Isolation and characterisation of suppressors of conditional histone mutants

ISOLATION AND CHARACTERISATION OF
SUPPRESSORS OF CONDITIONAL HISTONE
MUTANTS

LEE SHU YI, LINDA
(B. Sci. (Hons.), NUS)

A THESIS SUBMITTED
FOR THE DEGREE OF
MASTER OF SCIENCE (RSH-SOM)

DEPARTMENT OF MICROBIOLOGY

NATIONAL UNIVERSITY OF SINGAPORE

2012


Acknowledgements

I would like to express my deep gratitude to the following people who have made this
dissertation possible and because of whom my graduate experience will be cherished.

I have been fortunate to have Dr Norbert Lehming as my advisor, as he gave me
freedom to explore various areas on my own and always provided timely guidance
whenever I faltered. In addition, this project would not have been as smooth sailing as
it had been without the help and friendship of Zhao Jin, Wee Leng, Keven, Gary,
Edwin, Daniel, Mei Hui, Jia Hui and Agnes.

Most importantly, none of this would have been possible without the love and
patience of my two buddies, my family and Kian Sim. They have been a constant

source of love, concern, support and strength that encouraged me throughout this
endeavour.

Thank you once again to all.

i


Table of contents

1. Introduction
1.1 Epigenetics ............................................................................................................... 2
1.1.1 DNA methylation .............................................................................................. 2
1.1.2 RNA-associated silencing ................................................................................. 3
1.1.3 Histone modifications ....................................................................................... 3
1.2 Approaches utilised towards the study of epigenetics ............................................. 4
1.2.1 Model organism S. cerevisiae ........................................................................... 4
1.2.2 Alanine-scanning mutagenesis .......................................................................... 5
1.2.3 Phenotype testing .............................................................................................. 5
1.2.3.1 Sensitivity to 3-AT ..................................................................................... 6
1.2.3.2 Sensitivity to antimycin A .......................................................................... 7
1.2.3.3 Sensitivity to temperature ........................................................................... 7
1.2.4 Suppression ....................................................................................................... 7
1.2.4.1 Suppression via over-expression of genes involved in affected pathway .. 8
1.2.4.2 Suppression via extragenic mutation .......................................................... 9
1.2.5 Chromatin immunoprecipitation (ChIP) ........................................................... 9
1.3 Aims of this study .................................................................................................. 11

2. Literature review
2.1 Nucleosomal structure ........................................................................................... 13

2.1.1 Core histones ................................................................................................... 15
2.1.2 Core histones in S. cerevisiae .......................................................................... 16
2.2 Histone code hypothesis ........................................................................................ 16
2.2.1 ATP-dependent chromatin remodelling .......................................................... 18
2.2.2 Nucleosomal incorporation ............................................................................. 19
2.2.3 Post-translational modifications of histones ................................................... 21
2.2.3.1 Fundamental PTMs of histones ................................................................ 23
2.2.3.1.1 Histone acetylation............................................................................. 27
2.2.3.1.2 Histone methylation ........................................................................... 28
ii


2.2.3.1.3 Histone phosphorylation .................................................................... 30
2.2.3.2 Combinatorial PTMs of histones .............................................................. 30
2.2.3.3 Influences of histone H4 acetylation on transcription .............................. 32
2.3 Histone acetyltransferases ...................................................................................... 34
2.3.1 Gcn5 ................................................................................................................ 37
2.3.1.1 HIS3 as a model for the study of Gcn5 ..................................................... 42
2.3.2 Hpa1 (Elp3) ..................................................................................................... 44
2.3.3 Hpa2 and Hpa3 ................................................................................................ 45
2.4 Diseases.................................................................................................................. 46

3. Materials and methods
3.1 Project flowchart .................................................................................................... 50
3.2 Materials ................................................................................................................ 53
3.2.1 E. coli strains ................................................................................................... 53
3.2.2 S. cerevisiae strains ......................................................................................... 53
3.2.3 Plasmids .......................................................................................................... 55
3.2.3.1 Plasmids used for gene targeting .............................................................. 55
3.2.3.2 Plasmids used for genetic interaction analysis ......................................... 55

3.3 Methods.................................................................................................................. 57
3.3.1 Generation of plasmids.................................................................................... 57
3.3.1.1 Polymerase chain reaction (PCR) ............................................................. 57
3.3.1.2 Purification of extension products ............................................................ 68
3.3.1.3 Cloning and sub-cloning ........................................................................... 68
3.3.1.4 Purification of restriction digested products ............................................. 69
3.3.1.5 DNA ligation ............................................................................................ 69
3.3.1.6 Amplification of plasmid DNA ................................................................ 69
3.3.1.6.1 Chemical transformation into DH5α E. coli ...................................... 70
3.3.1.6.2 Electroporation into DH10β E. coli ................................................... 71
3.3.1.7 Miniprep for purification of plasmid DNA from E. coli .......................... 71
3.3.1.8 Agarose gel electrophoresis ...................................................................... 72
3.3.1.9 Sequencing reaction and purification of extension products .................... 73
3.3.2 Generation of S. cerevisiae strains .................................................................. 74
3.3.2.1 Production of competent S. cerevisiae ..................................................... 74
iii


3.3.2.2 Transformation of competent S. cerevisiae .............................................. 74
3.3.2.3 Generation of S. cerevisiae histone mutant strains — Plasmid shuffling 75
3.3.2.3.1 Titration — Droplet growth assay ..................................................... 77
3.3.2.4 Generation of S. cerevisiae mutant strains — Gene targeting .................. 77
3.3.2.5 Generation of S. cerevisiae glycerol stock ............................................... 79
3.3.3 Genomic library screening .............................................................................. 79
3.3.3.1 Transformation of competent S. cerevisiae with YEp13 library plasmids
.............................................................................................................................. 80
3.3.3.2 Extraction of genomic or plasmid DNA — Yeast breaking ..................... 81
3.3.4 Quantitative real-time PCR analysis ............................................................... 82
3.3.4.1 Purification of total ribonucleic acid (RNA) ............................................ 82
3.3.4.2 Quantitation of total RNA ........................................................................ 83

3.3.4.3 Formaldehyde agarose (FA) gel electrophoresis of total RNA ................ 84
3.3.4.4 DNaseI treatment of DNA contaminants.................................................. 85
3.3.4.5 Reverse transcription (RT) PCR ............................................................... 86
3.3.4.6 Quantitative real-time PCR ...................................................................... 86
3.3.5 Protein analysis ............................................................................................... 87
3.3.5.1 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
.............................................................................................................................. 87
3.3.5.2 Western blot .............................................................................................. 88
3.3.6 Chromatin immunoprecipitation (ChIP) ......................................................... 89
3.3.6.1 Culturing and crosslinking of sample ....................................................... 89
3.3.6.2 Cell lysis and sonication ........................................................................... 90
3.3.6.3 Analysis of chromatin fragment size ........................................................ 91
3.3.6.4 Immunoprecipitation ................................................................................ 92
3.3.6.5 PCR and quantitative real-time PCR analysis .......................................... 93

iv


4. Results

Chapter I Genomic library screening of histone H4 mutant strains Y51A, E53A
and Y98A
4I.1 Phenotype testing of histone H4 mutant strains Y51A, E53A and Y98A ............ 97
4I.2 Suppression studies via over-expression for observable phenotypes of histone H4
mutant strains Y51A, E53A and Y98A ....................................................................... 98
4I.3 Suppressor gene knock out studies ..................................................................... 103

Chapter II Characterisation of histone H4 tyrosine residues
4II.1 Alanine-scanning mutagenesis of histone H4 tyrosine residues ....................... 107
4II.1.1 Phenotype testing of histone H4 tyrosine residue mutant strains Y51A,

Y88A and Y98A..................................................................................................... 108
4II.2 Characterisation of histone H4 tyrosine residue Y98........................................ 109
4II.2.1 Phenotype testing of histone H4 mutant strains Y98A and Y98F .............. 111

Chapter III Directed screening of histone H4 mutant strain Y98A
4III.1 Suppression studies via over-expression of HATs for AT phenotype of histone
H4 mutant strain Y98A .............................................................................................. 113
4III.1.1 Suppression of the AT phenotype of the H4Y98A mutant strain by the
over-expression of HATs ....................................................................................... 116
4III.1.2 HATs phenotype specificity and strain specificity ................................... 119
4III.2 Suppressor gene knock out studies .................................................................. 121
4III.2.1 GCN5, HPA1, HPA2 and HPA3 single gene knock out studies ............... 121
4III.2.1.1 Suppression studies via over-expression in GCN5 and HPA1 single
gene knock out mutant strains ............................................................................ 122
4III.2.2 GCN5, HPA1, HPA2 and HPA3 double gene knock out studies .............. 124
4III.3 Quantitative real-time PCR analysis ................................................................ 124

v


Chapter IV Characterisation of histone H4 Y98A AT phenotype suppressors —
Gcn5, Hpa1 and Hpa2
4IV.1 Phenotype testing of an histone H4 N-terminal deletion strain ....................... 129
4IV.2 Alanine- and arginine-scanning mutagenesis of the histone H4 N-terminal
lysine residues ............................................................................................................ 130
4IV.2.1 Phenotype testing of the histone H4 N-terminal lysine residue mutant
strains ..................................................................................................................... 131
4IV.3 Alanine- and arginine-scanning mutagenesis of the histone H4 N-terminal
lysine residues in combination with H4Y98A ........................................................... 134
4IV.3.1 Phenotype testing of the histone H4 N-terminal lysine residue mutant

strains in combination with H4Y98A..................................................................... 136
4IV.3.2 Suppression studies via over-expression of HATs for AT phenotype of the
histone H4 N-terminal lysine residue mutant strains in combination with H4Y98A
................................................................................................................................ 138
4IV.4 Arginine-scanning mutagenesis of histone H4 N-terminal K8 and K16 residues
.................................................................................................................................... 141
4IV.4.1 Phenotype testing of the histone H4K8,16R double mutant strain ........... 142
4IV.4.2 Suppression of the AT phenotype of the histone H4K8,16R double mutant
strain by the over-expression of HATs .................................................................. 142
4IV.5 Alanine- and arginine-scanning mutagenesis of multiple histone H4 N-terminal
lysine residues without and in combination with H4Y98A ....................................... 143
4IV.5.1 Phenotype testing of the histone H4 N-terminal multiple lysine residues
mutant strains without and in combination with H4Y98A .................................... 146
4IV.6 Acetylation status of histone H4 N-terminal K8 and K16 residues................. 147
4IV.7 Chromatin immunoprecipitation (ChIP) .......................................................... 150
4IV.7.1 Histone H4 occupancy at the HIS3 promoter and ORF ............................ 153
4IV.7.2 Histone H4K16ac occupancy at the HIS3 promoter and ORF.................. 155
4IV.7.3 Gcn5 occupancy at the HIS3 promoter and ORF ...................................... 157

Chapter V Histone H3 and H4 crosstalk studies
4V.1 Plasmid shuffling of histone H3 and H4 ........................................................... 161
4V.1.1 Phenotype testing of cells expressing combinations of different histone H3
derivatives and WT histone H4 .............................................................................. 162
4V.1.2 Phenotype testing of cells expressing combinations of different histone H3
derivatives and histone H4Y98A ........................................................................... 163

vi


5. Discussion

5.1 Preface.................................................................................................................. 166
5.2 Histone H4 amino acid residues Y51, E53 and Y98 ........................................... 168
5.3 Histone H4 tyrosine residues Y51, Y72, Y88 and Y98 ....................................... 170
5.3.1 Histone H4 tyrosine residue Y98 .................................................................. 173
5.3.2 Histone H4 tyrosine residue Y98 in relation to the HATs Gcn5, Hpa1 and
Hpa2 ....................................................................................................................... 176
5.3.3 Histone H4 tyrosine residue Y98 and N-terminal lysine residues ................ 178
5.3.4 Histone H4 tyrosine residue Y98 and N-terminal lysine residues K8 and K16
in relation to the HATs Gcn5, Hpa1 and Hpa2 ...................................................... 181
5.3.4.1 Recruitment of Gcn5 to the HIS3 locus is dependent on H4Y98 ........... 183
5.4 Histone H3 and H4 crosstalk ............................................................................... 185

6. Conclusion and future studies
6.1 Conclusion and future studies .............................................................................. 188

7. Bibliography……………………………………………...…………………..189

8. Appendices
8.1 Gene derivatives of Bank 13 (YEp13) tested in the phenotypic assay ................ 210
8.2 Genes inserted into PactT424 and PactT424-HA tested in the phenotypic assay210
8.3 HHF1 WT and mutant genes inserted into YCplac22 tested in the phenotypic
assay ........................................................................................................................... 210
8.4 HHT1 WT and mutant genes inserted into YCplac111 tested in the phenotypic
assay ........................................................................................................................... 211
8.5 HHF1 WT and mutant genes inserted into YCplac111 tested in the phenotypic
assay ........................................................................................................................... 211
8.6 Genes inserted into YEplac181 tested in the phenotypic assay ........................... 212
8.7 Primers used for amplification of candidate suppressor genes in one-step PCR . 213
8.8 Preparation of DH5α E. coli ................................................................................ 213
8.9 Preparation of LB media ...................................................................................... 214

8.10 Preparation of DH10β E. coli ............................................................................ 215
vii


8.11 Preparation of miniprep solutions ...................................................................... 215
8.12 Preparation of 10X loading dye ......................................................................... 216
8.13 Preparation of yeast extract peptone dextrose adenine (YPDA) ....................... 216
8.14 Preparation of glucose/galactose complete or selective media .......................... 216
8.15 Preparation of 0.1 M LiAc ................................................................................. 217
8.16 Preparation of 40 % PEG ................................................................................... 218
8.17 Preparation of yeast breaking buffer .................................................................. 218
8.18 Preparation of FA gel solutions ......................................................................... 218
8.19 Preparation of SDS polyacrylamide denaturing gel........................................... 219
8.20 Preparation of 5X Western blot transfer buffer ................................................. 219
8.21 Preparation of TBST .......................................................................................... 219
8.22 Preparation of Coomassie Blue staining solution and destaining solution ........ 220
8.23 Preparation of yeast lysis buffer ........................................................................ 220
8.24 Preparation of pronase working buffer .............................................................. 220
8.25 Preparation of immunoprecipitation buffers ...................................................... 220
8.26 Data for HIS3 mRNA expression levels ............................................................ 221
8.27 Data for ImageJ quantification of the acetylation status of H4K8..................... 222
8.28 Data for ImageJ quantification of the acetylation status of H4K16................... 222
8.29 Data for histone H4 occupancy at the HIS3 locus..............................................223
8.30 Data for histone H4K16ac occupancy at the HIS3 locus...................................225
8.31 Data for Gcn5 occupancy at the HIS3 locus.......................................................227

viii


List of abbreviations and symbols

Symbol
∆
°C
µl
µM
Number
3-AT
5-FOA
5-FU
6AU-NAM
(phenotype)

Delta, knock out or deleted for
degree Celsius
Microlitre
Micromoles per litre

3-amino-1,2,4-triazole
5-fluoro-orotic acid
5-fluorouracil
Sensitivity to 6-azauracil and nicotinamide

A
A (Amino acid)
A. thaliana
aa
AA
AA (phenotype)
ACT1

Ahc1
Amp
AmpR
APS
AT (phenotype)
ATC1

Alanine
Arabidopsis thaliana
Amino acid
Antimycin A
Sensitivity to antimycin A
Actin
ADA HAT complex component 1
Ampicillin
Ampicillin resistant
Ammonium persulphate
Sensitivity to 3-amino-1,2,4-triazole
Aip three complex

B
BLAST
bp
BSA

Basic local alignment search tool
Base pair
Bovine serum albumin

C

CCT6
cDNA
ChIP
Chl
ChlR
CSE4
CuSO4

Chaperonin-containing TCP-1
Complementary DNA
Chromatin immunoprecipitation
Chloramphenicol
Chloramphenicol resistant
Chromosome segregation
Copper sulphate

ix


D
D (Amino acid)
D. melanogaster
DNA
DNMT
E
E (Amino acid)
E. coli
EAF7
EDTA
ELM1

ELP3
ESA1
EtOH
EUROSCARF

Aspartic acid
Drosophila melanogaster
Deoxyribonucleic acid
DNA methyltransferase

Glutamic acid
Escherichia coli
Esa1-associated factor
Ethylenediaminetetraacetic acid
Elongated morphology
Elongator protein 3
Catalytic subunit of the histone acetyltransferase complex
NuA4
Ethanol
EUROpean Saccharomyces cerevisiae ARchive for Functional
Analysis

F
F (Amino acid)
FA
FS DNA

Phenylalanine
Formaldehyde agarose
Fish sperm DNA

G
GAL4
GCN4 / GCN5
GNAT

Galactose metabolism
General control nonderepressible
Gcn5-related acetyltransferase

H
h
H (Amino acid)
HA
HAT (enzyme)
HAT1 / HAT2
HDAC
HDM
HHF1 / HHF2
HHT1 / HHT2
HHTF
HIS3
HKMT
HMT
HPA1 / HPA2 /
HPA3
HTA1 / HTA2
HTB1 / HTB2
HU (phenotype)

Hour (time)
Histidine
Haemagglutinin
Histone acetyltransferase
Histone acetyltransferase
Histone deacetylase
Histone demethylase
Histone H Four
Histone H Three
Histone H Three and H Four
Histidine
Histone lysine methyltransferase
Histone methyltransferase
Histone and other protein acetyltransferase
Histone H Two A
Histone H Two B
Sensitivity to hydroxyurea

x


K
K (Amino acid)
KAR4
kb
kDa
kV

Lysine
Karyogamy

Kilobase
Kilodalton
Kilovolt

L
L
L (Amino acid)
LB
LEU2
LiAc
LiCl
LYS2

Litre
Leucine
Luria-Bertani
Leucine biosynthesis
Lithium acetate
Lithium chloride
Lysine requiring

M
M
M (Amino acid)
MALDI-TOF
MCK1
MDa
MET3
mg
min

ml
mM
MMS (phenotype)
MOPS
MRPS18
MSC3
MYST

Moles per litre
Methionine
Matrix-assisted laser desorption ionisation time-of-flight
Meiotic and centromere regulatory ser, tyr-kinase
Megadalton
Methionine requiring
Milligram
Minute (time)
Millilitre
millimolar
Sensitivity to methyl-methanesulfonate
3-[N-morpholino]propanesulfonic acid
Mitochondrial ribosomal protein, small subunit
Meiotic sister-chromatid recombination
MOZ-Ybf2/Sas3-Sas2-Tip60

N
NaAc
NaOH
ng
nm

Sodium acetate
Sodium hydroxide
Nanogram
Nanometer

O
OD600
OMP
ORF

Optical density measured at a wavelength of 600 nm
Orotidine-5'-phosphate
Open reading frame

xi


P
PCAF
PCR
PEG
PHD finger
PLP1
PMSF
PRMT
PTM

p300/CREB-binding protein associated factor
Polymerase chain reaction
Polyethylene glycol

Plant homeodomain finger
Phosducin-like protein
Phenylmethanesulphonylfluoride
Protein arginine methyltransferase
Post-translational modification

R
R (Amino acid)
RNA
RNAi
rpm
RT
RTT109

Arginine
Ribonucleic acid
RNA interference
Revolutions per minute
Reverse transcription
Regulator of Ty1 transposition

S
s
S. cerevisiae
S. pombe
SAGA
SAS2 / SAS3
SDS
SDS-PAGE
SET

SFG1
SIP5
siRNA
SKI8
SLH1
SPS4
Spt (phenotype)
SUF2
SUMO

Second (time)
Saccharomyces cerevisiae
Schizosaccharomyces pombe
Spt-Ada-Gcn5 acetyltransferase
Something about silencing
Sodium dodecyl sulphate
Sodium dodecyl sulphate polyacrylamide gel electrophoresis
Su(var)3-9, Enhancer of zeste and Trithorax
Superficial pseudohyphal growth
Snf1 interacting protein
Small interfering RNA
Superkiller
Synthetic lethal with Hnt1
Sporulation specific transcript
Suppressor of Ty phenotype
Suppression of frameshift mutation
Small ubiquitin related modifier

T
T. gondii

T. thermophila
TAF1
TBST
TEMED
TRP1
TS (phenotype)

Toxoplasma gondii
Tetrahymena thermophila
TATA-binding protein-associated factor
Tris-buffered Saline Tween-20
N,N,N’,N’-tetramethyl-1,2-diaminoethane
Tryptophan requiring
Sensitivity to temperature

xii


U
U (Amino acid)
UMP
URA3
UV

Uracil
Uridine monophosphate
Uracil requiring
Ultraviolet

W

W (Amino acid)
WT

Tryptophan
Wild type

Y
Y (Amino acid)
YAP1
YPDA

Tyrosine
Yeast AP-1
Yeast extract peptone dextrose adenine

xiii


List of tables

Table 2.1
Table 2.2
Table 2.3
Table 3.1
Table 3.2
Table 3.3
Table 3.4
Table 3.5
Table 3.6
Table 3.7

Table 3.8
Table 3.9
Table 3.10
Table 3.11
Table 3.12
Table 3.13
Table 3.14
Table 3.15
Table 3.16
Table 3.17
Table 3.18
Table 3.19
Table 4.1
Table 4.2

Table 4.3
Table 4.4

Some known sites of PTMs of histones
23
Some proposed functions of PTMs of core histones carried out by
24
different histone modifying enzymes
PTMs of histone H4 N-terminal histone tail in different organisms 33
E. coli strains used
53
Parental S. cerevisiae strains used
53
S. cerevisiae knock out strains used
54

S. cerevisiae double knock out strains used
54
Plasmids used for genetic interaction analysis
55
Primers used for amplification of selected histone
57
acetyltransferases in one-step PCR
Primers used for amplification of selected gene promoter and
58
terminator sequences in one-step PCR
Primers used for amplification of selected histone
59
acetyltransferases in two-step PCR
Primers and PCR strategy used for amplification of HHF1 WT
60
Primers and PCR strategy used for amplification of HHF1
61
mutants at positions Y51, Y72, Y88 and Y98
Primers and PCR strategy used for amplification of HHF1 single
62
alanine mutants in combination with Y98A
Primers and PCR strategy used for amplification of HHF1 single
63
arginine mutants in combination with Y98A
Primers and PCR strategy used for amplification of HHF1
64
multiple alanine mutants in combination with Y98A
Primers and PCR strategy used for amplification of HHF1
65
multiple arginine mutants in combination with Y98A

Primers used for sequencing reactions
73
Primers used for quantitative real-time PCR
87
Primary and secondary antibodies used in Western blotting
88
Antibodies used in immunoprecipitation
93
Primers used for PCR and quantitative real-time PCR
94
Tabulation of observable phenotypes of the H4Y51A, H4E53A
98
and H4Y98A mutant strains
Details of YEp13 suppressor plasmids isolated for each of the
100
observable phenotypes of histone H4 mutant strains Y51A, E53A
and Y98A
Suppressors identified from H4Y51A AT phenotype suppression
102
studies
Suppressors identified from H4E53A TS phenotype suppression
103
studies
xiv


Table 4.5
Table 4.6
Table 4.7
Table 4.8

Table 5.1
Table 8.1
Table 8.2
Table 8.3
Table 8.4
Table 8.5
Table 8.6
Table 8.7
Table 8.8
Table 8.9
Table 8.10
Table 8.11
Table 8.12
Table 8.13
Table 8.14
Table 8.15
Table 8.16
Table 8.17
Table 8.18
Table 8.19
Table 8.20
Table 8.21
Table 8.22
Table 8.23
Table 8.24
Table 8.25
Table 8.26
Table 8.27
Table 8.28

Table 8.29
Table 8.30
Table 8.31

Suppressors identified from H4Y98A AT phenotype suppression
studies
HATs selected for H4Y98A AT phenotype suppression studies
Acetylation of core histones carried out by the HATs Gcn5, Hpa1
and Hpa2
Tabulation of observable AT phenotype of site-directed alanine
and arginine mutagenesis of the histone H4 N-terminal lysine
residues
Histone H4 amino acid sequence identity between S. cerevisiae
(S) and humans (H)
Gene derivatives of Bank 13 (YEp13)
Genes inserted into PactT424 and PactT424-HA
HHF1 WT and mutant genes inserted into YCplac22
HHT1 WT and mutant genes inserted into YCplac111
HHF1 WT and mutant genes inserted into YCplac111
Genes inserted into YEplac181
Primers used for amplification of candidate suppressor genes in
one-step PCR
Preparation of TFBI and TFBII solutions
Preparation of LB media
Preparation of miniprep solution I (cell suspension buffer)
Preparation of miniprep solution II (cell lysis buffer)
Preparation of miniprep solution III (cell neutralisation buffer)
Preparation of 10X loading dye
Preparation of YPDA
Preparation of glucose/galactose media

Preparation of 0.1 M LiAc
Preparation of 40 % PEG
Preparation of yeast breaking buffer
Preparation of 10X FA gel buffer
Preparation of 1X FA gel running buffer
Preparation of 4 % stacking gel
Preparation of resolving gels of varying percentages
Preparation of 5X Western blot transfer buffer
Preparation of TBST
Preparation of Coomassie Blue staining solution
Preparation of destaining solution
Preparation of yeast lysis buffer
Preparation of pronase working buffer
Preparation of yeast lysis buffer with 0.5 M NaCl
Preparation of ChIP wash buffer
Preparation of 1X TE buffer

103
114
129
134

167
210
210
210
211
211
212
213

214
214
215
215
216
216
216
216
217
218
218
218
218
219
219
219
219
220
220
220
220
220
221
221
xv


Table 8.32
Table 8.33
Table 8.34

Table 8.35
Table 8.36
Table 8.37
Table 8.38
Table 8.39
Table 8.40
Table 8.41

Preparation of ChIP elution buffer
HIS3 mRNA expression levels
ImageJ quantification of the acetylation status of H4K8
ImageJ quantification of the acetylation status of H4K16
Histone H4 occupancy at the HIS3 promoter
Histone H4 occupancy at the HIS3 ORF
Histone H4K16ac occupancy at the HIS3 promoter
Histone H4K16ac occupancy at the HIS3 ORF
Gcn5 occupancy at the HIS3 promoter
Gcn5 occupancy at the HIS3 ORF

221
221
222
222
223
224
225
226
227
228

xvi


List of figures

Figure 1.1
Figure 2.1
Figure 2.2
Figure 2.3
Figure 2.4
Figure 2.5
Figure 3.1
Figure 3.2
Figure 3.3
Figure 3.4
Figure 3.5
Figure 4.1
Figure 4.2
Figure 4.3

Figure 4.4
Figure 4.5

Figure 4.6

Figure 4.7
Figure 4.8
Figure 4.9
Figure 4.10
Figure 4.11

Schematic diagram of the X-ChIP and N-ChIP protocols
X-ray crystal structure of the nucleosome core particle
Schematic diagram of mammalian histone variants
Schematic diagram of PTMs of histones
The dynamic role of nucleosomes in transcriptional regulation
may be influenced by the PTMs of histones
Schematic diagram of Gcn5 homologues and their sizes
Schematic diagram of the two-step PCR
Schematic diagram of the URA3 marker’s positive and negative
selections
Schematic diagram of plasmid shuffling and URA3 marker’s
counter selection involved
Schematic diagram of gene targeting involving the hisG-URA3hisG cassette present in NKY1009 targeting vector
Schematic diagram of gene targeting involving the LEU2 marker
present in puc8+LEU2 targeting vector
Observable phenotypes of the H4Y51A, H4E53A and H4Y98A
mutant strains
Observable phenotypes of gene knock out strains of the genes
identified as multi-copy phenotypic suppressors
Plasmid shuffling and complementation of histone H4 genomic
deletion of cells expressing histone H4 tyrosine-alanine singlepoint mutant proteins
Observable phenotypes of the H4Y51A, H4Y88A and H4Y98A
mutant strains
Plasmid shuffling and complementation of histone H4 genomic
deletion of cells expressing histone H4 tyrosine-phenylalanine
and tyrosine-aspartic acid single-point mutant proteins
Plasmid shuffling and complementation of histone H4 genomic
deletion of cells expressing histone H4 tyrosine-phenylalanine
and tyrosine-aspartic acid single-point mutant proteins

Observable phenotypes of the H4Y98A and H4Y98F mutant
strains
Over-expression of the HATs in the H4Y98A mutant strain
Gcn5 suppression of the AT phenotype of the H4Y98A mutant
strain
Hpa1 and Hpa2 suppression of the AT phenotype of the H4Y98A
mutant strain
Hpa3 non-suppression of the AT phenotype of the H4Y98A
mutant strain

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xvii


Figure 4.12
Figure 4.13
Figure 4.14
Figure 4.15
Figure 4.16
Figure 4.17
Figure 4.18
Figure 4.19
Figure 4.20
Figure 4.21
Figure 4.22

Figure 4.23

Figure 4.24
Figure 4.25
Figure 4.26

Figure 4.27

Figure 4.28

Figure 4.29
Figure 4.30

Figure 4.31

Esa1, Hat1, Hat2, Rtt109 and Sas2 non-suppression of the AT
phenotype of the H4Y98A mutant strain
HATs phenotype specificity to the AT phenotype of the H4Y98A
mutant strain
Gcn5, Hpa1 and Hpa2 strain specificity and phenotype specificity
Observable AT phenotype of the ∆GCN5, ∆HPA1, ∆HPA2 and
∆HPA3 deletion strains
HATs over-expression in the ∆GCN5 deletion strain
HATs over-expression in the ∆HPA1 deletion strain
Observable AT phenotype of the ∆GCN5, ∆GCN5∆HPA1,
∆GCN5∆HPA2 and ∆GCN5∆HPA3 deletion strains
Integrity and size distribution of total RNA purified after the
extraction procedure
Over-expression of multi-copy phenotypic suppressors and the
correlation to the activation level of the HIS3 gene
Observable AT phenotype of an histone H4 N-terminal deletion
strain
Plasmid shuffling and complementation of histone H4 genomic
deletion of cells expressing histone H4 N-terminal lysine to
alanine single-point mutant proteins
Plasmid shuffling and complementation of histone H4 genomic
deletion of cells expressing histone H4 N-terminal lysine to
arginine single-point mutant proteins
Observable AT phenotype of the histone H4 N-terminal lysine to
alanine single-point mutant strains

Observable AT phenotype of the histone H4 N-terminal lysine to
arginine single-point mutant strains
Plasmid shuffling and complementation of histone H4 genomic
deletion of cells expressing histone H4 N-terminal lysine to
alanine single-point mutant proteins in combination with
H4Y98A
Plasmid shuffling and complementation of histone H4 genomic
deletion of cells expressing histone H4 N-terminal lysine to
arginine single-point mutant proteins in combination with
H4Y98A
Observable AT phenotype of the histone H4 N-terminal lysine to
alanine single-point mutant strains in combination with H4Y98A
Observable AT phenotype of the histone H4 N-terminal lysine to
arginine single-point mutant strains in combination with H4Y98A
Suppression by Gcn5, Hpa1 and Hpa2 of observable AT
phenotype of the histone H4 N-terminal lysine to alanine singlepoint mutant strains in combination with H4Y98A
Suppression by Gcn5, Hpa1 and Hpa2 of observable AT
phenotype of the histone H4 N-terminal lysine to arginine singlepoint mutant strains in combination with H4Y98A

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xviii


Figure 4.32

Figure 4.33
Figure 4.34
Figure 4.35

Figure 4.36

Figure 4.37

Figure 4.38

Figure 4.39
Figure 4.40
Figure 4.41
Figure 4.42
Figure 4.43
Figure 4.44
Figure 4.45
Figure 4.46
Figure 4.47
Figure 4.48
Figure 4.49
Figure 4.50
Figure 4.51
Figure 4.52
Figure 4.53

Figure 4.54

Plasmid shuffling and complementation of histone H4 genomic
deletion of cells expressing histone H4 N-terminal K8 and K16
residues lysine to arginine double mutant proteins without and in
combination with H4Y98A
Observable AT phenotype of the histone H4K8,16R double
mutant strain
The over-expression of the HATs Gcn5, Hpa1 and Hpa2 did not
suppress the AT phenotype of the H4K8,16R double mutant strain
Plasmid shuffling and complementation of histone H4 genomic
deletion of cells expressing histone H4 N-terminal lysine to
alanine multiple point mutant proteins without and in combination
with H4Y98A

Plasmid shuffling and complementation of histone H4 genomic
deletion of cells expressing histone H4 N-terminal lysine to
arginine multiple point mutant proteins
Plasmid shuffling and complementation of histone H4 genomic
deletion of cells expressing histone H4 N-terminal lysine to
arginine multiple point mutant proteins in combination with
H4Y98A
Observable AT phenotype of the histone H4 N-terminal lysine to
alanine multiple point mutant strains without and in combination
with H4Y98A
Observable AT phenotype of the histone H4 N-terminal lysine to
arginine multiple point mutant strains
Acetylation status of H4K8
ImageJ quantification of the acetylation status of H4K8
Acetylation status of H4K16
ImageJ quantification of the acetylation status of H4K16
Sonication over a time course to identify the optimum sonication
conditions
PCR to check for presence of DNA in samples obtained for WT
histone H4 strain
PCR to check for presence of DNA in samples obtained for the
H4Y98A mutant strain
Histone H4 occupancy at the HIS3 promoter
Histone H4 occupancy at the HIS3 ORF
Histone H4K16ac occupancy at the HIS3 promoter
Histone H4K16ac occupancy at the HIS3 ORF
Gcn5 occupancy at the HIS3 promoter
Gcn5 occupancy at the HIS3 ORF
Plasmid shuffling and complementation of histone H3 and H4
genomic deletion of cells expressing combinations of different

histone H3 and histone H4 derivatives
Observable AT phenotype of cells expressing combinations of
different histone H3 derivatives and WT histone H4

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xix


Figure 4.55
Figure 5.1
Figure 5.2

Observable AT phenotype of cells expressing combinations of
different histone H3 derivatives and histone H4Y98A
Locations of tyrosine residues in histone binding sites within the
nucleosome core particle
Tyrosine residues in the interfaces between the (H3-H4)2
heterotetramer and the flanking H2A-H2B heterodimers

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xx


Summary

Histone H4 is one of four core histone proteins that make up the nucleosome, the
smallest building block of chromosomes. Alanine-scanning mutagenesis of histone
H4 had determined that the three mutant proteins H4Y51A, H4E53A and H4Y98A

conferred sensitivity to 3-aminotriazole (AT), antimycin A and high temperature
when expressed in place of endogenous histone H4. Multi-copy phenotypic
suppressor screens were performed and the histone acetyltransferases Gcn5, Hpa1 and
Hpa2 were isolated as multi-copy suppressors of the AT sensitivity of the H4Y98A
mutant strain. Chromatin immunoprecipitation studies carried out at the HIS3 gene
showed that the histidine starvation-induced histone eviction was reduced in the
H4Y98A mutant strain and restored back to the WT levels upon the over-expression
of Gcn5. By controlling all aspects of DNA biology, histones play an important role
in human diseases, and the homologous human proteins of the isolated suppressors
might become interesting drug targets in the future.

(149 words)

xxi


1. Introduction

1


1.1 Epigenetics
Epigenetics, by definition, is the study of all mitotically and meiotically heritable
changes in phenotype that do not result from changes in the genomic
deoxyribonucleic acid (DNA) nucleotide sequence (Petronis, 2010; Zhu and Reinberg,
2011). Several important cellular processes were found to be fundamentally regulated
by epigenetic modifications, such as gene expression, DNA-protein interactions,
suppression

of

transposable

element

mobility,

cellular

differentiation

and

embryogenesis. Thus, several major pathologies, including cancer, syndromes
associated with chromosomal alterations and neurological diseases, often arise due to
the occurrence of aberrant epigenetic modifications. Within cells, there are at least
three mechanisms of epigenetic modifications that can interact and stabilise one
another to lead to the expression or silencing of genes — DNA methylation,
ribonucleic acid (RNA)-associated silencing and histone modifications (Egger et al.,
2004).

1.1.1 DNA methylation
DNA methylation is one of the most-studied epigenetic modifications because it plays
an important role in several key processes, such as genomic imprinting,
X chromosome inactivation and suppression of repetitive element transcription and
transposition (Jin et al., 2011), where it ensures the proper regulation of gene
expression and stable gene silencing (Khavari et al., 2010; Kulis and Esteller, 2010).
DNA methylation involves the covalent addition of a methyl group (-CH3) to DNA,
specifically at the carbon-5 position of the cytosine ring. DNA methyltransferases
(DNMTs) establish and maintain the methylation pattern, which occurs generally

within CpG dinucleotides where a cytosine nucleotide is linked by a phosphate

2


directly to a guanine nucleotide. DNA methylation is often associated with gene
silencing as it blocks the binding of transcription factors and also promotes the
recruitment of methyl-CpG-binding domain proteins, which then help to recruit
histone-modifying complexes and chromatin-remodelling complexes (Khavari et al.,
2010; Kulis and Esteller, 2010; Jin et al., 2011).

1.1.2 RNA-associated silencing
In living cells, RNA can have a regulatory effect on DNA and the expression profile
of the genome (Morris, 2005). RNA may affect gene expression by causing the
formation of heterochromatin or by triggering DNA methylation and histone
modification (Egger et al., 2004). RNA-associated silencing is achieved through a
RNA interference (RNAi)-based mechanism, which is mediated by small interfering
RNAs (siRNAs) that can specifically direct epigenetic modifications to targeted loci
to silence target genes (Egger et al., 2004; Morris, 2005).

1.1.3 Histone modifications
Histones are proteins, which together with non-histone chromosomal proteins,
associate with DNA to form chromatin. Four core histones, H2A, H2B, H3 and H4,
make up an octameric complex, around which 147 base pairs (bp) of double stranded
super helical DNA winds to form the nucleosome (Millar and Grunstein, 2006).
Initially, histones were regarded only as static, non-participating structural elements
of the nucleosome for DNA packaging (Felsenfeld and McGhee, 1986). However,
experimental evidence has shown histones to be dynamic and integral in regulating
chromatin condensation and DNA accessibility, where histones can undergo multiple
types of post-translational modifications. This is important for the regulation of all

3