Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1

<p>Abstract</p> <p>Background</p> <p>Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that plays critical functions in many biological processes, including DNA repair and gene transcription. The main function of PARP-1 is to catalyze the transfer of ADP-ribose units from nicotinamide adenine dinucleotide (NAD⁺) to a large array of acceptor proteins, which comprises histones, transcription factors, as well as PARP-1 itself. We have previously demonstrated that transcription of the PARP-1 gene essentially rely on the opposite regulatory actions of two distinct transcription factors, Sp1 and NFI. In the present study, we examined whether suppression of PARP-1 expression in embryonic fibroblasts derived from PARP-1 knockout mice (PARP-1^-/-) might alter the expression and/or DNA binding properties of Sp1 and NFI. We also explored the possibility that Sp1 or NFI (or both) may represent target proteins of PARP-1 activity.</p> <p>Results</p> <p>Expression of both Sp1 and NFI was found to be considerably reduced in PARP-1^-/-cells. Co-immunoprecipitation assays revealed that PARP-1 physically interacts with Sp1 in a DNA-independent manner, but neither with Sp3 nor NFI, in PARP-1^+/+cells. In addition, <it>in vitro </it>PARP assays indicated that PARP-1 could catalyze the addition of polymer of ADP-ribose to Sp1, which also translated into a reduction of Sp1 binding to its consensus DNA target site. Transfection of the PARP-1 promoter into both PARP-1^+/+and PARP-1^-/-cells revealed that the lack of PARP-1 expression in PARP-1^-/-cells also results in a strong increase in PARP-1 promoter activity. This influence of PARP-1 was found to rely on the presence of the Sp1 sites present on the basal PARP-1 promoter as their mutation entirely abolished the increased promoter activity observed in PARP-1^-/-cells. Subjecting PARP-1^+/+cells to an oxidative challenge with hydrogen peroxide to increase PARP-1 activity translated into a dramatic reduction in the DNA binding properties of Sp1. However, its suppression by the inhibitor PJ34 improved DNA binding of Sp1 and led to a dramatic increase in PARP-1 promoter function.</p> <p>Conclusion</p> <p>Our results therefore recognized Sp1 as a target protein of PARP-1 activity, the addition of polymer of ADP-ribose to this transcription factor restricting its positive regulatory influence on gene transcription.</p

Differential binding of the transcription factors Sp1, AP-1, and NFI to the promoter of the human α5 integrin gene dictates its transcriptional activity

Purpose. Damage to the corneal epithelium results in the massive secretion of fibronectin (FN) shortly after injury and induces the expression of its integrin receptor α5β1. The authors reported previously that FN induces α5 expression in human corneal epithelial cells and rabbit corneal epithelial cells by altering the binding of the transcription factor (TF) Sp1 to a regulatory element from the α5 promoter that it is also flanked by binding sites for the TFs NFI and AP-1. Here, they assessed the function of NFI and AP-1 on α5 gene expression and evaluated the contribution of FN to their overall regulatory influence. Methods. TF binding to the α5 promoter was evaluated in vitro by electrophoretic mobility shift assays and in vivo by ligation-mediated PCR or chromatin immunoprecipitation. TFs expression was monitored by Western blot, whereas their influence was assessed by transfection and RNAi analyses. Results. Coexpression of Sp1, NFI, and AP-1 was demonstrated in all cell types, and each TF was shown to bind efficiently to the α5 promoter. Whereas both AP-1 and Sp1 activated expression directed by the α5 promoter, NFI functioned as a potent repressor of that gene. Interestingly, FN could either promote or repress α5 promoter activity in a cell density–dependent manner by differentially altering the ratio of these TFs. Conclusions. These results suggest that α5 gene expression is likely dictated by subtle alterations in the nuclear ratio of TFs that either repress (NFI) or activate (Sp1 and AP-1) α5 transcription in corneal epithelial cells

Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1-0

<p><b>Copyright information:</b></p><p>Taken from "Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1"</p><p>http://www.biomedcentral.com/1471-2199/8/96</p><p>BMC Molecular Biology 2007;8():96-96.</p><p>Published online 25 Oct 2007</p><p>PMCID:PMC2175517.</p><p></p>e position of the 120 kDa and 60 kDa proteins used as molecular mass markers is indicated. The asterisk indicates the position of the typical NFI complex whereas the arrowhead designates NFI complexes with a reduced electrophoretic mobility that predominated in the extract from PARP-1cells. Data of one from three similar experiments are presented

Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1-9

<p><b>Copyright information:</b></p><p>Taken from "Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1"</p><p>http://www.biomedcentral.com/1471-2199/8/96</p><p>BMC Molecular Biology 2007;8():96-96.</p><p>Published online 25 Oct 2007</p><p>PMCID:PMC2175517.</p><p></p>igh affinity binding site for either Sp1 (left) or NFI (right). Formation of DNA/protein complexes was then monitored by EMSA on an 8% (Sp1) and 10% (NFI) native polyacrylamide gel and their position revealed through autoradiography. The position of both the Sp1/Sp3 and NFI DNA-protein complexes are shown, as well as that of the free probe (U). P: labeled probe alone. () Sp1 competition experiment in EMSA. The Sp1 labeled probe used in panel A was incubated with nuclear proteins (5 μg) from both PARP-1and PARP-1cells in the presence of either no (-) or 100- and 500-fold molar excesses of unlabeled competitor oligonucleotides (either Sp1 or NFI). Formation of DNA/protein complexes was then monitored by EMSA on an 8% native gel. () NFI competition experiment in EMSA. Same as in panel B except that the NFI double-stranded oligonucleotide was 5'-end labeled and used as probe for the assay. () Supershift experiment in EMSA. Crude nuclear proteins from both PARP-1and PARP-1cells were incubated with the either the Sp1 (5 μg proteins were used) or NFI (10 μg proteins were used) labeled probe in the presence of either no (-), or 2 μl of a polyclonal antibody directed against Sp1 (Sp1Ab) or Sp3 (Sp3Ab) and added either individually or in combination (Sp1+Sp3Ab) (left), or with a polyclonal antibody directed against NFI (right). Formation of both the Sp1/Sp3 and NFI complexes, as well as their corresponding supershifted complexes (SSC) is indicated. P: labeled probe alone; U: unbound fraction of the labeled probe

Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1-8

<p><b>Copyright information:</b></p><p>Taken from "Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1"</p><p>http://www.biomedcentral.com/1471-2199/8/96</p><p>BMC Molecular Biology 2007;8():96-96.</p><p>Published online 25 Oct 2007</p><p>PMCID:PMC2175517.</p><p></p>e position of the 120 kDa and 60 kDa proteins used as molecular mass markers is indicated. The asterisk indicates the position of the typical NFI complex whereas the arrowhead designates NFI complexes with a reduced electrophoretic mobility that predominated in the extract from PARP-1cells. Data of one from three similar experiments are presented

Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1-3

<p><b>Copyright information:</b></p><p>Taken from "Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1"</p><p>http://www.biomedcentral.com/1471-2199/8/96</p><p>BMC Molecular Biology 2007;8():96-96.</p><p>Published online 25 Oct 2007</p><p>PMCID:PMC2175517.</p><p></p>re incubated with the Sp1 Ab (sc-59) and the Sp1-protein complexes recovered by the addition of protein-A-Sepharose. The resulting immunoprecipitated proteins were then SDS-gel fractionated before being membrane-transferred and Western blotted with antibodies against Sp1, PARP-1 (C-2-10) and PAR (LP-9610). Ctl-: protein A-Sepharose added to crude nuclear proteins in the absence of Sp1 Ab and used as a negative control. IgG-Ab: normal rabbit IgG incubated with nuclear proteins prior to addition of protein A-Sepharose as a negative control. () Immunoprecipitation of the PARP-1-protein complexes in PARP-1and PARP-1nuclear extracts. Same as in panel A except that the immunoprecipitation was conducted using the PARP-1 F-123 Ab. The blotted, PARP-1-immunoprecipitated proteins were then analyzed with the PARP-1 (422), Sp1 (sc-59), Sp3 (sc-644), and PAR (LP-9610) antibodies. Negative controls (Ctl- and IgG-Ab) are as in panel A. TE: total cell extract that has not been immunoprecipitated with the PARP-1 Ab

The tissue-engineered human cornea as a model to study expression of matrix metalloproteinases during corneal wound healing

Corneal injuries remain a major cause of consultation in the ophthalmology clinics worldwide. Repair of corneal wounds is a complex mechanism that involves cell death, migration, proliferation, differentiation, and extracellular matrix (ECM) remodeling. In the present study, we used a tissue-engineered, twolayers (epithelium and stroma) human cornea as a biomaterial to study both the cellular and molecular mechanisms of wound healing. Gene profiling on microarrays revealed important alterations in the pattern of genes expressed by tissue-engineered corneas in response to wound healing. Expression of many MMPs-encoding genes was shown by microarray and qPCR analyses to increase in the migrating epithelium of wounded corneas. Many of these enzymes were converted into their enzymatically active form as wound closure proceeded. In addition, expression of MMPs by human corneal epithelial cells (HCECs) was affected both by the stromal fibroblasts and the collagen-enriched ECM they produce. Most of all, results from mass spectrometry analyses provided evidence that a fully stratified epithelium is required for proper synthesis and organization of the ECM on which the epithelial cells adhere. In conclusion, and because of the many characteristics it shares with the native cornea, this human two layers corneal substitute may prove particularly useful to decipher the mechanistic details of corneal wound healing

Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1-5

<p><b>Copyright information:</b></p><p>Taken from "Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1"</p><p>http://www.biomedcentral.com/1471-2199/8/96</p><p>BMC Molecular Biology 2007;8():96-96.</p><p>Published online 25 Oct 2007</p><p>PMCID:PMC2175517.</p><p></p>ane 5). The reaction mixture was subjected to Western blot analysis with the PARP-1 (C-2-10), Sp1 (sc-59) and PAR (LP-9610) antibodies. When indicated, the PARP inhibitor PJ34 was added to the reaction mixture with purified PARP-1 alone (lane 3) or in the presence of recombinant Sp1 (lane 6). When indicated, samples from the PARP assay were electrophoresed and electrotransfered onto nitrocellulose membranes. The PAR covalently linked onto the automodified PARP-1 and Sp1 proteins was then erased by incubation with PARG and the proteins analyzed by Western blotting with the same antibodies as detailed above (lane 8). Lane 1: PARP-1 alone; lane 2: PARP-1 incubated with NAD+; lane 3: same as in lane 2 plus PJ34; lane 7: same as in lane 5 but incubated in PARG buffer without addition of PARG-1. The position of modified PARP-1 (PARP-1Mod) and Sp1 (Sp1Mod) is indicated (left) along with the appropriate molecular mass marker (right). () Recombinant Sp1 was incubated in reaction buffer containing 200 μM NAD+ and nicked DNA either alone (+SP1; lane 3) or with purified bovine PARP-1 (1 unit) (+Sp1/PARP-1; lane 4). A sample (16 μl) from the reaction mixture was then incubated with the 5'-end labeled Sp1 oligonucleotide and formation of DNA-protein complexes monitored by EMSA as in Figure 2. As a control, the PARP-1 inhibitor PJ34 was added to the reaction mixture containing PARP-1/NAD/Sp1 (+Sp1/PARP-1/PJ34; lane 5). Lane 1: labeled probe alone in reaction mix (P); Lane 2: labeled probe incubated in buffer D with PARP-1 but in the absence of NAD and Sp1 (+PARP-1). The position of both the Sp1 complex (Sp1) and the free probe (U) is indicated