RAD51 foci and exhibit sister chromatid exchanges suggesting homologous recombination repair. Finally, we report elevated expression of genes involved in DNA damage signaling, checkpoint function, and repair of various types of DNA lesions in ES and iPS cells relative to their differentiated counterparts.\ud \ud CONCLUSIONS/SIGNIFICANCE: High degrees of similarity in DNA damage responses between ES and iPS cells were found. Even though reprogramming did not alter checkpoint signaling following DNA damage, dramatic changes in cell cycle structure, including a high percentage of cells in the S phase, increased radiosensitivity and loss of DNA damage-induced G(1)/S cell cycle arrest, were observed in stem cells generated by induced pluripotency.\ud \u

A Key Commitment Step in Erythropoiesis Is Synchronized with the Cell Cycle Clock through Mutual Inhibition between PU.1 and S-Phase Progression

During red blood cell development, differentiation and cell cycle progression are intimately and uniquely linked through interdependent mechanisms involving the erythroid transcriptional suppressor PU.1 and the cyclin-dependent kinase inhibitor p57KIP2