G3: Genes|Genomes|Genetics Early Online, published on October 16, 2017 as doi:10.1534/g3.117.300252
1
Dissecting nucleosome function with a comprehensive
2
histone H2A and H2B mutant library
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Shuangying Jiang1,2,#, Yan Liu1,#, Caiyue Xu1,$, Yun Wang3,4, Jianhui Gong3,4, Yue Shen3,4, Qingyu Wu1, Jef D. Boeke5 and Junbiao Dai1,2* 1
MOE Key laboratory of Bioinformatics and Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University, Beijing 100084, PR China 2 Center for Synthetic Biology Engineering Research, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China 3 China National GeneBank, BGI-Shenzhen, Shenzhen 518120, China 4 BGI-Shenzhen, Shenzhen, 518083, China 5 Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, New York University Langone Medical Center, New York, New York 10011, USA
Running title: Histone H2A and H2B mutagenesis Keywords: histone; heterochromatin gene silencing; DNA damage, Post-translational Modification #
These authors contributed to this work equally $ Current address: Epigenetics Program, Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA Corresponding author: Dr. Junbiao Dai Center for Synthetic Biology Engineering Research, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China. Phone: 86-755-86585244; Email:
[email protected]
33 34
1 © The Author(s) 2013. Published by the Genetics Society of America.
35
ABSTRACT
36
Using a comprehensive library of histone H2A and H2B mutants, we assessed
37
the biological function of each amino acid residue involved in various stress
38
conditions including exposure to different DNA damage-inducing reagents,
39
different growth temperatures and other chemicals. H2B N- and H2A C-termini
40
were critical for maintaining nucleosome function and mutations in these
41
regions led to pleiotropic phenotypes. Additionally, two screens were
42
performed using this library, monitoring heterochromatin gene silencing and
43
genome stability, to identify residues which could compromise normal function
44
when mutated. Many distinctive regions within the nucleosome were revealed.
45
Furthermore, we used the bar-seq method to profile the mutant composition of
46
many libraries in one high-throughput sequencing experiment, greatly reducing
47
the labor and increasing the capacity. This study not only demonstrates the
48
applications of the versatile histone library, but also revealed many previously
49
unknown functions of histone H2A and H2B.
50
2
51
INTRODUCTION
52
In eukaryotes, genomic DNA is compacted into chromatin to fit into the nucleus.
53
As the functional unit of chromatin, a nucleosome consists of histone octamers,
54
including two copies each of the four core histones H2A, H2B, H3 and H4, and
55
about 147bp DNA (LUGER et al. 1997). Histones are known to be modified at
56
multiple positions and numerous studies have been carried out in recent years
57
to understand the function and regulation of these post-translational
58
modifications (PTMs) (MORRISON et al. 2004; KEOGH et al. 2005; MASUMOTO et
59
al. 2005; SHILATIFARD 2006; KOUZARIDES 2007; CHANG AND PILLUS 2009; DANG
60
et al. 2009; MOSAMMAPARAST
61
WURTELE et al. 2012; PATEL AND WANG 2013; WATANABE et al. 2013; ZENTNER
62
AND
63
Currently, about 20 types of PTMs, which are distributed among more than 100
64
residues of core histones, have been identified (TAN et al. 2011; HUANG et al.
65
2014). Although several “popular” modifications have been well studied, the
66
functions of many newly discovered histone marks remain poorly understood.
67
In addition, critical functions of residues without any PTMs have also been
68
reported (LUO et al. 2010; YU et al. 2011), underscoring the importance of each
69
histone residue.
70
Compared to histone H3 and H4, histone H2A and H2B are less conserved
71
from yeast to human, especially in the N terminal tails. In addition, H3-H4
72
tetramers form the core of the nucleosomes while H2A and H2B are more
AND
HENIKOFF 2013; TESSARZ
SHI 2010; ZHOU et al. 2011; RANDO 2012;
AND
KOUZARIDES 2014; SEN et al. 2015).
3
73
readily displaced (KIMURA AND COOK 2001; KIREEVA et al. 2002). Perhaps for
74
these reasons, fewer studies have focused on histones H2A and H2B than for
75
H3 and H4. Although modifications at more than 50 residues of H2A and H2B
76
were characterized (TAN et al. 2011; HUANG et al. 2014), only a few modified
77
residues have been well characterized. For example, ubiquitination of histone
78
H2B K123, mediated by Rad6, stimulates the methylation of H3K4 and H3K79
79
and plays important roles in transcription regulation and DNA damage
80
checkpoint signaling (ROBZYK et al. 2000; NG et al. 2002; SUN AND ALLIS 2002;
81
GIANNATTASIO et al. 2005; PAVRI et al. 2006; FLEMING et al. 2008; MOYAL et al.
82
2011; HUNG et al. 2017). The functions and regulation of most modifications
83
remain unknown. Until now, only two versions of the histone H2A and H2B
84
mutant library were available for S. cerevisiae (MATSUBARA et al. 2007;
85
NAKANISHI et al. 2008; SAKAMOTO et al. 2009), and both of these libraries
86
consist of exclusively alanine substitutions and can only be used in an
87
episomal plasmid format.
88
In this study, we used a comprehensive new library of histone H2A/H2B
89
mutants to systematically probe the functions of histone H2A/H2B residues in
90
different biological processes, including DNA damage repair, temperature
91
tolerance, genome stability and heterochromatic gene silencing, revealing
92
many interesting features of these less well-known histones. In addition, the
93
presence of two unique barcodes on every mutant allowed adoption of a
94
barcode sequencing method to dissect complex phenotypic assays which
4
95
otherwise would be labor intensive and time-consuming. Other applications,
96
such as construction of an interaction network among the core histone
97
surfaces, can be envisioned.
98 99
MATERIALS AND METHODS
100
Yeast strains, plasmids and media
101
Strains in BY-H2ML1&2 library were used for high-throughput phenotype
102
analysis under different stress conditions with serial dilution. Linearized
103
plasmids of H2ML1 by BciVI were transformed into JDY187 (a derivative of
104
strain GRF167) and SHY15 (a derivative of W303) to screen the mutants
105
which affect heterochromatin silencing and genome stability, respectively.
106
Strains in BY-H2ML1 library were used for MMS assay by bar-seq or serial
107
dilution. Genotype of all strains are listed in Table S1.
108
Synthetic complete (SC) medium contained 0.17% (w/v) yeast nitrogen base
109
without amino acids and ammonium sulfate (YNB, USBio), 0.5% (w/v)
110
ammonium sulfate, 2% (w/v) glucose, and 2% (w/v) bacto-agar, supplemented
111
with 0.2% (w/v) amino acids drop-out powder. The concentration of 5-FOA in
112
synthetic media was 1 g/l. Drug-containing media were prepared by
113
supplementing YPD with 200 mM HU, 0.03% (v/v) MMS, 10 μg/ml benomyl, 8
114
μg/ml CPT, 2 μg/ml nocodazole, 25 nM rapamycin, or 0.3% (v/v) acetic acid,
115
respectively.
116
Chromosome loss assay
5
117
The reporter strain (SHY015) was derived from SBY8054, which contains an
118
artificial chromosome III fragment with SUP11 and HIS3 markers (NG et al.
119
2013). The HTA2-HTB2 locus was knocked out. ura3-1 and leu2-3 were
120
corrected to ura3∆0 and leu2∆0 to generate the final strain. Individual histone
121
mutations were integrated at the endogenous HTA1-HTB1 locus and
122
confirmed by colony PCR as described above. Chromosome loss assay was
123
done as described (NG et al. 2013). At least two independent isolates were
124
tested for each mutant.
125
Heterochromatin silencing assay
126
The reporter strain (JDY187) was derived from YNB12, containing two makers,
127
MET15 and ADE2, within the rDNA repeat and the subtelomeric region on
128
chromosome V respectively (SMITH et al. 1999). Genomic HTA1-HTB1 and
129
HTA2-HTB2 were knocked out and the cells were supplied with pJD78 to
130
support viability, which was removed after a histone mutant was introduced. At
131
least two independent isolates were tested for each mutant. For rDNA
132
silencing, diluted fresh cells were plated on lead plates (1 g/l) for one week at
133
30° before we scored the colony color (DAI et al. 2010). For telomeric silencing,
134
cells were spotted on SC–Leu plates and incubated at 30° for 3 days. Pictures
135
of the red colony color were taken after storing the cells at 4° for 4 days.
136
Identify MMS-sensitive alleles with barcode sequencing
137
Viable H2A/H2B mutants were pooled together and stored in 15% glycerol at –
138
80°. For the MMS assay, cells of H2A/H2B pool were inoculated in 50 ml of
6
139
YPD for 4 hours at 30°. 10 ml of cell cultures were transferred into two new
140
tubes: one with MMS (final concentration at 0.05%) and the other without. The
141
cells were cultured at 30° for 2 hours, precipitated by centrifugation and
142
washed with ddH2O twice before inoculating into 50 ml YPD for overnight
143
growth at 30°. The cell density of the overnight cultures was measured and
144
2.5X108 cells from both cultures were harvested for genomic DNA preparation.
145
For barcode sequencing, genomic DNA was isolated and used as the
146
templates for two rounds of PCR to construct the sequencing libraries. At first,
147
the TAG regions of each histone mutant were amplified using the primers with
148
same annealing sequences (Table S6) (DAI et al. 2008). To sequence different
149
samples, a five-digit index sequence, among which at least two nucleotides
150
were different from each other, were introduced. Next, the Illumina sequencing
151
adaptors (P5/P7) were added by second round of PCR using diluted first PCR
152
products as templates. These PCR products were mixed, gel purified and
153
subjected to Illumina GAII pair-end sequencing. We got over 95k pairs of reads
154
per sample. Statistics of log2 and p-value with FDR/FWER false positive
155
control were used to compare the sequencing data for different mutants in
156
MMS treated and YPD group. The mutants with log2 ratio