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    Research Grants Awarded

    Cell cycle-dependent regulation of human BRCA2 gene expression by SLUG and PRDX5C

    Study Section:
    Tumor Cell Biology I

    Scientific Abstract:
    BACKGROUND. The expression of the tumor suppressor protein BRCA2 is strictly regulated depending upon the growth stage of the breast cell. It is not expressed in the non-dividing human breast cells and it is only expressed in the dividing stages of the cells. The notion is that balanced expression of BRCA2 in the rapidly dividing human breast cells is critical to prevent the malignant transformation of these cells. We recently have identified SLUG as the repressor of human BRCA2 gene expression in the non-dividing cells which works through an Alu repeat-containing transcriptional silencer located upstream of human BRCA2 gene promoter. Our unpublished preliminary data suggest that the DNA binding nuclear isotype of peroxiredoxin 5 (PRDX5) plays a critical role in the desilencing of BRCA2 gene expression in the dividing human breast cells. The HYPOTHESIS is that BRCA2 gene expression is silenced in the non-dividing human breast cells by the repressor protein SLUG and is desilenced in the dividing cells by the sequestration of the binding site of SLUG by PRDX5 at the BRCA2 gene silencer. The SPECIFIC AIMS to test the hypotheses are: (i) to determine the cell cycle stage dependent binding of SLUG and PRDX5 to the BRCA2 gene silencer in vivo by chromatin immunoprecipitation (ChIP) assays; (ii) to determine whether a specific isotype of PRDX5 preferentially binds to the silencer; (iii) to characterize which domain of PRDX5 isotypes binds to what sequence of the silencer DNA; and (iv) to evaluate the levels of acetylated histones at the BRCA2 gene promoter at different growth stages of cultured human breast cells with or without knock down of SLUG or PRDX5 gene expression using specific siRNAs. STUDY DESIGN. We will verify potential variations in the cell cycle dependent binding of SLUG and PRDX5 to the BRCA2 gene silencer in vivo by ChIP assays followed by real time PCR to quantitate the silencer DNA in the chromatin immunoprecipitates. We will evaluate in vitro binding of recombinant PRDX5 variants with the silencer DNA by electrophoretic mobility shift assay. We will evaluate in vivo binding of over expressed FLAG-tagged PRDX5 variants to the silencer by ChIP assay. We will selectively knock-down different isotypes of PRDX5 using specific siRNAs followed by evaluation of the binding of the remaining subtypes by ChIP assay. We will evaluate whether PRDX5 indeed blocks the suppressor functions of SLUG by inhibiting the recruitments of co-repressors and HDACs and thus increasing acetylated histones at the BRCA2 gene promoter at the dividing stage of cultured human breast cells. POTENTIAL OUTCOME. Understanding the silencing/desilencing mechanism of BRCA2 gene expression in detail, we should be able to effectively intervene in the pathway and thus be able to efficiently combat the onset of human breast cell malignancy caused by this malfunction.

    Lay Abstract:
    Existing data and our studies suggest that proportionate expression of BRCA2 in the human breast cells is critical for the survival and growth of human breast cells. Because of its growth suppressor activity, over-expression of BRCA2 in the non-dividing cells may be toxic to these cells. We recently found that SLUG, which binds to the transcriptional silencer at the upstream of the BRCA2 gene, silences BRCA2 gene expression in the quiescent cells. The objective of the proposed study is to understand how BRCA2 silencer is inactivated in the dividing cells. Our notion is that a variant of PRDX5 goes to the nucleus in the dividing breast cells, sequester the binding site of SLUG on the BRCA2 gene silencer and thus prevent SLUG from silencing the expression of BRCA2 gene. The research proposed will study in details how PRDX5 competes with SLUG to bring about the desilencing of BRCA2 gene expression in the dividing human breast cells. According to our hypothesis, SLUG should bind to the silencer in the quiescent cells whereas PRDX5 should be the major binder to the silencer of the dividing cells. We will verify these expected variations in the cell cycle stage dependent binding of SLUG and PRDX5 to the BRCA2 gene silencer in vivo. PRDX5 has three subtypes and all of them are expressed in human breast cells (unpublished data). We postulate that PRDX5C, which lacks catalytic activity (active site Cys is missing) and is smallest in size, may preferentially enter the nucleus and bind to the silencer in the dividing cells. To verify this we will evaluate in vitro binding of recombinant PRDX5 variants with the silencer DNA; in vivo binding of over expressed PRDX5 variants to the silencer and will selectively knock-down different isotypes of PRDX5 using specific siRNAs followed by evaluation of the binding of the remaining subtypes. We will also determine which subtype of PRDX5 best competes with SLUG for the binding with the silencer DNA. The objective of this part of the research activity is to understand (i) What is the exact binding site for PRDX5 to the silencer DNA? and, (ii) which part of PRDX5 protein is responsible for this binding? The footprint of recombinant PRDX5A was previously determined for the Alu promoter. From sequence similarity we have predicted the probable site of PRDX5 binding to the silencer DNA. We will experimentally verify this binding site. This study will help us to understand the mechanism of desilencing of human BRCA2 gene expression by PRDX5 at the molecular level. These data may also give us some clue to design molecular decoys to intervene this process to accomplish clinical or research objectives.