Date of Award
Fall 2022
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Genetics
First Advisor
Xiao, Andrew
Abstract
Stem cells are the parental cells from which all other cells of the organism originate. Their diverse functions range from producing an embryo from a single-celled zygote and replenishing cells that are lost during adulthood. Over the past decades, there has been growing interest in understanding how stem cells regulate their cell fate decisions, whether to self-renew or differentiate into daughter cell types. One mechanism controlling these decisions is epigenetic regulation, which involves stable changes to gene expression that do not change the DNA sequence. Studying the epigenetic pathways that regulate stem cells will provide a better understanding of development, tissue homeostasis, and disease pathology. In this dissertation, we examine the epigenetic function of the DNA modification N(6)-methyladenine (N6-mA) in mammalian stem cell self-renewal and differentiation. The discovery of N6-mA in mammalian genomes led to multiple studies describing its genomic distribution, effects on gene expression, and cellular functions. However, the broader biological role of N6-mA and the specific molecular pathways that exert its function remain poorly understood. We first demonstrate that N6-mA changes the epigenetic landscape during stem cell differentiation in early development. We initially observe that N6-mA becomes upregulated in mouse trophoblast stem cells (TSCs), specifically at regions of DNA that are predicted to become single-stranded under topological stress, called stress-induced DNA double helix destabilization (SIDD). These SIDD regions have roles in genome organization through their interaction with DNA-binding proteins, such as SATB1. We demonstrate N6-mA in DNA reduces the binding of SATB1 at SIDD sequences by more than 500-fold. Deposition of N6-mA also prevents SATB1 binding to chromatin in TSCs and thereby prevents TSC differentiation through this interaction. We propose a new molecular mechanism for N6-mA function via SATB1 inhibition during trophoblast development. Next, we explore a mechanism for N6-mA regulation by its demethylase ALKBH1 in ssDNA structures. We use genome-wide sequencing to profile single-stranded DNA (ssDNA) in embryonic stem (ES) cells and show that it is prevalent in gene bodies and retrotransposons. We also show that in the TSC system of N6-mA upregulation, ssDNA is present in the future N6-mA-methylated regions. This relationship between N6-mA and ssDNA is investigated further by assessing whether DNA secondary structures containing N6-mA are potential substrates for ALKBH1. Our biochemical experiments reveal that that ALKBH1 is most active towards a DNA substrate containing 6 to 16 unpaired nucleotides, hereafter called a DNA bubble. Structurally, ALKBH1 requires both double-stranded DNA for binding and the ssDNA bubble for access to the methylated adenine for catalysis. Collectively, these results suggest that ALKBH1 regulates N6-mA in regions predisposed to form short DNA bubbles that may be prevalent in mammalian ES cells. Finally, we investigate ALKBH1 and N6-mA in hematopoiesis due to the known importance of SATB1 in regulating the lineage potential of hematopoietic stem and progenitor cells (HSPCs). To this end, we conditionally knockout Alkbh1 in mouse HSPCs. We show that competitively transplanted bone marrow from knockout mice have decreased T cell reconstitution, but normal myeloid production, similar to Satb1 deficient mice. However, there is no change to HSPC populations in the unperturbed bone marrow, suggesting a compensatory mechanism. In addition to these findings, we also develop and validate a flow cytometry method to quantify total N6-mA methylation in HSPC populations. We hypothesize that N6-mA inhibits SATB1 and the lymphoid potential of the lymphoid-primed multipotent progenitors during stress hematopoiesis. In summary, N6-mA is an epigenetic pathway that is increasingly being recognized as a mediator of mammalian stem cell function. This dissertation work concludes that the N6-mA pathway regulates early embryonic development and hematopoiesis through its inhibition of SATB1 binding and proposes new connections between DNA methylation, DNA secondary structure, and chromatin organization in stem cells.
Recommended Citation
Nelakanti, Raman Venkat, "Epigenetic Regulation of Stem Cell Fate Decisions by DNA N(6)-Methyladenine" (2022). Yale Graduate School of Arts and Sciences Dissertations. 785.
https://elischolar.library.yale.edu/gsas_dissertations/785
