MABE308 Sigma-AldrichAnti-ICBP90/UHRF1 Antibody, clone 1RC1C-10

Poly(ADP-ribose) polymerase 1 (PARP1, also known as ARTD1) is an abundant nuclear enzyme that plays important roles in DNA repair, gene transcription and differentiation through the modulation of chromatin structure and function. In this work we identify a physical and functional poly(ADP-ribose) mediated interaction of PARP1 with the E3 ubiquitin ligase UHRF1 (also known as NP95, ICBP90) that influences two UHRF1-regulated cellular processes. On one hand, we uncover a cooperative interplay between PARP1 and UHRF1 in the accumulation of the heterochromatin repressive mark H4K20me3. The absence of PARP1 leads to reduced accumulation of H4K20me3 onto pericentric heterochromatin that coincides with abnormally enhanced transcription. The loss of H4K20me3 is rescued by the additional depletion of UHRF1. In contrast, although PARP1 also seems to facilitate the association of UHRF1 with DNMT1, its absence does not impair the loading of DNMT1 onto heterochromatin nor the methylation of pericentric regions possibly owing to a compensating interaction of DNMT1 with PCNA. On the other hand, we show that PARP1 controls the UHRF1-mediated ubiquitination of DNMT1 to timely regulate its abundance during S and G2. Together, this report identifies PARP1 as a novel modulator of two UHRF1-regulated heterochromatin-associated events : the accumulation of H4K20me3 and the clearance of DNMT1.

Effective treatment for metastatic prostate cancer is critically needed. The present study was aimed at identifying metastasis-driving genes as potential targets for therapy (oncotargets). A differential gene expression profile of metastatic LTL-313H and non-metastatic LTL-313B prostate cancer tissue xenografts, derived from one patient's specimen, was subjected to integrative analysis using the Ingenuity Upstream Regulator Analysis tool. Six candidate master regulatory genes were identified, including GATA2, a gene encoding a pioneer factor, a special transcription factor facilitating the recruitment of additional transcription factors. Elevated GATA2 expression in metastatic prostate cancer tissues correlated with poor patient prognosis. Furthermore, GATA2 gene silencing in human prostate cancer LNCaP cells led to a marked reduction in cell migration, tissue invasion, focal adhesion disassembly and to a dramatic change in cell transcriptomes, indicating that GATA2 plays a critical role in prostate cancer metastasis. As such, GATA2 could represent a prostate cancer metastasis-driving gene and a potential target for therapy of metastatic prostate cancer.

Ubiquitin-like containing PHD and RING finger 1 (UHRF1) contributes to silencing of tumor suppressor genes by recruiting DNA methyltransferase 1 (DNMT1) to their hemi-methylated promoters. Conversely, demethylation of these promoters has been ascribed to the natural anti-cancer drug, epigallocatechin-3-gallate (EGCG). The aim of the present study was to investigate whether the UHRF1/DNMT1 pair is an important target of EGCG action. Here, we show that EGCG down-regulates UHRF1 and DNMT1 expression in Jurkat cells, with subsequent up-regulation of p73 and p16(INK4A) genes. The down-regulation of UHRF1 is dependent upon the generation of reactive oxygen species by EGCG. Up-regulation of p16(INK4A) is strongly correlated with decreased promoter binding by UHRF1. UHRF1 over-expression counteracted EGCG-induced G1-arrested cells, apoptosis, and up-regulation of p16(INK4A) and p73. Mutants of the Set and Ring Associated (SRA) domain of UHRF1 were unable to down-regulate p16(INK4A) and p73, either in the presence or absence of EGCG. Our results show that down-regulation of UHRF1 is upstream to many cellular events, including G1 cell arrest, up-regulation of tumor suppressor genes and apoptosis.

Aberrant DNA methylation is often associated with cancer and the formation of tumors; however, the underlying mechanisms, in particular the recruitment and regulation of DNA methyltransferases remain largely unknown. In this study, we identified USP7 as an interaction partner of Dnmt1 and UHRF1 in vivo. Dnmt1 and USP7 formed a soluble dimer complex that associated with UHRF1 as a trimeric complex on chromatin. Complex interactions were mediated by the C-terminal domain of USP7 with the TS-domain of Dnmt1, whereas the TRAF-domain of USP7 bound to the SRA-domain of UHRF1. USP7 was capable of targeting UHRF1 for deubiquitination and affects UHRF1 protein stability in vivo. Furthermore, Dnmt1, UHRF1 and USP7 co-localized on silenced, methylated genes in vivo. Strikingly, when analyzing the impact of UHRF1 and USP7 on Dnmt1-dependent DNA methylation, we found that USP7 stimulated both the maintenance and de novo DNA methylation activity of Dnmt1 in vitro. Therefore, we propose a dual role of USP7, regulating the protein turnover of UHRF1 and stimulating the enzymatic activity of Dnmt1 in vitro and in vivo.

The aim of this study on a series of biopsies diagnosed as normal, metaplastic, low-grade squamous intraepithelial lesions (LSILs), and high-grade squamous intraepithelial lesions (HSILs) was dual: to determine the chronology of cell cycle and proliferation abnormalities after human papillomavirus infection during the development of squamous intraepithelial lesions and to determine the best diagnostic indicator(s) linked to the appearance of an HSIL. Ninety-nine cervical biopsies, 18 normal, 9 with metaplastic changes, 29 LSIL, and 43 HSIL (23 cervical intraepithelial neoplasia 2 and 20 cervical intraepithelial neoplasia 3), were analyzed by image cytometry for DNA ploidy and p16INK4A determination, AgNOR counting, MIB-1, and ICBP90 immunostaining quantification. The human papillomavirus status had been previously determined on corresponding cytological smears with the Hybrid Capture II test. Suspect DNA profile and p16INK4A staining were the first significant events that preceded the increase of cell proliferation. Indeed, these markers were the best tests for the detection of a lesion, whatever its grade (positive predictive values of 90% and 100%, respectively). The presence of MIB-1- or ICBP90-positive cells in the upper two thirds of the epithelium was a very accurate feature to select HSIL (sensitivity, 100% for MIB-1) but with a low specificity. The sensitivity of a suspect DNA profile associated with a positive MIB-1 or ICPB90 immunostaining for the detection of an HSIL was, respectively, 92.8% and 92.7%; their specificities were 54.2% and 44%; their positive predictive values were 78% and 73%; their negative predictive values were 81.2% and 78.6%; and the global values were 78.8% and 74.3%. Thus, the most accurate test to distinguish an LSIL from an HSIL was the association of a suspect DNA profile and the presence of MIB-1- or ICBP90-positive cells in the upper two thirds of the epithelium.

We have recently identified a novel CCAAT box binding protein (ICBP90) involved in the regulation of topoisomerase IIalpha gene expression. We have observed that it is expressed in non-tumoral proliferating human lung fibroblast cells whereas in HeLa cells, a tumoral cell line, ICBP90 was still present even when cells were at confluence. In the present study, we have determined the ICBP90 gene structure by screening of a human placenta genomic library and PCR analysis. We report that the ICBP90 gene spans about 35.8 kb and contains six coding exons named A to F. In the 5' upstream sequence of the region containing the coding exons, two additional exons (I and II) were found. Additionally, an internal splicing site was found in exon A. A promoter region, including three putative Sp1 binding sites between exons I and A, was identified by transient transfection. Northern blot analysis of several cancer cell lines revealed the existence of two ICBP90 mRNA species of 5.1 and 4.3 kb that are transcribed from the gene. The relative amounts of these mRNAs depended on the cell type. In MOLT-4 cells and Burkitt's lymphoma Raji cells, the 4.3 kb or the 5.1 kb transcripts were mainly observed, respectively. In other cell lines, such as HL-60 cells, chronic myelogenous leukaemia K-562, lung carcinoma A549, HeLa or colorectal SW480, both 4.3 and 5.1 kb forms of ICBP90 mRNA could be detected. Interestingly, western blot analysis showed several ICBP90 protein bands in HeLa but only a single band in MOLT-4 cell extracts. Taken together our results are consistent with the ICBP90 gene exhibiting alternative splicing and promoter usage in a cell-specific manner.

The one-hybrid system with an inverted CCAAT box as the DNA target sequence was used to identify proteins acting on key DNA sequences of the promoter of the topoisomerase IIalpha gene. Screening of cDNA libraries from the leukemia Jurkat cell line and from the adult human thymus resulted in the isolation of a novel protein of 793 amino acids (89,758 Da). This protein has in vitro CCAAT binding properties and has been called ICBP90. Adult thymus, fetal thymus, fetal liver, and bone marrow, known as active tissues in terms of cell proliferation, are the tissues richest in ICBP90 mRNA. In contrast, highly differentiated tissues and cells such as the central nervous system and peripheral leukocytes are free of ICBP90 mRNA. Western blotting experiments showed a simultaneous expression of topoisomerase IIalpha and ICBP90 in proliferating human lung fibroblasts. Simultaneous expression of both proteins has also been observed in HeLa cells, but in both proliferating and confluent cells. Overexpression of ICBP90 in COS-1-transfected cells induced an enhanced expression of endogenous topoisomerase IIalpha. Immunohistochemistry experiments showed that topoisomerase IIalpha and ICBP90 were coexpressed in proliferating areas of paraffin-embedded human appendix tissues and in high-grade breast carcinoma tissues. We have identified ICBP90, which is a novel CCAAT binding protein, and our results suggest that it may be involved in topoisomerase IIalpha expression. ICBP90 may also be useful as a new proliferation marker for cancer tissues.