Characterising the Roles of Zinc Finger Proteins CTCF and ZRANB2 in Modulating Alternative Splicing

Abstract

Zinc finger (ZF) proteins constitute the most abundant protein class and are involved in multiple biological processes including development, differentiation, tumour suppression and apoptosis. CTCF and ZRANB2 are two ZF proteins that have been recently linked to modulation of alternative splicing (AS). AS is a complex biological process enriching transcriptome and proteome diversity by facilitating the production of multiple mRNA and protein isoforms from individual genes. However, the genome-wide impact of Ctcf and Zranb2 dosage on AS has not been investigated. The present study examined the effect of Ctcf haploinsufficiency and Zranb2 deficiency on gene expression and AS in Ctcf hemizygous (Ctcf+/-) and conditional Zranb2 knockout (Zranb2-/-) mice, respectively. Reduced Ctcf and Zranb2 levels caused distinct differences in gene expression and AS. In Ctcf+/- mice, these differences were tissue-specific and exhibited a significant increase in intron retention in Ctcf+/- liver and kidney. Interestingly, Ctcf binding sites were enriched proximal to the genomic regions of the intron-retaining genes in Ctcf+/- liver. Proteomic analysis of Ctcf interacting partners in five mouse tissues identified the Small RNA Binding Exonuclease Protection Factor La (Ssb) as a novel Ctcf interactor in all the examined tissues. Tissue-specific protein interactions with Ctcf were also observed. In the brain, co-immunoprecipitation was used to experimentally validate Ctcf interactions with Tra2β, C1qbp, Cpsf6 as well as Atxn1, which are known to be involved in pre-mRNA splicing and brain development, respectively. This study provides new insights into effects of Ctcf haploinsufficiency and Zranb2 deficiency on the transcriptome and highlights a new role for Ctcf in mediating tissue-specific intron retention and protein interactions.