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ORCID

http://orcid.org/0000-0001-8799-0729

Date of Award

Winter 12-15-2018

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Human & Statistical Genetics)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

The gene that encodes de novo DNA methyltransferase 3A (DNMT3A) is frequently mutated in acute myeloid leukemia genomes. Point mutations at position R882 have been shown to cause a dominant negative loss of DNMT3A methylation activity, but 15% of DNMT3A mutations are predicted to produce truncated proteins that could either have dominant negative activities or cause loss of function and haploinsufficiency. Here, we demonstrate that 3 of these mutants produce truncated, inactive proteins that do not dimerize with WT DNMT3A, strongly supporting the haploinsufficiency hypothesis. We therefore evaluated hematopoiesis in mice heterozygous for a constitutive null Dnmt3a mutation. With no other manipulations, Dnmt3a+/– mice developed myeloid skewing over time, and their hematopoietic stem/progenitor cells exhibited a long-term competitive transplantation advantage. Dnmt3a+/– mice also spontaneously developed transplantable myeloid malignancies after a long latent period, and 3 of 12 tumors tested had cooperating mutations in the Ras/MAPK pathway. The residual Dnmt3a allele was neither mutated nor downregulated in these tumors. The bone marrow cells of Dnmt3a+/– mice had a subtle but statistically significant DNA hypomethylation phenotype that was not associated with gene dysregulation. These data demonstrate that haploinsufficiency for Dnmt3a alters hematopoiesis and predisposes mice (and probably humans) to myeloid malignancies by a mechanism that is not yet clear.

Mutations in the DNMT3A gene are the most common cause of the clonal expansion of hematopoietic stem/progenitor cells in older individuals, and are the most common initiating events for acute myeloid leukemia (AML). Dnmt3a deficiency, and the DNMT3AR882H mutation, both cause a focal, canonical DNA hypomethylation phenotype that may be relevant creating the epigenetic state that contributes to clonal expansion. To determine whether this phenotype is reversible, we developed an inducible system to restore expression of DNMT3A in transplanted bone marrow cells from Dnmt3a-/- mice. Remethylation of differentially methylated regions was detected within a week, and was essentially complete within 6 months. Remethylation was accurate, dynamic, and highly ordered, suggesting that differentially methylated regions have unique properties that may be relevant for their function.

Language

English (en)

Chair and Committee

Timothy J. Ley

Committee Members

Daniel C. Link, Christopher A. Maher, David H. Spencer, Matthew J. Walter,

Comments

Permanent URL: https://doi.org/10.7936/mxsq-4719

Available for download on Thursday, December 15, 2118

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