Kitra Cates

ORCID

http://orcid.org/0000-0001-7716-153X

Date of Award

Spring 5-15-2023

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Genetics & Genomics)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

The development of techniques to model adult-onset neurodegeneration are increasingly important as the global aging population rises. Neurodegenerative disease modeling using cellular reprogramming requires the generation of neurons that recapitulate molecular signatures of neuronal cell identity, maturity, and pathogenesis. Ectopic expression of microRNAs-9/9* and microRNA-124 (miR-9/9*-124) directly convert human adult dermal fibroblasts (hDF) into microRNA-induced neurons (miN). This direct conversion provides a platform to study adult-onset neurodegeneration, as it bypasses pluripotency and maintains molecular age-associated properties of patient-derived cells. In coordination with transcription factors, miR-9/9*-124 reprogramming can generate subtype-specific neurons, which can be used for the study of genetic processes and cellular pathologies causing adult-onset neurodegenerative diseases. However, the molecular cascade underlying the ability of miR-9/9*-124 to evoke neuronal identity across somatic cells remained incomplete. In my thesis work, I identify the stepwise cellular and molecular fate dynamics by which miR-9/9*-124 orchestrate neuronal reprogramming. First, I uncovered the fate trajectory of hDF during miR-9/9*-124-mediated reprogramming. My study showed that miR-9/9*-124 first act post-transcriptionally to erase existing fibroblast cell fate, then activate a uniform transition to neuronal cell fate over the course of 20 days. This work demonstrates the sufficiency of miR-9/9*-124 alone to act as potent reprogramming effectors, showing that microRNA-mediated reprogramming does not rely on transcription factor activity to dictate reprogramming dynamics. Further, this study identified the repression of Kruppel-like factors KLF4 and KLF5 as key targets of miR-9/9*-124 for the closure of fibroblast chromatin landscape and silencing of fibroblast gene programs. Conversely, I found that activation of the small non-coding RNA 7SK is critical for the opening of neuronal chromatin loci and initiation of neuronal gene expression. Together, this work pinpoints the cellular dynamics of miR-9/9*-124 reprogramming and essential molecular determinants in the reprogramming cascade. Following this study, I sought to understand the precise gene networks that miR-9/9*-124 erase to silence the pre-existing somatic cell fate. Upon studying the conversion of hDF with variable reprogramming efficiencies, I found that the primary limiting step in miR-9/9*-124-mediated conversion is erasure of starting cell fate. To identify the core gene networks that are erased for successful neuronal conversion, I compared gene network silencing across multiple somatic cell types expressing miR-9/9*-124, including dura fibroblasts, astrocytes, pericytes, and smooth muscle cells. I concluded that networks enriched for function in cell cycle, adhesion, metabolism, chromatin organization, and autophagy are central to miR-9/9*-124-mediated fate erasure. I perturbed the top predicted upstream regulators of fate erasure and observed that TP53 was a critical inhibitory target of reprogramming fate erasure, with inhibition of P53 accelerating reprogramming processes. Overall, my thesis work uncovers the molecular dynamics of miR-9/9*-124-mediated neuronal reprogramming and identifies the key downstream molecules that govern successful neuronal reprogramming.

Language

English (en)

Chair and Committee

Andrew S. Yoo

Committee Members

Harrison Gabel, Charles Kaufman, Kristen Kroll, Thorold Theunissen,

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