Date of Award

4-2026

Embargo Period

4-9-2028

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Regenerative Medicine and Cell Biology

College

College of Graduate Studies

First Advisor

David T. Long

Second Advisor

Stephen A. Duncan

Third Advisor

Robin Muise-Helmericks

Fourth Advisor

Jorge O. Munera

Fifth Advisor

Stefano Berto

Abstract

The liver arises from the ventral foregut endoderm through a tightly regulated developmental program governed by transcription factor (TF) networks and inductive signals. Commitment to hepatic fate requires coordinated transcriptional regulation and chromatin remodeling. A central regulator of this process is Hepatocyte Nuclear Factor 1 Beta (HNF1B). In humans, heterozygous mutations in HNF1B cause multisystem disease, while in mice, loss of Hnf1b leads to early embryonic lethality due to defective visceral endoderm differentiation and impaired liver bud formation. Despite its importance, the molecular mechanisms by which HNF1B controls the endoderm-to-hepatic transition in humans remain unclear.

To address this, we used human induced pluripotent stem cells (iPSCs) to model hepatogenesis. Expression profiling showed that HNF1B is activated prior to key hepatic regulators, including HNF4A. Using CRISPR-Cas9, we generated iPSC lines with biallelic HNF1B or HNF4A knockouts and inducible rescue systems. Loss of HNF1B blocked hepatoblast formation, while its reintroduction restored hepatic differentiation. In contrast, re-expression of HNF4A in HNF1B-deficient cells failed to rescue hepatic fate. Transcriptomic analyses revealed largely non-overlapping regulatory programs, with HNF1B acting upstream of HNF4A.

Temporal RNA-seq and ATAC-seq integration showed that HNF1B establishes chromatin accessibility at hepatic regulatory regions during definitive endoderm, including HNF4A loci and enhancers enriched for GATA motifs. Consistent with this, HNF1B ChIP-seq demonstrated direct binding of HNF1B at HNF4A promoters. These findings support a model in which HNF1B functions as a chromatin gatekeeper that primes hepatic competence by creating a permissive epigenetic landscape in coordination with pioneer factors such as GATA6 and FOXA2.

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Copyright is held by the author. All rights reserved.

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