Elucidating gene regulatory mechanisms of postnatal maturation and regeneration in the mammalian liver

Bangru, Sushant

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Description

Title

Elucidating gene regulatory mechanisms of postnatal maturation and regeneration in the mammalian liver

Author(s)

Bangru, Sushant

Issue Date

2021-08-19

Director of Research (if dissertation) or Advisor (if thesis)

Kalsotra, Auinash

Doctoral Committee Chair(s)

Kalsotra, Auinash

Committee Member(s)

• Martinis, Susan
• Prasanth, KV
• Yang, Jing
• Zhang, Kai

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

post-transcriptional gene regulation, alternative splicing, rna binding proteins, liver regeneration, polyploidy

Abstract

The mammalian liver is a major metabolic organ responsible for a variety of functions and has an exceptional capacity for regeneration. To enable functional competency in postnatal and adult stages, enable survival in response to injury and support extensive remodeling in regeneration hepatocytes undergo dramatic changes in gene regulatory networks. My thesis focuses on understanding the mechanisms of gene regulation, specifically post-transcriptional gene regulation, within hepatocytes in postnatal maturation, regeneration and disease using the murine model system. At the outset, I begin by exploring gene expression in chronic toxin-mediated liver injury and repair. I use genomewide transcriptomic methodologies to probe gene expression, alternative splicing and translation within the regenerating hepatocytes and find extensive remodeling at all stages of gene regulation. We find that RNA binding proteins especially splicing factors are regulated at translation level, leading to redeployment of a postnatal splicing programme. Furthermore, to overcome limitation of bulk methods in revealing subtleties in population behavior we employed single-cell genomics in acute liver injury. In Chapter three, we probed and analyzed single cell gene expression from ~22,000 liver cells post-2/3rd partial hepatectomy to reveal the division of labor within hepatocytes in regeneration to simultaneously support proliferation and metabolism. Furthermore, we show evidence using cumulative labelling methods that the major source of new hepatocytes in acute injury is midlobular zone within the liver lobule. From analysis in Chapter two as well as previous work in the lab, we had identified Epithelial splicing regulatory protein 2 (ESRP2) as an important splicing factor that has a postnatal onset in hepatocytes and is dynamically regulated in regeneration and disease. In chapter four, I begin by exploring the genomewide splicing regulation by ESRP2 using a loss-of-function mouse model and find extensive regulation of both developmentally regulated events as well as an adult stage specific program. We also find extensive defects in hepatic proliferation, cell size and polyploidization in ESPR2 KO mice. Furthermore, we developed an acute hepatocyte specific ESRP2 loss-of-function model revealing that ESRP2 is necessary to not only bring about but also maintain the adult hepatic splicing program. Using a hepatocyte specific inducible overexpression model for ESRP2 we showed by in young pups that ESRP2 is sufficient for early induction of postnatal maturation and revealed the importance of temporal regulation of ESRP2 expression. Lastly, to identify ESRP2’s genomewide binding landscape we developed, using CRISPR-Cas9 methodology, a 2xFLAG-tagged transgenic mouse and performed eCLIP to reveal both intronic as well as surprisingly 3’UTR binding by ESRP2. Lastly, we identify miR-122 biogenesis as a direct target of ESRP2 regulation leading to reduced mature miR-122 levels in ESRP2 loss-of-function mice with eventual loss of proper postnatal hepatic polyploidization. In chapter five, I explore the role of ESRP2 in chronic liver injury wherein it is dynamically downregulated via translational regulation. Using gain- and loss-of-function models we find that ESRP2 levels directly regulate hepatocyte proliferation rates. We identify 4 cassette exons harbored within proteins of the Hippo pathway, namely, NF2, CSKN1D, YAP1 and TEAD1 that are regulated by ESRP2. These exons are upregulated in development, and in regeneration their inclusion is reduced and this correlates directly with ESRP2 expression. We show using cell culture of primary hepatocytes that exclusion of these exons augments hepatocyte proliferative potential, and this occurs due to attenuation of Hippo signaling leading to YAP1 translocation to nucleus and transcriptional activation. In conclusion, I have identified a cell-type specific and temporally coordinated gene expression program operative in postnatal liver development and regeneration. Furthermore, I demonstrated a direct role for ESRP2 in regulating a post-transcriptional gene regulatory network to support postnatal hepatocyte maturation and regeneration.

Graduation Semester

2021-12

Type of Resource

Thesis

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Copyright 2021 Sushant Bangru

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