Silencing p300 in MCF7 cells to study expression and alternative splicing

Splicing of precursor mRNA (pre-mRNA) is an important regulatory step in gene expression. Recent evidence points to a regulatory role of chromatin-related proteins in alternative splicing (AS) regulation. Using an unbiased approach, we have identified the acetyltransferase p300 as a key chromatin-related regulator of AS. P300 promotes genome-wide exon inclusion in both a transcription-dependent and ‑independent manner. Using CD44 as a paradigm, we found that p300 regulates AS by modulating the binding of splicing factors to pre-mRNA. Employing a tethering strategy, we found that binding of p300 to the CD44 promoter region promotes CD44v exon inclusion independently of RNAPII transcriptional elongation rate. Promoter-bound p300 regulates AS by acetylating splicing factors, leading to exclusion of hnRNP M from CD44 pre-mRNA and activation of Sam68. P300-mediated CD44 AS reduces cell motility and promotes epithelial features. Our findings reveal a mechanism through which chromatin-related proteins regulate AS and show the impact of this mechanism on cell function. MCF7 cells were transfected with non-targeting siRNA (siNT) and sip300 for 72 h, each in triplicates. RNA was extracted, libraries prepared and RNA-seq was conducted.