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Ubiquitylation of the PAF Oncoprotein and Interaction with PCNA is Essential for Hematopoietic Stem Cell Function and Development

by
Megha Shettigar
M.S., Mumbai University - 2011

Thesis Advisor: Lisa K. Denzin, Ph.D.
Graduate Program in Microbiology & Molecular Genetics

Child Health Institute of New Jersey (CHINJ)
Room 3209
New Brunswick

Thursday, December 1, 2016
10:00 a.m.


Abstract

Hematopoietic stem cells (HSCs) are critical for the lifelong production and maintenance of all blood cell types. The molecular mechanisms that guide this process remain poorly understood. The 15kDa proliferating cell nuclear antigen (PCNA) associated factor (Paf) is a potent oncogene that is over-expressed in most cancers. We have previously shown that Paf is essential for hematopoietic stem cell (HSC) and progenitor function and development. Paf deficient mice (Paf-/-) are leukopenic due to reduced number of HSCs and committed progenitors. Paf-/- HSCs failed to maintain quiescence, to self-renew and to support long term hematopoietic reconstitution. To determine the in vivo molecular interactions and pathways by which PAF functions to mediate hematopoiesis, we introduced mutant versions of PAF into the Paf-/- mice. These unique mouse models allowed us to show that PAF-PCNA interactions and PAF ubiquitylation are both essential for hematopoiesis and maintaining long-term hematopoietic stem cell (LT-HSC) quiescence. Furthermore, biochemical analyses of cells from these mice showed that PAF interaction with PCNA was essential for nuclear localization and proper ubiquitylation of PAF. Also, Paf-/- LT-HSCs, short term HSCs (ST-HSCs), multi potent progenitors (MPPs) and lymphoid primed multipotent progenitors (LMPPs) were found to accumulate more DNA damage than their Paf+/+ counterparts. Collectively, therefore, our studies show that PAF function in the development and differentiation of HSCs is dependent upon its ability to interact with PCNA and that the ubiquitylation of PAF regulates PAF function during hematopoiesis. Our studies also shed light on the importance of PAF in maintaining genomic integrity in HSCs and committed progenitors.


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