Date of Award

5-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Cancer and Developmental Biology

Research Advisor

Janet F. Partridge, Ph.D.

Committee

Eric J. Enemark, Ph.D. Linda M. Hendershot, Ph.D. Ronald N. Laribee, Ph.D. Lawrence M. Pfeffer, Ph.D.

Keywords

Clr3, DDX3X, Ded1, HDAC, Heterochromatin, Sir2

Abstract

The fission yeast Schizosaccharomyces pombe provides a good model system to quickly study basic mechanisms underlying biological pathways conserved in higher eukaryotes. Here we utilized fission yeast to study the roles of the histone deacetylases (HDACs) Sir2 and Clr3 in heterochromatin formation and cancer associated mutations of the DEAD-box RNA helicase DDX3X, homolog of fission yeast Ded1, in translational control. Heterochromatin in fission yeast is characterized by hypoacetylation as well as methylation of histone H3 on lysine 9 (H3K9me). Heterochromatin assembly can now be separated into three distinct steps: heterochromatin establishment, spreading, and maintenance. These steps involve the actions of the histone H3K9 methyltransferase Clr4 along with the RNAi pathway. HDACs are also required for heterochromatin assembly, with the H3K9 HDAC Sir2 and H3K14 HDAC Clr3 participating in the processes of heterochromatin establishment and maintenance. Here, we show that both a serine rich patch within the N-terminal domain of Sir2 and the HDAC activity of Sir2 are required for proper recruitment of Sir2 to chromatin and for heterochromatin establishment. We also report that Sir2 shares similarity in global transcriptional control with Mit1 and Chp2, components of the Snf2/HDAC-containing repressor complex (SHREC), suggesting a connection between Sir2 and SHREC in transcriptional regulation. We also identified additional sites that are deacetylated by the SHREC HDAC Clr3 in addition to H3K14, specifically H2B K5, 6, 10, and 15. Using fission yeast, this study also examined the biological consequence of medulloblastoma associated mutations of the DEAD-box ATP-dependent RNA helicase DDX3X. In fission yeast, Ded1 is an essential protein with connections to both the RNAi pathway and translation of transcripts with complex UTRs. Here we show that human DDX3X can functionally complement for growth defects observed in thermosensitive ded1 mutants while specific cancer-associated DDX3X mutants (A222P, G302V, G325E, and P568L) cannot complement this growth defect. Fission yeast bearing these specific DDX3X mutants exhibit defects in the expression of specific proteins, suggesting the mutant proteins impair translational control.

ORCID

http://orcid.org/0000-0002-4253-0494

DOI

10.21007/etd.cghs.2017.0436

Comments

One year embargo expires May 2018.

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