ORCID

0000-0002-6683-9322

Date of Award

4-26-2024

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

Direct reprogramming, the direct forced conversion of one somatic cell type to another has widespread applications in regenerative medicine. Owing to its highly complex nature, discovery and validation of new factors for direct reprogramming is challenging and many current protocols suffer from low conversion efficiency, failing to create mature cell types comparable to their in vivo counterparts. Recent advances in single-cell have coupled cell ancestry and transcriptomic state measurement in single-cell RNA sequencing (scRNA-seq) assays. In reprogramming and development, these have allowed mapping lineage specific transcriptional changes both before and during cell fate specification. These methods work by encoding lineage into heritable, transcribed DNA barcodes that are captured alongside single-cell transcriptomes. While ingenuous, this approach limits lineage tracing to single-cell transcriptomic assays. On the other hand, cell fate conversions, both artificial and natural, are driven by complex gene regulatory mechanisms and precise control and modification the cellular epigenomic state. As a result, the assay of lineage paired with cellular epigenetic state could provide significant insights into fate specifying epigenetic changes, in a lineage specific manner. We enabled this via the development of CellTag-multi - an assay to directly capture lineage barcodes across both single-cell transcriptomic and epigenomic assays. CellTag-multi build on our original single-cell transcriptional lineage tracing method, CellTagging. With the first iteration of CellTag-multi, we extended lineage tracing to single-cell epigenomics by pairing lineage barcode capture with single-cell genome-wide accessible chromatin profiles - measured via single-cell Assay of Transposase Accessible Chromatin by Sequencing (scATAC-seq). We validated CellTag-multi by applying it to in vitro hematopoiesis, a well characterized system of multi-lineage differentiation, where we use it to recapitulate known signatures of lineage specific epigenetic priming in differentiating progenitor cells. Additionally, we compared the degree of lineage priming across transcriptional and epigenomic state and demonstrate the existence of non-redundant fate specifying information across the two modalities. Next, we applied CellTag-multi to a less defined system of fate conversion - the direct reprogramming of Mouse Embryonic Fibroblasts (MEFs) to induced Endoderm Progenitors (iEPs). iEP reprogramming yields a heterogenous cell population comprised of on-target reprogrammed, epithelialized cells, and off-target cells characterized by partial retention of fibroblast-like identity and activation of certain imprinted genes. Beyond mapping fate-specific cis-regulatory elements, CellTag-multi identified several features delineating cells destined for off- target reprogramming including the failure of reprogramming TFs to engage with their genomic targets and broad activation of mesenchymal gene programs. This analysis also revealed distinct sets of TFs driving fate-specific gene programs in both on-target and off-target destined cells, suggesting lineage dependent re-wiring of gene regulatory networks during the early stages of reprogramming. Lastly, we experimentally validated the function of one such TF, Zfp281, in driving off-target cell identity. We further developed the technical capabilities of CellTag-multi, enabling lineage profiling with single-cell assay of genome-wide histone occupancy profiles. We demonstrated the feasibility of this assay through a series of benchmarking experiments in clonally expanded iEPs. Finally, we switched focus from mechanisms driving on-target vs off-target fates in iEP reprogramming to those governing the in vivo engraftment fates of reprogrammed cells. We observed epigenetic and transcriptional signatures suggestive of fate priming towards either liver or colon in vivo fates, through multiomic clonal analysis, and design a framework for in vivo clonal tracking of these clones to test these findings. In summary, I present CellTag-multi as a platform for lineage tracing across an increasingly diverse array of single cell sequencing assays and demonstrate its utility in revealing fate specifying gene regulatory changes across multiple paradigms of cell fate conversion.

Language

English (en)

Chair and Committee

Samantha Morris

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