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Identification of Id4 as a regulator of BRCA1 expression by using a ribozyme-library-based inverse genomics approach.

ABSTRACT: Expression of the breast and ovarian cancer susceptibility gene BRCA1 is down-regulated in sporadic breast and ovarian cancer cases. Therefore, the identification of genes involved in the regulation of BRCA1 expression might lead to new insights into the pathogenesis and treatment of these tumors. In the present study, an "inverse genomics" approach based on a randomized ribozyme gene library was applied to identify cellular genes regulating BRCA1 expression. A ribozyme gene library with randomized target recognition sequences was introduced into human ovarian cancer-derived cells stably expressing a selectable marker [enhanced green fluorescence protein (EGFP)] under the control of the BRCA1 promoter. Cells in which BRCA1 expression was upregulated by particular ribozymes were selected through their concomitant increase in EGFP expression. The cellular target gene of one ribozyme was identified to be the dominant negative transcriptional regulator Id4. Modulation of Id4 expression resulted in inversely regulated expression of BRCA1. In addition, increase in Id4 expression was associated with the ability of cells to exhibit anchorage-independent growth, demonstrating the biological relevance of this gene. Our data suggest that Id4 is a crucial gene regulating BRCA1 expression and might therefore be important for the BRCA1 regulatory pathway involved in the pathogenesis of sporadic breast and ovarian cancer.

SUBMITTER: Beger C

PROVIDER: S-EPMC14556 | biostudies-literature | 2001 Jan

REPOSITORIES: biostudies-literature

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Identification of Id4 as a regulator of BRCA1 expression by using a ribozyme-library-based inverse genomics approach.

Beger C C Pierce L N LN Kruger M M Marcusson E G EG Robbins J M JM Welcsh P P Welch P J PJ Welte K K King M C MC Barber J R JR Wong-Staal F F

Proceedings of the National Academy of Sciences of the United States of America 20010101 1

Expression of the breast and ovarian cancer susceptibility gene BRCA1 is down-regulated in sporadic breast and ovarian cancer cases. Therefore, the identification of genes involved in the regulation of BRCA1 expression might lead to new insights into the pathogenesis and treatment of these tumors. In the present study, an "inverse genomics" approach based on a randomized ribozyme gene library was applied to identify cellular genes regulating BRCA1 expression. A ribozyme gene library with randomi ...[more]

PMID: 11136250

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Project description:A miRNAs profiling on a group of familial and sporadic breast cancers showed that miRNA-342 was significantly associated with estrogen receptor (ER) levels. To investigate at functional level the role of miR-342 in the pathogenesis of breast cancer, we focused our attention on its "in silico" predicted putative target gene ID4, a transcription factor of the helix-loop-helix protein family whose expression is inversely correlated with that of ER. ID4 is expressed in breast cancer and can negatively regulate BRCA1 expression. Our results showed an inverse correlation between ID4 and miR-342 as well as between ID4 and BRCA1 expression. We functionally validated the interaction between ID4 and miR-342 in a reporter Luciferase system. Based on these findings, we hypothesized that regulation of ID4 mediated by miR-342 could be involved in the pathogenesis of breast cancer by downregulating BRCA1 expression. We functionally demonstrated the interactions between miR-342, ID4 and BRCA1 in a model provided by ER-negative MDA-MB-231 breast cancer cell line that presented high levels of ID4. Overexpression of miR-342 in these cells reduced ID4 and increased BRCA1 expression, supporting a possible role of this mechanism in breast cancer. In the ER-positive MCF7 and in the BRCA1-mutant HCC1937 cell lines miR-342 over-expression only reduced ID4. In the cohort of patients we studied, a correlation between miR-342 and BRCA1 expression was found in the ER-negative cases. As ER-negative cases were mainly BRCA1-mutant, we speculate that the mechanism we demonstrated could be involved in the decreased expression of BRCA1 frequently observed in non BRCA1-mutant breast cancers and could be implicated as a causal factor in part of the familial cases grouped in the heterogeneous class of non BRCA1 or BRCA2-mutant cases (BRCAx). To validate this hypothesis, the study should be extended to a larger cohort of ER-negative cases, including those belonging to the BRCAx class.

Project description:BackgroundKaposi sarcoma-associated herpes virus (KSHV) is one of the most common causal agents of Kaposi Sarcoma (KS) in individuals with HIV-infections. The virus has gained attention over the past few decades due to its remarkable pathogenic mechanisms. A group of genes, ORF71, ORF72, and ORF73, are expressed as polycistronic mRNAs and the functions of ORF71 and ORF72 in KSHV are already reported in the literature. However, the function of ORF73 has remained a mystery. The aim of this study is to conduct comprehensive exploratory experiments to clarify the role of ORF73 in KSHV pathology and discover markers of AIDS-associated KSHV-induced KS by bioinformatic approaches.Methods and resultsWe searched for homologues of ORF-73 and attempted to predict protein-protein interactions (PPI) based on GeneCards and UniProtKB, utilizing Position-Specific Iterated BLAST (PSI-BLAST). We applied Gene Ontology (GO) and KEGG pathway analyses to identify highly conserved regions between ORF-73 and p53to help us identify potential markers with predominant hits and interactions in the KEGG pathway associated with host apoptosis and cell arrest. The protein p53 is selected because it is an important tumor suppressor antigen. To identify the potential roles of the candidate markers at the molecular level, we used PSIPRED keeping the conserved domains as the major parameters to predict secondary structures. We based the FUGE interpretation consolidations of the sequence-structure comparisons on distance homology, where the score for the amino acids matching the insertion/deletion (indels) detected were based on structures compared to the FUGE database of structural profiles. We also calculated the compatibility scores of sequence alignments accordingly. Based on the PSI-BLAST homologues, we checked the disordered structures predicted using PSI-Pred and DISO-Pred for developing a hidden Markov model (HMM). We further applied these HMMs models based on the alignment of constructed 3D models between the known structure and the HMM of our sequence. Moreover, stable homology and structurally conserved domains confirmed that ORF-73 maybe an important prognostic marker for AIDS-associated KS.ConclusionCollectively, similar variants of ORF-73 markers involved in the immune response may interact with targeted host proteins as predicted by our computational analysis. This work also suggests the existence of potential conformational changes that need to be further explored to help elucidate the role of immune signaling during KS towards the development of therapeutic applications.

Dataset Information

Identification of Id4 as a regulator of BRCA1 expression by using a ribozyme-library-based inverse genomics approach.

Publications

Identification of Id4 as a regulator of BRCA1 expression by using a ribozyme-library-based inverse genomics approach.

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