Date of Award

12-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Cancer and Developmental Biology

Research Advisor

Joseph T. Opferman, Ph.D.

Committee

Mondira Kundu, MD, Ph.D. David R. Nelson, Ph.D. Martine F. Roussel, Ph.D. Gerard P. Zambetti, Ph.D.

Keywords

Anti-apoptosis, BCL-2 family, CRISPR-Cas9, MCL-1, Non-canonical role, Stem cell differentiation

Abstract

MCL-1, a well-known pro-survival BCL-2 family member, is indispensable for the survival of various cellular lineages and is also among the most frequently amplified genes in a variety of human malignancies. Gene ablation studies previously revealed that Mcl-1 deficiency leads to embryonic lethality around E3.5 during peri-implantation stage. Strikingly, the study did not detect any increase in apoptotic cells of the blastocyst, indicating a function of MCL-1 beyond regulating apoptosis. Our previous studies revealed an unrecognized role of MCL-1 in promoting mitochondrial physiology, which is independent of its classical anti-apoptotic function and requires being imported into the mitochondrial matrix. In order to understand the role of MCL-1 in early embryonic development, we used CRISPR-Cas9 to target Mcl-1’s start codon on established embryonic stem cells (ESCs). This approach resulted in the establishment of ESCs in which MCL-1’s N-terminus was truncated. Biochemical evaluation revealed that Nterminal- deleted MCL-1 retains anti-apoptotic function. However, this truncated MCL-1 is restricted to the mitochondrial outer membrane and functionally these mutated ESCs showed a dramatic defect in differentiation into the three embryonic germ layers- ectoderm, mesoderm, and ectoderm. These data suggest that in addition to MCL-1’s required antagonism of cell death by the C-terminal region, MCL-1’s N-terminus is required for efficient cellular differentiation, potentially by facilitating MCL-1’s import into the mitochondrial matrix.

ORCID

http://orcid.org/0000-0001-8557-4342

DOI

10.21007/etd.cghs.2017.0443

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