Mechanisms in Ascl1-mediated reprogramming of fibroblasts into neurons
- Lineage reprogramming of somatic cells, i.e. the conversion of one cell type into another, unrelated cell type, has innovated the fields of stem cell research and translational medicine. The goal of my thesis is to understand the molecular mechanisms of induced lineage reprogramming to improve efficiencies and thereby enable its translation for clinical applications. Previous studies have shown that during reprogramming of fibroblasts into induced pluripotent stem cells (iPSCs) using transcription factors Oct4, Sox2, Klf4 and c-Myc (OSKM), OSK act cooperatively as pioneering factors to first bind fibroblast enchancers to shut down the donor program before moving to activate the pluripotency circuit. It was also shown that iPSC reprogramming involves an initial stochastic phase to activate a subpopulation of cells that eventually enter a late hierarchical phase that activates the endogenous pluripotent circuitry. In contrast, studies in direct reprogramming of fibroblasts into induced neuronal (iN) cells and muscles by Ascl1 and Myod1 respectively have shown that Ascl1 and Myod1 bind immediately to their endogenous binding sites and activate their respective target programs. This leads us to hypothesize that the mechanism of direct reprogramming differs from iPSC reprogramming. Using single cell and bulk RNA sequencing (RNA-seq), Assay of Transposase Accessibly Chromatin with sequencing (ATAC-seq) and Chromatin Immunoprecipitation followed by sequencing (ChIP-seq) to observe Ascl1 binding and changes in gene expression and chromatin accessibility during iN cell reprogramming, we found that in contrast to iPSCs, there is an initial homogenous response to Ascl1 within 2 days of transgene induction. There is a corresponding increase in accessibility of chromatin regions bound by Ascl1 within 12 hours, which leads to the up-regulation of a series of Ascl1 target genes that act in concert to facilitate a major cell fate transition between 2 -- 5 days. Finally, a maturation phase between occurs after 5 days whereby the cells begin to activate neuronal and synapse maturation genes to become fully functional neurons. Surprisingly, we also found a small fraction of cells that got redirected to an alternate myogenic program, and showed that it can be attributed to unexpected similarities in Ascl1 and Myod1 DNA-binding affinities. Finally, we found that the myogenic program can by suppressed by pro-neuronal Myt1l to both increase Ascl1-mediated iN cell reprogramming efficiency and also redirect Myod1-expressing cells towards a neurogenic fate. These observations underscore the different molecular mechanisms between iN cell and iPSC reprogramming, reveal specific properties of transcription factors able to induce reprogramming, and also show the underlying the importance of endogenous co-factors in regulating cell fate during development.
|Type of resource
|electronic; electronic resource; remote
|1 online resource.
|Lee, Qian Yi
|Stanford University, Department of Bioengineering.
|Quake, Stephen Ronald
|Quake, Stephen Ronald
|Statement of responsibility
|Qian Yi Lee.
|Submitted to the Department of Bioengineering.
|Thesis (Ph.D.)--Stanford University, 2017.
- © 2017 by Qian Yi Lee
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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