Alternative Splicing Reporters Integrated Throughout the Genome Measure Splice Pattern Sensitivity to Local Chromatin Context

Abstract

Alternative splicing (AS) of transcripts into one or more isoforms is a major contributor to eukaryotic transcriptional diversity. Regulation of one splice isoform over another is important for downstream expression, and sequence and protein binding determinants of alternative splicing have been thoroughly studied. While gene position within the genome has been shown to be a powerful regulator of general transcription, the relationship between AS and epigenetic mechanisms like gene position or nuclear compartmentalization is still not well studied. To better understand position effects on AS, I apply a massively parallel reporter assay to integrate thousands of alternatively splicing reporters across the genome of K562 cells and measure alternative splicing activity by location. Five separate gene designs with different alternatively splicing exons were utilized to observe positional effects on single exons with baseline inclusions ranging from high to low. By associating insertion locations with known DNA elements, epigenetic markers, and domain labels, the effects of general elements on a uniform gene's expression and splicing patterns within the same domain could be measured. Ultimately, measurements of integrated reporters across thousands of positions demonstrated little variance in alternative splicing and more minor variance in expression than previously observed with non-splicing reporters. Together, these results demonstrate the greater relative strength of exon, intron, and promoter sequence on transcriptional regulation. This thesis provides the groundwork for position effects on alternative splicing in the human genome, and highlights the need for such integrated studies with a wider variety of gene types.