Gene Regulatory Mechanisms Controlling Tissue Maturation and Polyploidization

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I. ABSTRACT Polyploidy—a state in which cells carry more than two sets of chromosomes—is frequently observed in nature, yet, the genetic mechanisms controlling ploidy and its functional significance remain enigmatic. The liver, in particular, gains a high percentage of polyploid hepatocytes during postnatal period of development; and the frequency and extent of hepatic polyploidization are further increased following injury, DNA damage, and oxidative stress, but are decreased in hepatocellular carcinoma. Recent evidence suggests that polyploidy safeguards the liver from tumorigenesis by slowing the proliferative capacity of hepatocytes and maintaining a reservoir of tumor suppressors. However, there is minimal understanding of the molecular events that govern the postnatal initiation/promotion of hepatic polyploidization or how differences in chromosomal ploidy affect the transcriptional and posttranscriptional activities of hepatocytes. We have previously demonstrated that the RNA binding protein ESRP2 is a key developmentally regulated factor, which activates an adult splicing program to facilitate terminal differentiation, functional competence, and maturation of hepatocytes. The goals of this proposal are to (i) determine the physiological necessity/sufficiency of ESRP2 and its splicing- regulatory-network in driving hepatocyte polyploidy, and (ii) define the quantitative and qualitative impact of ploidy on hepatocyte transcriptional output. Aim 1 will use ESRP2 gain-and loss-of-function mouse models to determine if programmed changes in RNA splicing through ESRP2 activation are crucial for the polyploidization of hepatocytes. In Aim 2, we will generate high-resolution transcriptomes from diploid and polyploid murine hepatocytes to investigate how ploidy influences the steady-state levels and alternative splicing patterns of hepatic transcripts at a genome-wide scale. The proposed aims will examine new gene regulatory mechanism(s) controlling polyploidization while uncovering previously unrecognized links between alternative splicing and cellular polyploidy.