Transcriptional and post-transcriptional regulation in tissue regeneration and disease

Valiya Chembazhi, Ullas

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https://hdl.handle.net/2142/120527 Copy

Description

Title

Transcriptional and post-transcriptional regulation in tissue regeneration and disease

Author(s)

Valiya Chembazhi, Ullas

Issue Date

2023-04-26

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

Kalsotra, Auinash

Doctoral Committee Chair(s)

Kalsotra, Auinash

Committee Member(s)

• Shapiro, David J.
• Prasanth, Kannanganattu V.
• Kieffer, Collin K.

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• RNA
• Regeneration
• Liver
• Intestine
• Alcoholic liver diseases
• Transcriptomics
• ESRP2
• PTBP1

Abstract

The central focus of this thesis is to understand healthy regeneration in mammalian tissues and delineate why it fails in disease/stress conditions. The first part of my thesis describes mechanisms that confer adult liver with exceptional ability to regenerate while maintaining its specialized functions. We used partial-hepatectomy as a model of mice liver regeneration in tandem with single cell transcriptomics to study the transitions of liver cells through the initiation, progression, and termination phases of regeneration. Cumulative EdU labeling and immunostaining of metabolic, portal, and central vein-specific markers revealed that hepatocyte proliferation after PHx initiates in the midlobular region before proceeding towards the periportal and pericentral areas. We discovered that following partial hepatectomy, a subset of hepatocytes transiently reactivates an early-postnatal-like gene expression program to proliferate, while a distinct population of metabolically hyperactive cells appears to compensate for any temporary deficits in liver function. Subsequent chapters of the thesis provide an account of transcriptional and post-transcriptional mis-regulation in the pathology of liver diseases and addresses why they fail to mount a normal regenerative program. We focused on alcohol-associated liver disease (ALD), which is the primary cause of liver-related mortality worldwide. Drastic deterioration of hepatocellular function and survival is seen as ALDs progress from fatty liver to steatohepatitis (ASH), and then to largely irreversible cirrhosis (AC). We performed comprehensive multi-omic profiling of normal, ASH, and AC patient livers using RNA-seq and ATAC-seq from over 27,000 single nuclei, in parallel with bulk and long read direct RNAseq analyses. We report that alcohol-associated decompensation in human livers induces incomplete transitions to immature state in transcriptional and chromatin states. Strikingly, we identified predominant misregulation in expression levels of several RNA binding proteins (RBPs), including ESRP, PTBP, and SR families of proteins. Our findings, for the first time, report that widespread RBP expression changes in ASH lead to extensive misregulation of developmentally controlled RNA splicing programs and thereby contribute to metabolic dysregulation in patients. Notably, we discovered that adult to fetal switch in alternative splicing patterns of Tcf4, Slk, and several other genes in ASH directly alters their nuclear localization and activities, disrupting signaling networks including WNT and Hippo signaling pathway. Analyzing cellular interaction networks, we reveal that aberrations in RBP levels are induced by disease-associated inflammatory and pro-proliferative signaling. Importantly, we establish suppression of ESRP2 by inflammatory cytokines and TGF-beta in ASH as a major mechanism that disrupts normal adult splicing programs in hepatocytes, leading to a replacement of functional parenchyma with fetal-like cells that lack liver-specific functions. The last chapter presents a distinct scenario of stem-cell mediated regeneration that is critical for homeostasis of the intestinal lining. Intestinal stem cells reside in the crypt bottom and provide the driving force for intestinal epithelial regeneration. We report that polypyrimidine tract binding protein 1 (PTBP1, also known as HNRNP I) is critical for controlling the survival and stemness of intestinal stem cells in mice. Depletion of PTBP1 in mouse intestinal epithelia results in death of intestinal stem cells and subsequent failure of the intestinal epithelial regeneration. We present evidence that PTBP1 maintains the survival of intestinal stem cells at least in part by repressing the expression of Phlda3, an inhibitor of Akt signaling activity that promotes cell apoptosis. Mechanistically, PTBP1 destabilizes Phlda3 mRNA through binding to its 3’ UTR. Moreover, we demonstrate that PTBP1 inhibits a neuronal-like splicing program in crypt cells, which maintains the multipotency of intestinal stem cells. This function is achieved at least in part through inhibiting PTBP2, a paralog of PTBP1 that promotes neuronal differentiation. We demonstrate that PTBP1 inhibits PTBP2 expression in the intestinal crypt cells by repressing the inclusion of alternative exon 10 to the Ptbp2 transcripts. Our results thus reveal a novel mechanism whereby PTBP1 maintains the survival and stemness of intestinal stem cells in vivo through post-transcriptional regulatory mechanisms.

Graduation Semester

2023-05

Type of Resource

Thesis

Copyright and License Information

Copyright 2023 Ullas Valiya Chembazhi

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