Genome-Wide Profiling of PARP1 Reveals an Interplay with Gene Regulatory Regions and DNA Methylation.

Poly (ADP-ribose) polymerase-1 (PARP1) is a nuclear enzyme involved in DNA repair, chromatin remodeling and gene expression. PARP1 interactions with chromatin architectural multi-protein complexes (i.e. nucleosomes) alter chromatin structure resulting in changes in gene expression. Chromatin structure impacts gene regulatory processes including transcription, splicing, DNA repair, replication and recombination. It is important to delineate whether PARP1 randomly associates with nucleosomes or is present at specific nucleosome regions throughout the cell genome. We performed genome-wide association studies in breast cancer cell lines to address these questions. Our studies show that PARP1 associates with epigenetic regulatory elements genome-wide, such as active histone marks, CTCF and DNase hypersensitive sites. Additionally, the binding of PARP1 to chromatin genome-wide is mutually exclusive with DNA methylation pattern suggesting a functional interplay between PARP1 and DNA methylation. Indeed, inhibition of PARylation results in genome-wide changes in DNA methylation patterns. Our results suggest that PARP1 controls the fidelity of gene transcription and marks actively transcribed gene regions by selectively binding to transcriptionally active chromatin. These studies provide a platform for developing our understanding of PARP1's role in gene regulation.

VorinostatSAHA Promotes Hyper-Radiosensitivity in Wild Type p53 Human Glioblastoma Cells.

Glioblastoma multiforme (GBM) is a very aggressive and locally invasive tumor. The current standard of care is partial brain radiation therapy (60 Gy) concurrently with the alkylating agent temozolomide (TMZ). However, patients' survival remains poor (6-12 months) mainly due to local and diffuse (distant) recurrence. The possibility to promote hyper radiosensitivity (HRS) with low dose radiation may contribute to improve outcome. Here, we evaluated the effect of VorinostatSAHA and TMZ on glioblastoma cells' sensitivity to low dose radiation. Clonogenic survivals were performed on D54 (p53 and PTEN wild type) and U118 (p53 and PTEN mutants) cells exposed to clinically relevant doses of VorinostatSAHA and TMZ and increasing radiation doses. Apoptosis was measured by the activation of caspase-3 and the role of p53 and PTEN were evaluated with the p53 inhibitor pifithrin α and the PI3K/AKT pathway inhibitor LY29002. VorinostatSAHA promoted HRS at doses as low as 0.25 Gy in the D54 but not the U118 cells. Killing efficiency was associated with caspase-3 activation, delayed H2AX phosphorylation and abrogation of a radiation -induced G2 arrest. Inhibiting p53 function with pifithrin α prevented the promotion of HRS by VorinostatSAHA. Moreover, LY29002, a PI-3K inhibitor, restored promotion of HRS by VorinostatSAHA in the p53 mutant U118 cells to levels similar to the p53 wild type cells. TMZ also promoted HRS at doses as low as 0.15 Gy. These finding indicate that HRS can be promoted in p53 wild type glioblastoma cells through a functional PTEN to delay DNA repair and sensitize cells to low dose radiation. Promotion of HRS thus appears to be a viable approach for GBM that could be used as a basis to develop new Phase I/II studies.

The chromatin architectural proteins HMGD1 and H1 bind reciprocally and have opposite effects on chromatin structure and gene regulation.

BACKGROUND: Chromatin architectural proteins interact with nucleosomes to modulate chromatin accessibility and higher-order chromatin structure. While these proteins are almost certainly important for gene regulation they have been studied far less than the core histone proteins. RESULTS: Here we describe the genomic distributions and functional roles of two chromatin architectural proteins: histone H1 and the high mobility group protein HMGD1 in Drosophila S2 cells. Using ChIP-seq, biochemical and gene specific approaches, we find that HMGD1 binds to highly accessible regulatory chromatin and active promoters. In contrast, H1 is primarily associated with heterochromatic regions marked with repressive histone marks. We find that the ratio of HMGD1 to H1 binding is a better predictor of gene activity than either protein by itself, which suggests that reciprocal binding between these proteins is important for gene regulation. Using knockdown experiments, we show that HMGD1 and H1 affect the occupancy of the other protein, change nucleosome repeat length and modulate gene expression. CONCLUSION: Collectively, our data suggest that dynamic and mutually exclusive binding of H1 and HMGD1 to nucleosomes and their linker sequences may control the fluid chromatin structure that is required for transcriptional regulation. This study provides a framework to further study the interplay between chromatin architectural proteins and epigenetics in gene regulation.

Archaeal nucleosome positioning in vivo and in vitro is directed by primary sequence motifs.

BACKGROUND: Histone wrapping of DNA into nucleosomes almost certainly evolved in the Archaea, and predates Eukaryotes. In Eukaryotes, nucleosome positioning plays a central role in regulating gene expression and is directed by primary sequence motifs that together form a nucleosome positioning code. The experiments reported were undertaken to determine if archaeal histone assembly conforms to the nucleosome positioning code. RESULTS: Eukaryotic nucleosome positioning is favored and directed by phased helical repeats of AA/TT/AT/TA and CC/GG/CG/GC dinucleotides, and disfavored by longer AT-rich oligonucleotides. Deep sequencing of genomic DNA protected from micrococcal nuclease digestion by assembly into archaeal nucleosomes has established that archaeal nucleosome assembly is also directed and positioned by these sequence motifs, both in vivo in Methanothermobacter thermautotrophicus and Thermococcus kodakarensis and in vitro in reaction mixtures containing only one purified archaeal histone and genomic DNA. Archaeal nucleosomes assembled at the same locations in vivo and in vitro, with much reduced assembly immediately upstream of open reading frames and throughout the ribosomal rDNA operons. Providing further support for a common positioning code, archaeal histones assembled into nucleosomes on eukaryotic DNA and eukaryotic histones into nucleosomes on archaeal DNA at the same locations. T. kodakarensis has two histones, designated HTkA and HTkB, and strains with either but not both histones deleted grow normally but do exhibit transcriptome differences. Comparisons of the archaeal nucleosome profiles in the intergenic regions immediately upstream of genes that exhibited increased or decreased transcription in the absence of HTkA or HTkB revealed substantial differences but no consistent pattern of changes that would correlate directly with archaeal nucleosome positioning inhibiting or stimulating transcription. CONCLUSIONS: The results obtained establish that an archaeal histone and a genome sequence together are sufficient to determine where archaeal nucleosomes preferentially assemble and where they avoid assembly. We confirm that the same nucleosome positioning code operates in Archaea as in Eukaryotes and presumably therefore evolved with the histone-fold mechanism of DNA binding and compaction early in the archaeal lineage, before the divergence of Eukaryotes.

Cancer cells' epigenetic composition and predisposition to histone deacetylase inhibitor sensitization.

Normal cells are up to ten times more resistant to histone deacetylase inhibitors (HDACis)-induced cell death compared with transformed cells. The molecular processes underlying this selectivity for cancer cells are still not well understood. Although a differential response to oxidative stress and capacity to repair damaged DNA have been described in some systems, these cannot fully account for the sensitivity of cancer cells to HDACis since the heterogeneity of cancer cells prompts differential sensitivities to reactive oxygen species and generates a panoply of defective DNA repair mechanisms within given histologies, cancer cell lines and tumor xenografts. It seems also unlikely that the influence of HDACis on cancer treatments reside primarily on gene transcription, since gene-expression profiling aimed at defining correlation with response to HDACis in cancer cells indicates that less than 5% to approximately 20% of transcribed genes are altered by HDACis treatment. Moreover, the altered genes vary from cell line to cell line and between different HDACis. Therefore, no consistent picture of a target(s) or pathway(s) modulated by HDACis has emerged. One consistent parameter that has however been observed in peripheral blood mononuclear cells of patients treated with HDACi is the accumulation of acetylated histones. Because one of the primary functions of histone acetylation is to increase chromatin accessibility, this article will explore the possibility that intrinsic molecular and structural characteristics of cancer cells provide a selective advantage for HDACis sensitivity.

Over Expression of Nucleophosmin and Nucleolin Contributes to the Suboptimal Activation of a G2/M Checkpoint in Ataxia Telangiectasia Fibroblasts.

Ataxia Telangiectasia (AT) cells exhibit suboptimal activation of radiation-induced cell cycle checkpoints despite having a wild type p53 genotype. Reducing or eliminating this delay could restore p53 function and reinstate normal cellular response to genotoxic stress. Here we show that the levels of Nuclephosmin (NPM), NPM phosphorylated at Serine 125, p53, p53 phosphorylated at Serine 15 and Serine 392 and the levels of Nucleolin (NCL) are high in AT fibroblasts compared to normal cells. Transfection of a functional ATM into AT fibroblasts reduced p53, phospo-p53, phospho-NPM and NCL levels to wild type fibroblasts levels. Our data indicate that ATM regulates phospho-NPM and NCL indirectly through the Protein Phosphatase 1 (PP1). Both, NPM and NCL interact with p53 and hinder its phosphorylation at Serine 15 in response to bleomycin. Moreover, NPM and NCL are phosphorylated by several of the same kinases targeting p53 and could potentially compete with p53 for phosphorylation in AT cells. In addition, our data indicate that down regulation of NCL and to a lesser extent NPM increase the number of AT cells arrested in G2/M in response to bleomycin. Together this data indicate that the lack of PP1 activation in AT cells result in increased NPM and NCL protein levels which prevents p53 phosphorylation in response to bleomycin and contributes to a defective G2/M checkpoint.

The Nucleolus Takes Control of Protein Trafficking Under Cellular Stress.

The nucleolus is a highly dynamic nuclear substructure that was originally described as the site of ribosome biogenesis. The advent of proteomic analysis has now allowed the identification of over 4500 nucleolus associated proteins with only about 30% of them associated with ribogenesis (1). The great number of nucleolar proteins not associated with traditionally accepted nucleolar functions indicates a role for the nucleolus in other cellular functions such as mitosis, cell-cycle progression, cell proliferation and many forms of stress response including DNA repair (2). A number of recent reviews have addressed the pivotal role of the nucleolus in the cellular stress response (1, 3, 4). Here, we will focus on the role of Nucleolin and Nucleophosmin, two major components of the nucleolus, in response to genotoxic stress. Due to space constraint only a limited number of studies are cited. We thus apologize to all our colleagues whose works are not referenced here.

Inhibitory effects of arresten on bFGF-induced proliferation, migration, and matrix metalloproteinase-2 activation in mouse retinal endothelial cells.

PURPOSE: The potential role of arresten (alpha1(IV)NC1) as an endogenous angiogenesis inhibitor in the prevention of bFGF mediated retinal angiogenesis and regulation of matrix metalloproteinase-2 activation has not been explored. METHODS: Mouse retinal endothelial cells (MREC) were cultured on type IV collagen and treated with basic fibroblast growth factor (bFGF) alone or in the presence of arresten at concentrations ranging from 1 to 10 microg/ml. The proliferation of MRECs were evaluated using 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay, and bFGF stimulated endothelial cell migration was assessed using Boyden chamber. Expression of matrix metalloproteinase-2 (MMP-2) was assessed by reverse transcription polymerase chain reaction (RT-PCR) analysis using RNA isolated from MRECs. Secretion and activation of MMP-2 in arresten-treated conditioned MREC growth medium was determined by gelatin zymography and Western blotting. RESULTS: Different doses of bFGF induced MREC proliferation was significantly inhibited upon arresten treatment (P < 0.005). The bFGF-induced migration was significantly inhibited by arresten at 1 and 10 microg/ml concentrations (P < 0.01). The bFGF stimulated expression of MMP-2 mRNA and secretion of MMP-2 in MREC was not affected and interestingly activation of MMP-2 was suppressed by arresten in a dose and time dependent manner. CONCLUSIONS: Inhibitory effects of arresten on proliferation, migration and MMP-2 activation but not on expression and secretion of MMP-2 in MREC; this early work with arresten supports potential therapeutic action in retinal neovascularization dependent disorders.

FAK and p38-MAP kinase-dependent activation of apoptosis and caspase-3 in retinal endothelial cells by alpha1(IV)NC1.

PURPOSE: To determine the impact of the antiangiogenic factor alpha1(IV)NC1 on vascular endothelial growth factor-mediated proangiogenic activity in mouse retinal endothelial cells (MRECs). METHODS: Primary culture of MRECs was established as previously described and was used to determine the effects of alpha1(IV)NC1 on the proangiogenic activity of VEGF. Cell proliferation was evaluated using [(3)H]-thymidine incorporation and 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide colorimetric assays. Cell migration was determined using modified Boyden chamber and scratch wound assays and tube formation was assessed on basement membrane matrix (BMM). Intracellular signaling events Bcl-2/Bcl-x(L) and caspase-3/poly (ADP-ribose) polymerase (PARP) activities were evaluated in cells stimulated with VEGF and plated on type IV collagen-coated dishes. Apoptosis was assessed by measuring caspase activity and by performing quantitative fluorescence analysis using fluorescence-activated cell sorting assay. Subcutaneously injected VEGF induced in vivo neovascularization was studied with the BMM plug assay. RESULTS: VEGF-induced subconfluent MREC proliferation, migration, and tube formation were significantly inhibited by alpha1(IV)NC1 at 1 muM (P < 0.001). alpha1(IV)NC1 induced MREC apoptosis is mediated by inhibition of Bcl-2 and Bcl-x(L) expression and activation of caspase-3/PARP through FAK/p38-MAPK signaling. In addition, alpha1(IV)NC1 dose dependently inhibited VEGF-mediated neovascularization in vivo. CONCLUSIONS: alpha1(IV)NC1 inhibited VEGF-mediated angiogenesis by promoting apoptosis and caspase-3/PARP activation and by negatively impacting FAK/p38-MAPK phosphorylation, Bcl-2, and Bcl-x(L) expression leading to MREC death. The endothelial-specific inhibitory actions of recombinant alpha1(IV)NC1 may be of benefit in the treatment of a variety of eye diseases with a neovascular component.

Sense p16 and antisense uPAR bicistronic construct inhibits angiogenesis and induces glioma cell death.

High-grade gliomas comprise the most malignant type of primary brain tumor and are relatively frequent in adults. Recent studies have indicated that the loss of p16, an inhibitor of CDK4, promotes the acquisition of malignant characteristics in gliomas. A correlation between overexpression of urokinase-type plasminogen activator receptor (uPAR) and glioblastoma invasion has also been established. Moreover, uPAR/integrin binding has been shown to initiate or potentiate integrin signaling through focal adhesion kinase and/or src kinases. Our previous studies demonstrated that downregulation of uPAR expression and restoration of p16 regress glioma growth in nude mice and downregulate alphavbeta3 integrin receptor expression. Here, we show the effect of a bicistronic construct on alphavbeta5 integrin receptor expression, angiogenesis and the biochemical pathway that causes glioma cell death. The U251 glioblastoma and a glioblastoma xenograft cell line transduced with a recombinant replication-defective adenovirus vector containing the cDNA of wild-type p16 and antisense RNA of uPAR significantly inhibited human mammary epithelial cell capillary formation and vascular endothelial growth factor (VEGF) expression. Inactivation of anti-apoptotic molecules such as Akt, PARP, activation of caspases and accumulation of heteroduplex chromosomal DNA in pre-G1 phase of the cell cycle was demonstrated by Western blotting, caspase activity assay and FACS analysis. Nuclear DNA fragmentation upon induction of apoptosis was scored using the TUNEL assay. Significant downregulation of alphavbeta5 integrin receptor expression was also confirmed by FACS analysis, immunoprecipitation and RT-PCR. Taken together, the results demonstrate that the sense p16 and anti-sense uPAR bicistronic construct significantly inhibits angiogenesis, induces apoptosis by deregulation of the PI3K-Akt pathway and downregulates alphavbeta5 integrin receptor expression.

Identification of calpain cleavage sites in the G1 cyclin-dependent kinase inhibitor p19(INK4d).

Calpains are a large family of Ca2+-dependent cysteine proteases that are ubiquitously distributed across most cell types and vertebrate species. Calpains play a role in cell differentiation, apoptosis, cytoskeletal remodeling, signal transduction and the cell cycle. The cell cycle proteins cyclin D1 and p21(KIP1), for example, have been shown to be affected by calpains. However, the rules that govern calpain cleavage specificity are poorly understood. We report here studies on the pattern of mu-calpain proteolysis of the p19(INK4d) protein, a cyclin-dependent kinase 4/6 inhibitor that negatively regulates the mammalian cell cycle. Our data show new characteristics of calpain action: mu-calpain cleaves p19(INK4d) immediately after the first and second ankyrin repeats that are structurally less stable compared to the other repeats. This is in contrast to features observed so far in the specificity of calpains for their substrates. These results imply that calpain may be involved in the cell cycle by regulating the cell cycle regulatory protein turnover through CDK inhibitors and cyclins.

NMR and mass spectrometry studies of putative interactions of cell cycle proteins pRb and CDK6 with cell differentiation proteins MyoD and ID-2.

Cell growth and differentiation require precise coordination of cell cycle and differentiation proteins. This can be achieved by direct interactions between proteins, by indirect interaction in multiprotein complexes, or by modulation of gene expression levels of partner proteins. Contradictory data abound in the literature regarding the binding between some central cell cycle proteins, pRb, and CDK6, with myogenic differentiation promoting, MyoD, and inhibiting, Id-2, factors. We have tested these interactions using pure proteins and in vitro biophysical and biochemical methods, which included mass spectrometry, nuclear magnetic resonance (NMR), the affinity chromatography pull-down assays, and gel filtration chromatography. Using this multimethod approach, we were able to document interactions between pRb and HPV-E7, pRb and SV40 large T antigen, CDK6 and p19, and MyoD and DNA. Using the same methods, we could unambiguously show that there is no direct protein-protein interaction in vitro between the small pocket domain of pRb and the bHLH domain of MyoD, the small pocket domain of pRb and Id-2, and CDK6 and a 15-amino-acid peptide from the C-terminal domain of MyoD. Indirect interactions, through additional binding partners in multiprotein complexes or modulation of gene expression levels of these proteins, are therefore their probable mode of action.