Functional characterization of SMARCA2 gene.

My PhD project is based on the characterization of the SMARCA2 gene. On one hand, I focused my attention on the role of SMARCA2 product, the protein brahma (BRM), in the regulation of alternative pre-mRNA splicing. On the other hand, I investigated the transcriptional regulation of SMARCA2 expression. My first project derived from a comparative evaluation and validation of microarray data from two mitochondrial stress models. The first model represented by an acute mitochondrial stress is constituted by human SH-SY5Y neuroblastoma cells treated with Paraquat (PQ), while the second model is a chronic model constituted from the same cell line stably overexpressing the Superoxide Dismutase 1 carrying the most common mutation found in familiar ALS (SOD1 G93A). The merge of these microarrays data showed that oxidative stress affects the choice of specific alternative last exons (ALEs) increasing the production of transcripts variants terminating at a more proximal ALE. Moreover, oxidative stress induces the transcriptional downregulation of the SMARCA2 gene product BRM, one of the two alternative ATPase subunits of the SWI/SNF complex. I found that in normal condition BRM is enriched on the proximal ALE. In addition, I observed the accumulation of BARD1, a protein that forms a functional heterodimer with BRCA1, which has E3 ubiquitin-ligase activity and interacts with the 50 kDa subunit of CstF inhibiting 3’ end processing. Consistent with these observations, I detected an ubiquitinated pool of CstF50 and showed that ubiquitination is mediated by BARD1/BRCA1. Taken together, these results suggested that the presence of BRM on the proximal exon leads to the BARD1/BRCA1-mediated ubiquitination of CstF50 and the inhibition of 3’ end processing at the proximal poly(A). This in turn allows transcription to proceed to the distal terminal exon. In the same microarray data used as a starting point for my first project we detected a shift in SMARCA2 expression towards shorter mRNA isoforms upon oxidative stress. Thus, my second project dealt with the characterization of these transcripts. Bioinformatic analysis revealed that the shorter mRNA variants are evolutionarily conserved and are most likely generated from an internal promoter. Interestingly, in zebrafish the short isoform is produced as an independent gene on the same chromosome of the long isoform but in its reverse strand. This peculiar genomic organization hints to a potentially relevant function for this alternative isoform of the BRM protein. Bioinformatic analyses revealed that the short isoform encode a protein that lacks the N-terminal, catalytic ATP-ase domain but shares the C-terminal region that contains a Bromodomain, a protein motif that is known to bind to acetylated histones. First, I identified the potential alternative promoter region using bioinformatic tools and cloned this region. Using a luciferase reporter system I demonstrated the existence of the alternative promoter. Next, I cloned the short, most conserved isoform and I tested it for interaction with known partners of the full-length BRM protein by Co-Immunoprecipitation (Co-IP). I discovered that BRM-s interacts with histone H3 but not with core component of the SWI/SNF complex. Considering that short isoform does not display an ATPase domain, the Co-IP suggests a possible “dominant negative” role for this protein. If short isoforms works as negative dominant of Brm, the ratio between long and short expressed proteins could become very important for BRM target genes in development and differentiation. In particular, this alteration of ratio could have a negative effect on the cells since several tumor cell lines show a very low level of the long protein. (i.e. human lung tumor cell lines).