Exploiting a Saccharomyces cerevisiae Model to Characterize Dysfunctional DNA Repair in a Pediatric High-Grade Glioma-Causing Oncohistone Restricted; Files Only

Abstract

DNA is packaged into chromatin through wrapping around histone proteins. Certain somatic missense mutations in genes encoding histones transform them into oncohistones, promoting cancer development. How these oncohistone mutants impact histone function and molecular pathways is still not well characterized. To understand how specific missense mutations drive oncogenesis, we used a budding yeast model, which has 2 genes encoding histone H3 while humans have 15 genes. Critically, yeast and human histone H3 proteins, establishing that our findings in yeast will likely be relevant in humans. Oncohistone mutants often result in changing the profile of histone post-translational modifications, which are critical for determining DNA accessibility for various functions such as gene expression and DNA repair. Oncohistone mutants H3G34R and H3G34V are implicated in gliomas. It is known that H3G34V causes dysregulation of DNA repair pathways in fission yeast and causes increased levels of genomic instability in humans. To that end, we conducted a high-copy suppressor screen to identify genetic suppressors that alleviate or mitigate the growth defects caused by H3G34 mutations in the presence of phleomycin, a drug that induces double- strand breaks. identified a number of candidate suppressors, SUPP 76 (ROM1), SUPP 80 (RPD3) and SUPP 91 (TRA1), that suppress the growth sensitivity of a budding yeast H3G34V oncohistone model. Our ultimate intention is to discover targets that may translate into therapeutically actionable items for patients with glioblastoma.

Table of Contents

INTRODUCTION .......................................................................................................................................... 1

INTRODUCTION TO HISTONES ...................................................................................................................... 1

HISTONE GENE MUTATIONS ASSOCIATED WITH CANCER ..............................................................................2

GLIOMA-CAUSING ONCOHISTONES..............................................................................................................4

H3G34 MUTANTS DISPLAY DISRUPTED DNA REPAIR .................................................................................. 8

SACCHAROMYCES CEREVISIAE AS AN ONCOHISTONE MODEL ........................................................................ 9

STUDY OBJECTIVE ...................................................................................................................................10

METHODS .................................................................................................................................................11

S. CEREVISIAE STRAINS AND PLASMIDS ..................................................................................................... 11

HIGH COPY SUPPRESSOR SCREEN ...........................................................................................................12

GROWTH ASSAYS.....................................................................................................................................13

RESULTS................................................................................................................................................... 14

H3G34 MUTANTS ARE SENSITIVE TO INDUCED DNA DOUBLE-STRAND BREAK ............................................. 14

OPTIMIZATION FOR HIGH COPY SUPPRESSOR SCREEN...............................................................................16

IDENTIFYING CANDIDATE SUPPRESSORS ...................................................................................................20

IDENTIFYING AND VALIDATING GENES ON CANDIDATE SUPPRESSORS..........................................................23

DISCUSSION ............................................................................................................................................. 26

BIBLIOGRAPHY ........................................................................................................................................ 31

SUPPLEMENTAL TABLES AND FIGURES.............................................................................................37

About this Honors Thesis

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School	Emory College
Department	Neuroscience and Behavioral Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Genetics Chemistry, Biochemistry
Mot-clé	mutation yeast DNA repair epigenetic oncohistones glioblastoma cancer
Committee Chair / Thesis Advisor	Anita Corbett, Emory University
Committee Members	Jennifer Spangle, Emory University Gillian Hue, Emory University

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