Identifying Functional Enhancers For Fibrotic Gene Regulation In Liver Fibrosis

Author

Parnaz Merikhian, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical SciencesFollow

Author ORCID Identifier

https://orcid.org/0000-0001-7482-836X

Date of Graduation

8-2023

Document Type

Thesis (MS)

Program Affiliation

Genetics and Epigenetics

Degree Name

Masters of Science (MS)

Advisor/Committee Chair

Wenbo Li PhD

Committee Member

Chunru Lin PhD

Committee Member

George A. Calin MD PhD

Committee Member

Dung-Fang Lee PhD

Committee Member

Zheng Sun PhD

Abstract

Liver fibrosis is characterized by progressive activation of proliferating and migrating myofibroblasts that lead to accumulation of extracellular matrix (ECM). These myofibroblasts most arise from activated liver-resident hepatic stellate cells (HSCs). There is an increasing number of patients suffering from liver fibrosis in developed countries including the United States, which is anticipated to continue to grow during 2023-2033 period. TGF-β1 is a key cytokine with a significant role in regulating cell differentiation and adhesion in liver fibrosis. TGF-β signaling triggers gene expression changes in HSCs, including that of fibrotic and EMT-related genes, which then functionally promote HSCs activation and fibrogenesis. The TGF-β pathway achieves this primarily by transcription factor SMAD3, which acts on specific genetic elements to regulate gene expression, most of which are distal enhancers. These suggest that enhancers play essential roles in HSC gene deregulation and liver fibrosis. However, it remains underexplored in terms of the category of active enhancers in human HSCs and their contribution to liver fibrosis. Here we used human HSC LX-2 cells after TGF-β treatment as a model, and utilized epigenetic methods such as ChIP-seq and nascent RNA-seq TT-Seq to define TGF-β activated enhancers in human HSC cells. We also identified the noncoding RNAs produced from active enhancers that we referred to as fibrotic enhancer-associated eRNAs. I further conducted an eRNA perturbation experiment that supported a role of key enhancer and eRNA in activating fibrotic genes in HSC cells. Findings here support the identification and interrogation of functional enhancers that drive fibrotic gene activation in human HSC cells, allowing their potential exploration for treatment of liver fibrosis.

Keywords

Liver fibrosis, Hepatic stellate cells, TGF-β, LX-2, Enhancer, eRNA, qPCR, RNA sequencing