understanding the role of the histone acetyltransferase gcn5 in the regulation of abiotic stress in

93 Pages·2016·2.38 MB·English

by  Hardev Brar

UNDERSTANDING THE ROLE OF THE HISTONE
ACETYLTRANSFERASE GCN5 IN THE REGULATION OF
ABIOTIC STRESS IN BRACHYPODIUM DISTACHYON

Hardev Brar

Department of Plant Science

McGill University, Montreal

DECEMBER 2015

A thesis submitted to McGill University in partial fulfillment of the
requirements of the degree of Masters of Science

© Hardev Brar 2015
Table of Contents

ABSTRACT .......................................................................................................................................................... III
RÉSUMÉ ................................................................................................................................................................ V
ACKNOWLEDGEMENTS ................................................................................................................................... VIII
PREFACE/CONTRIBUTION OF AUTHORS ........................................................................................................ IX
LIST OF ABBREVIATIONS ................................................................................................................................... X
LIST OF FIGURES AND TABLES ......................................................................................................................XVII
CHAPTER 1: OVERALL INTRODUCTION ........................................................................................................... 1
1.1 INTRODUCTION .................................................................................................................................................... 1
1.2 WORK HYPOTHESES .............................................................................................................................................. 3
1.3 RESEARCH GOALS ................................................................................................................................................. 3
CHAPTER II: LITERATURE REVIEW .................................................................................................................. 4
2.1 ABIOTIC STRESS ................................................................................................................................................... 4
2.1.1 Plant molecular response to cold stress ............................................................................................... 4
2.1.2 Plant molecular response to heat stress ............................................................................................... 6
2.2 TRANSCRIPTIONAL REGULATION ........................................................................................................................... 8
2.2.1 Plant promoters........................................................................................................................................ 8
2.2.2 Transcription factors ............................................................................................................................... 9
2.2.3 Epigenetics .............................................................................................................................................. 10
2.2.4 The nucleosome ...................................................................................................................................... 10
2.2.5 Epigenetics layers................................................................................................................................... 12
2.3 THE SAGA COMPLEX AND GCN5 ........................................................................................................................ 21
2.3.1 Roles and Functions of GCN5 ................................................................................................................ 24
2.4 BRACHYPODIUM DISTACHYON AS A MODEL ORGANISM ........................................................................................... 28
CHAPTER III: MATERIALS AND METHODS ..................................................................................................... 29
3.1 PLANT MATERIAL ............................................................................................................................................... 29
3.2 DATA MINING ..................................................................................................................................................... 30
3.3 IN SILICO ANALYSIS OF ALTERNATIVE SPLICING ..................................................................................................... 30
3.4 PRIMER DESIGN .................................................................................................................................................. 30
3.5 RNA AND GENOMIC DNA EXTRACTION ............................................................................................................... 31
3.6 CLONING OF GCN5 IN PGEM ............................................................................................................................... 31
3.7 QUANTITATIVE REAL TIME PCR .......................................................................................................................... 32
3.9 CLONING INTO PANIC VECTOR ............................................................................................................................ 32
3.10 CLONING INTO PAVA VECTOR ........................................................................................................................... 33
3.8 STATISTICAL ANALYSIS........................................................................................................................................ 33
CHAPTER IV: RESULTS ..................................................................................................................................... 34
4.1 SEQUENCE HOMOLOGY ........................................................................................................................................ 34
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4.2 IDENTIFICATION OF IMPORTANT DOMAINS WITHIN GCN5 .................................................................................... 34
4.3 IDENTIFICATION OF BDGCN5 SPLICE VARIANTS ................................................................................................... 35
4.4 GENOMIC AMPLIFICATION OF DELETION AND INSERTION REGION .......................................................................... 36
4.5 IN SILICO ANALYSIS OF POSSIBLE ALTERNATIVE SPLICE SITES ................................................................................. 37
4.6 TRANSCRIPT ACCUMULATION OF FULL LENGTH BDGCN5, S-GCN5 AND L-GCN5 UNDER HEAT AND COLD STRESS .. 38
4.7 CLONING S-GCN5 IN THE OVEREXPRESSION VECTOR PANIC 6A AND THE CELLULAR LOCALISATION VECTOR
PAVA321 ................................................................................................................................................................ 39
CHAPTER V: DISCUSSION ................................................................................................................................. 54
5.1 THE CONSERVED FUNCTION OF BDGCN5 ............................................................................................................. 55
5.2 ALTERNATIVE SPLICING OF BDGCN5 AND THE LOSS OF THE BROMODOMAIN ......................................................... 56
5.3 UNDERSTANDING STRESS-INDUCED ALTERNATIVE SPLICING ................................................................................. 58
CHAPTER VI: CONCLUDING STATEMENT ....................................................................................................... 60
6.1 SUMMARY ........................................................................................................................................................... 60
6.2 FUTURE WORK AND DIRECTIONS ......................................................................................................................... 61
6.3 CONTRIBUTIONS TO SCIENCE ............................................................................................................................... 61
CHAPTER VII: REFERENCES ............................................................................................................................. 62

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Abstract
Abiotic stresses such as drought, salinity, heavy metals, high temperature and low
temperature are largely responsible for crop yield losses. Plants have adapted various
coping mechanisms to overcome these adverse environmental conditions to sustain
growth, and reproduction. Stress responses, are predominantly regulated at the
transcriptional level. Plants respond to changes in environmental conditions by activating a
transcriptional cascade that relies on a set of transcription factors that regulate the
transcription of stress responsive genes. Recently, transcription has been shown to be
dependent on the chromatin compaction level. As the DNA tightens around the
nucleosomes, promoter accessibility to transcription factors decreases. On the other hand,
when the DNA structure loosens, accessibility increases and transcription factors can more
freely bind to specific promoters and initiate transcription. This dynamic process has been
shown to involve the Spt-Ada-GCN5- Acetyltransferase (SAGA) in multiple species. In this
project the role of a subunit of this complex, General Control of Non-repressible 5 (GCN5),
was characterized in the monocot model organism, Brachypodium distachyon. By
acetylating histone tails, GCN5 is able to favor an open chromatin state and ultimately
positively impact transcription and we hypothesize that when exposed to sub-optimal
environmental conditions, B.distachyon utilizes GCN5 as a crucial chromatin modifier to
precisely control key stress responsive genes through dynamic changes in chromatin
structure. To test this hypothesis, GCN5 was characterized in several ways. First, we
conducted a bioinformatics analysis and showed that a BdGCN5 gene locus exists in
Brachypodium and codes for a protein containing four HAT domains and a complete
bromodomain necessary for the acetylation process of histone lysines and the recognition
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of acetylated lysine residues. Further analysis, supported that BdGCN5 shares a great level
of homology with GCN5 proteins of other organisms which further emphasizes the
conserved nature and the functional importance of the GCN5 protein. Second, we cloned
the BdGCN5 cDNA and uncovered multiple putative variants of BdGCN5. One variant, s-
gcn5, had two deletions within its sequence while the other, l-gcn5, had an intronic
insertion. In both cases, premature stop codons that could prevent the translation of the
bromodomain of BdGCN5 were introduced in these variants. Thirdly, we then sought to
verify if BdGCN5, s-gcn5 and l-gcn5 were expressed in plants exposed to stressful
conditions. All three transcripts were detected and showed differential accumulation under
both heat and cold stress. We also verified if other genomic loci could be responsible for the
presence of the s-gcn5 and l-gcn5 variants and determined that these variant are most
likely generated by RNA processing mechanisms as only one locus seems to code for
BdGCN5, s-gcn5 and l-gcn5. Finally, s-gcn5 was cloned into the overexpression vector
pANIC6A and the protein localization vector pAVA321. This project can thus be viewed as a
stepping stone towards understanding the biological function of the histone
acetyltransferase GCN5 in monocot plants.

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Résumé
Les stress biotiques et abiotiques affectent grandement le rendement des cultures.
Les plantes répondent aux changements imposés par leurs environnements en activant une
cascade transcriptionnelle qui s’appuie sur des facteurs de transcriptions qui régulent la
transcription de gènes directement et indirectement impliqués dans la réponse aux stress.
Il a été démontré que l’activation de ces cascades transcriptionnelles est dépendante du
niveau de compaction de la chromatine. À mesure que l’ADN se compacte autour des
nucléosomes, l’accessibilité à l’ADN des facteurs de transcription se trouve réduite ce qui
entrave la transcription. À l’opposé, lorsque la structure de l’ADN se détend, l’accessibilité
des facteurs de transcription augmente ce qui favorise la transcription. Lors de ce projet,
nous avons caractérisé le rôle de la protéine Général Control of Amino Acid Synthesis-5
(GCN5) dans la réponse de la plante modèle Brachypodium distachyon aux conditions de
stress. Cette protéine fait partie du complexe Spt-Ada-GCN5-Acetyltransférase (SAGA) qui
est composé de quatre modules comprenant plusieurs protéines qui régulent ensemble la
transcription en modifiant la structure de la chromatine. GCN5 est un des composants
centraux du complexe SAGA qui acétyle les résidus lysine des queues d’histones et signale
ainsi le relâchement de la structure chromatinienne et l’activation de la transcription.
Notre hypothèse de travail était la suivante : lors de conditions de stress, Brachypodium
distachyon utilise GCN5 pour modifier la structure de la chromatine et activer la
transcription des gènes nécessaires à son adaptation. Afin de tester cette hypothèse, nous
avons réalisé plusieurs études. Premièrement, à l’aide d’analyses bioinformatiques nous
avons montré que BdGCN5 possède l’ensemble des motifs nécessaire à son bon
fonctionnement. Le gène BdGCN5 code pour une protéine contenant les cinq domaines HAT
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et un bromodomaine nécessaires à la reconnaissance des résidus lysine acétylés et au
processus d’acétylation. De plus, la protéine BdGCN5 démontre un haut taux d’homologie
de séquence avec d’autres protéines GCN5 de plantes, champignons et animaux, ce qui
démontre l’importance fonctionnelle de cette protéine. Deuxièmement, nous avons cloné
l’ADN complémentaire de BdGCN5 et nous nous sommes aperçus que BdGCN5 était
probablement épissée de façon alternative étant donné la présence de variants. Le premier
variant, s-gcn5 possède deux délétions dans sa séquence tandis que le second, l-gcn5
possède une insertion d’une section d’intron. Dans les deux cas, ces réarrangements
introduisent des codons stop prématurés dans la séquence protéique de ces variants ce qui
empêche la traduction du bromodomaine. Nous avons aussi tenté de déterminer si d’autres
loci génomiques pouvaient coder pour les deux variants et avons déterminé que s-gcn5 et l-
gcn5 étaient fort probablement codés par le locus BdGCN5. Troisièmement, nous avons
mesuré le niveau d’accumulation des transcrits de BdGCN5, s-gcn5 et l-gcn5 en conditions
normales et lors de conditions de stress à la chaleur et au froid. Nous avons donc démontré
que les transcrits de BdGCN5 ainsi que ceux de ses variants s’accumulent tous de façon
différentielle lors de conditions de stress. Toutes les formes de transcrit de BdGCN5
s’accumulent d’avantage lors d’une exposition prolongée à la chaleur. Par contre, lors d’une
exposition au froid nous avons mesuré une diminution des niveaux de transcrits BdGCN5,
une augmentation des transcrits s-gcn5 et aucune différence d’accumulation des transcrits
l-gcn5. Finalement, nous avons cloné l’ADN complémentaire de s-gcn5 dans le vecteur de
surexpression pANIC6A et de localisation cellulaire pAVA321 afin de générer des outils qui
nous permettront de caractériser la fonction de ce variant. L’ensemble des études constitue
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donc la pierre d’assise d’un vaste projet visant à caractériser le rôle du complexe SAGA lors
de la réponse des plantes aux stress environnementaux.

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Acknowledgements
I would like to thank my supervisor Dr. Jean-Benoit Charron for his expert guidance
and constant encouragement throughout the course of my study at McGill. He has very
kindly taught me different techniques and provided help in the preparation of this thesis. It
was a privilege to work with him for two years. During this period I learnt several aspects
of molecular biology and genetics. I also owe my special thanks to my committee member,
Dr. Jaswinder Singh for his guidance and support through the progression of my project. I
appreciate the help of Guy Rimmer who was kind enough to provide laboratory materials
as and when needed.
I acknowledge the support of my colleagues Boris Mayer, Alexandre Martel and
Purva Karia; Boris for teaching me rt-qPCR and helping me in troubleshooting experiments,
Alex for continuing my project and providing help with data analysis and Purva for
assisting me in the use of laboratory techniques. I also extend my thanks to my other
colleagues Steven, Gab, Bianca, Eliana and Jordon.
I owe my gratitude to my family for their unconditional love and support
throughout my studies. I am equally thankful to my younger sister, Rajdev, who has been
with me in McGill and has made my stay enjoyable. I also thank my friends in Montreal for
their love and help.
I owe my regards to my dear friend, Rustam, for his support and encouragement.
Last but not the least, I would like to give thanks to the faculty, students and staff of
the Plant Science Department of McGill University.
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Preface/Contribution of Authors
The thesis has been written by Hardev Brar as per the guidelines of McGill
University. The thesis is comprised of seven chapters. The first chapter deals with the
overall introduction and the literature review is included in Chapter 2. Material and
Methods are given in Chapter 3. The results of different experiments are given in Chapter 4
and are discussed in Chapter 5. Chapter 6 highlights the key findings and conclusions of the
study. All citations are given in Chapter 7, References. Dr. Jean Benoit Charron and Hardev
Brar designed the experiments and Hardev Brar conducted all experiments.

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