Klein, David Charles
(2023)
Cell fate decisions are altered through regulation and misregulation of gene-distal elements in embryonic stem cells and acute myeloid leukemia.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Cells with the capacity to mature into other effector cell types are faced with a constant, critical decision: whether to maintain their status as progenitor cells, or to undergo specific differentiation into various effector cells. These cell fate decisions are carried out at the individual cell level, leading to a tremendous diversity of differentiation states, from totipotent embryonic stem cells to terminally differentiated effector cells, such as platelets, erythrocytes, and macrophages. This diversity is maintained by specialized gene regulation at the level of individual cells. Gene expression is highly regulated, particularly through pre-transcriptional changes in DNA-bound factor localization, physical contact between genes and their distal regulatory regions, and general accessibility of the DNA composing these genes and gene regulatory regions. Our work examines these regulatory factors through a lens of cellular identity—in other words, how gene regulation shapes cell type, focusing largely on gene-distal gene regulation.
To identify the role of the FACT complex in maintaining murine embryonic stem cell identity, we depleted the FACT subunit SPT16, causing a failure to maintain pluripotency and resulting nonspecific differentiation to occur. To understand what the role of the FACT complex is in maintaining pluripotent cell identity, we performed genomic characterization assays on SPT16-depleted cells, identifying extensive co-regulation of genic and gene-distal elements with the core pluripotency factors OCT4, SOX2, and NANOG. FACT facilitates maintenance of open chromatin at enhancer regions, including those bound by the core pluripotency circuit, with implications on coding and non-coding transcription. These gene-distal regulatory regions are hotspots of nucleosome remodeling factor activity and gene regulation, and we identified novel occupancy of two understudied alternative nucleosome structures, overlapping dinucleosomes and fragile nucleosomes, at putative enhancer loci. These alternative nucleosome structures are enriched at sites bound by OCT4, SOX2, and NANOG, and are oppositely regulated by the esBAF ATPase BRG1. Finally, we examined the role of the BRD9 bromodomain in ncBAF-mediated regulation of acute myeloid leukemia (AML) cell identity. ncBAF sustains the characteristic differentiation block of AML, and inhibition of BRD9 derepresses myeloid lineage specification factors. BRD9 maintains accessible chromatin at distal enhancers to regulate hematopoietic transcription factor occupancy, thereby regulating myeloid cell identity.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
11 May 2023 |
| Date Type: |
Publication |
| Defense Date: |
29 March 2023 |
| Approval Date: |
11 May 2023 |
| Submission Date: |
3 April 2023 |
| Access Restriction: |
2 year -- Restrict access to University of Pittsburgh for a period of 2 years. |
| Number of Pages: |
303 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Biological Sciences |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
chromatin, transcription, nucleosomes, nucleosome remodeling, enhancers, genomics, pluripotency, differentiation |
| Date Deposited: |
11 May 2023 14:54 |
| Last Modified: |
11 May 2023 14:54 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/44412 |
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