Date of Award

2006

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biology and Pathobiology

College

College of Graduate Studies

First Advisor

Demetri Spyropoulos

Second Advisor

Craig C. Beeson

Third Advisor

Makio Ogawa

Fourth Advisor

Dennis Watson

Fifth Advisor

Daohong Zhou

Abstract

Ets1 is a member of the ETS transcription factor family with demonstrated roles in cell proliferation, migration, and survival. Alternative splicing of exon VII results in two distinct protein isoforms: full length Ets1 and Ets1ΔVII. These isoforms bear key distinctions regarding protein-protein interactions, DNA binding kinetics and transcriptional target specificity. Targeted disruption of both Ets1 isoforms (Ets1 null) in mice results in increased perinatal lethality, loss of detectable NK and NKT cell activity, and differentiation, proliferation and survival defects in B- and T-Iymphocytes. To further delineate the individual roles of Ets1 isoforms in vivo we have generated an allelic series of Ets1 gene targeted mice. One line expresses only the Ets1ΔVII isoform. These Ets1ΔVII mice present with increased perinatal lethality, thymomegaly, as well as variable splenic and peripheral lymphopenia. Rates of apoptosis were diminished in the thymus and increased in the spleen of homozygotes. Increased rates of cell proliferation were also observed in both spleen and thymus with concomitant reduction in expression of both Cdkn1b and Cdkn2a proteins. Statistically significant elevation in CD4-CD8+ and CD8+CD4+ thymocyte calculated cell mass was observed in homozygote thymuses. Diminished cellularity in the spleen was due to reductions in the numbers of lymphoid cells (CD19+, CD4+, and CD8+). Two putative Ets1 target genes important in thymopoiesis, CD44 and RUNX1 were found to be misexpressed in the thymuses of homozygous mutant mice. Collectively these results demonstrate that Ets1ΔVII results in apoptotic and differentiative defects in lymphocytes and these defects are associated with misregulation of the putative target genes CD44 and RUNX1. Another line of mice in this allelic series, Ets1S->A, contains serine to alanine conversions in a critical domain coded by exon VII. This mutation abrogates phosphorylation mediated inhibition of Ets1 DNA binding. These mice present with a minimal phenotype suggestive that this autoinhibitory process is not central to the phenotypes observed in Ets1ΔVII mice. Targeted ES cells have been generated for a third line of mice expressing only full-length Ets1. Our results have demonstrated that regulation of Ets1 isoforms represents a mechanism in the maintenance of normal lymphoid homeostasis.

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