BRCA1 interacts with dominant negative SWI/SNF enzymes without affecting homologous recombination or radiation-induced gene activation of p21 or Mdm2

BRCA1 is a tumor suppressor gene linked to familial breast and ovarian cancer. The BRCA1 protein has been implicated in a diverse set of cellular functions, including activation of gene expression by the p53 tumor suppressor and control of homologous recombination (HR) during DNA repair. Prior reports have demonstrated that BRCA1 can exist in cells in a complex with the BRG1-based SWI/SNF ATP-dependent chromatin remodeling enzymes and that SWI/SNF components contribute to p53-mediated gene activation. To investigate the link between SWI/SNF function and BRCA1 mediated effects on p53-mediated gene activation and on mechanisms of homologous recombination, we have utilized mammalian cells that inducibly express an ATPase-deficient, dominant negative SWI/SNF enzymes. Mutant SWI/SNF ATPases retain the ability to interact with BRCA1 in cells. We report that expression of dominant negative SWI/SNF enzymes does not affect p53-mediated induction of the p21 cyclin dependent kinase inhibitor or the Mdm2 E3 ubiquitin ligase that regulates p53 in cells exposed to UV or gamma irradiation. Similarly, integration of a reporter that monitors homologous recombination by gene conversion into these cells demonstrated no change in the recombination rate in the absence of functional SWI/SNF enzyme. We conclude that the SWI/SNF chromatin remodeling enzymes may contribute to but are not required for these processes.     

The RSC chromatin remodeling complex has a crucial role in the complete remodeler set for yeast PHO5 promoter opening

Although yeast PHO5 promoter chromatin opening is a founding model for chromatin remodeling, the complete set of involved remodelers remained unknown for a long time. The SWI/SNF and INO80 remodelers cooperate here, but nonessentially, and none of the many tested single or combined remodeler gene mutations could prevent PHO5 promoter opening. RSC, the most abundant and only remodeler essential for viability, was a controversial candidate for the unrecognized remodeling activity but unassessed in vivo. Now we show that remodels the structure of chromatin (RSC) is crucially involved in PHO5 promoter opening. Further, the isw1 chd1 double deletion also delayed chromatin remodeling. Strikingly, combined absence of RSC and Isw1/Chd1 or Snf2 abolished for the first time promoter opening on otherwise sufficient induction in vivo. Together with previous findings, we recognize now a surprisingly complex network of five remodelers (RSC, SWI/SNF, INO80, Isw1 and Chd1) from four subfamilies (SWI/SNF, INO80, ISWI and CHD) as involved in PHO5 promoter chromatin remodeling. This is likely the first described complete remodeler set for a physiological chromatin transition. RSC was hardly involved at the coregulated PHO8 or PHO84 promoters despite cofactor recruitment by the same transactivator and RSC’s presence at all three promoters. Therefore, promoter-specific chromatin rather than transactivators determine remodeler requirements.     

Expression of base excision repair key factors and miR17 in familial and sporadic breast cancer

Understanding of BRCA1/2 interaction with the base excision repair (BER) pathway could improve therapy based on ‘synthetic lethality'', whose effectiveness is based on homologous recombination deficiency in cells lacking functional BRCA genes. However, poly (ADP-ribose) polymerase (PARP) inhibitors failed in some patients and for this reason we explored BER key enzyme expression. In this study, the expression of BER enzymes (redox factor 1/apurinic-apyrimidinic endonuclease 1 (REF1/APEX1), NTH endonuclease III-like 1 (NTHL1), 8-oxoguanine DNA glycosylase (OGG1), PARP1) and of the scaffold protein XRCC1 (X-ray repair complementing defective repair in Chinese hamster cells 1) were investigated in familial (BRCA-related and not) and sporadic breast cancer cases. Furthermore, miR17 expression was measured because of its role in the epigenetic regulation of BRCA1. Gene expression was evaluated in BRCA1-mutated cell lines, SUM149PT and SUM1315MO2, and in a BRCA1-proficient triple-negative MDA-MB-231 cell line. A cohort of 27 familial and 16 sporadic breast cancer patients was then examined to confirm results obtained from the cell line model. APEX1/REF1 was found to be upregulated in familial BRCA-wild-type and sporadic cases, indicating this enzyme as a potential therapeutic target. Furthermore, XRCC1 was overexpressed in BRCAX patients; consequently, we suggest to test the effectiveness of inhibitors targeting two different BER components in preclinical studies. XRCC1, which is also involved in the non-homologous end-joining pathway, was found to be downregulated in BRCA2-related patients concurrently with no change in PARP1 expression. Interestingly, no difference in PARP1 and miR17 expression was found in BRCA-related and sporadic breast cancer cases. PARP1 and miR17 could therefore be further investigated as molecular biomarkers of ‘BRCAness'' phenotype, indicating patients which could really benefit from PARP inhibitor therapies.