Characterizing the role of recurrent histone H3 mutations in cancer initiation Restricted; Files Only

Abstract

Epigenetic dysregulation, a novel hallmark of cancer, plays an important role in cancer initiation. Of the proteins involved in epigenetic regulation, histones are at the core, packaging the genetic material into chromatin and controlling gene expression via post translational modifications (PTM). Across cancer types, gain-of-function missense mutations within core histones, including histone H3, drive cancer progression and lead to a poor prognosis, leading to the term oncohistones. Defined oncohistones (K27M, K36M, G34R/V) have amino acid changes at or near sites of lysine residues leading to reprogramming of the epigenome in these tumors. Epigenetic dysregulation in cells expressing oncohistones causes differential gene expression. For example, in H3K27M-associated brain cancers, many gene alterations are involved in development and differentiation. These genes include Dopamine Receptor D2 (DRD2), which is a target of the ONC201 inhibitor, which is currently in phase II clinical trials. These data lead us to hypothesize that additional oncohistone mutations are yet to be defined. Here, we utilize publicly available patient tumor data to identify six highly recurring histone H3 missense mutations- R42K, E50K, Q68K, E73K, E97K, and E105K- that are present within the globular domain of these histone proteins and are surface accessible for potential PTM, near sites of known PTMs, and are available for protein-protein or protein-DNA interactions. Using untransformed mammalian cell lines and a budding yeast model that expresses this panel of histone lysine mutants, we find that H3E50K, H3Q68K, H3E73K, and H3E97K enhance cellular proliferation and cancer-like transformation and alter proximal post-translational modification. In the budding yeast model, when the H3 X to K mutants are expressed in cells their growth was inhibited when exposed to caffeine, formamide, and hydroxyurea. This work provides the first evidence for novel H3 globular domain mutations that may convert histone protein into bona fide oncohistones and provides the reagents and tools to study possible mechanisms of action. 

Table of Contents

Table of Contents:

Introduction

1

     Histones and the nucleosome

2

    Post translational modifications of histones

5

    The role of histones in cancer

8

    Clinical relevance of oncohistones

11

    Research summary

11

Results

13

    Histone H3 X to K mutations

14

    Using mammalian cells and S. cerevisiae to model H3 X to K mutants

17

    H3 X to K mutations increase proliferation

20

    Some H3 X to K mutants enhance colony formation

22

    Expression of H3.3E50K, H3.3Q68K, and H3.3E73K alter histone PTMs

24

    S. cerevisiae models provides preliminary mechanistic insight

26

Discussion

29

    Future Directions

35

Materials and Methods

39

Works Cited

44

Appendix

50

    Protocols

51

    Plasmids and Primers

71

About this Master's Thesis

Rights statement

• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Cancer Biology
Degree	M.S.
Submission	Master's Thesis
Language	English
Research Field	Biology, Genetics Biology, Molecular Biology, General
Keyword	Cancer Breast Cancer S. Cerevisiae Histone Oncohistone
Committee Chair / Thesis Advisor	Andrew L. Hong, MD, Emory University Jennifer M. Spangle, PhD, Emory University Anita H. Corbett, PhD, Emory University David A. Frank, MD, PhD, Emory University

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