ORCID

http://orcid.org/0000-0001-8068-7348

Date of Award

Spring 5-15-2022

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Cell Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Hematopoiesis is strictly regulated to sustain blood production throughout adult life. De novo DNA methyltransferase 3-alpha (DNMT3A) is one of the major epigenetic regulators found to have an essential role in hematopoietic stem cell (HSC) differentiation. DNMT3A mutations are prevalent in myeloid diseases and malignancies that include acute myeloid leukemia (AML; ~22%), myelodysplastic syndrome (MDS; ~10%), and myeloproliferative neoplasm (MPN; ~8%). Importantly, DNMT3A mutations are not only the founding events of HSC malignant transformation, but they are also occurring as pre-leukemic lesions. Therefore, it is necessary to evaluate the molecular changes that occur in DNMT3A mutant HSCs, and how those changes promote clonal expansion and increase susceptibility to additional genetic lesions. However, the exact molecular mechanisms of how DNMT3A contributes to normal hematopoiesis and its mutations prime HSCs for leukemic formation are yet to be determined. We hypothesized that DNMT3A has novel functions other than DNA methylation that regulate HSC function. We first approached this by generating catalytically inactive and impaired Dnmt3a cDNA constructs that were used to ectopically express in Dnmt3a-null background to study the HSC function in vitro. We have shown that DNMT3A could regulate HSC function in cell-autonomous colony forming ability by DNA methylation independent pathway. This result has given us the rationale to generate germline mutant Dnmt3a knock-in mice to study DNMT3A function in hematopoiesis in a physiological setting. We further generated conditional mutant Dnmt3a knock-in mice, which allows temporal induction of one allele of Dnmt3a and expression of one mutant Dnmt3a. Our current results from in vivo suggest that DNA methylation activity of DNMT3A could be necessary for the HSC differentiation to produce blood cells, but DNMT3A may regulate HSC self-renewal to sustain the cell pool by novel mechanism apart from its catalytic activity. Further investigation is ongoing in an effort to validate our findings, and novel mechanisms will be explored. For the second part of my thesis, we glimpsed at the potential role of DNMT3A in HSC telomere regulation. Telomere shortening has been associated with diminishing HSC self-renewal potential. And Dnmt3a-null effect of stem cell telomere maintenance and the correlation between longer telomere and MDS patients with DNMT3A mutations have been described previously. We utilized Terc (RNA component of telomerase complex)-mutant mice crossbred with Dnmt3a-null mice to determine if loss of Dnmt3a could reverse the telomere shortening of Terc-mutant mice and increase the HSC self-renewal potential. We have shown the phenotypes of longer telomeres with elevated telomerase activity of Dnmt3a-null mice that correlate with hyper self-renewal potential. However, the question remains about whether there is cause and effect relationship between HSC telomere and functional regulations of DNMT3A. Further mechanistical study is needed. A large scope of my thesis project is to contribute to elucidating previously unknown function(s) of DNMT3A in HSC biology to dissect its molecular mechanisms in normal and malignant hematopoieses. The mouse models that we have generated with DNMT3A catalytic mutations could provide valuable tools to identify new therapeutic approaches for leukemia patients with DNMT3A mutations and provide evidence to suggest how DNMT3A mutations act as pre-leukemic lesions.

Language

English (en)

Chair and Committee

Grant A. Challen

Committee Members

Luis FZ Batista

Comments

Update embargo

Download

Share

COinS