G3: Genes|Genomes|Genetics Early Online, published on October 16, 2017 as doi:10.1534/g3.117.300252
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Dissecting nucleosome function with a comprehensive
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histone H2A and H2B mutant library
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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]
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1 © The Author(s) 2013. Published by the Genetics Society of America.
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ABSTRACT
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Using a comprehensive library of histone H2A and H2B mutants, we assessed
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the biological function of each amino acid residue involved in various stress
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conditions including exposure to different DNA damage-inducing reagents,
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different growth temperatures and other chemicals. H2B N- and H2A C-termini
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were critical for maintaining nucleosome function and mutations in these
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regions led to pleiotropic phenotypes. Additionally, two screens were
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performed using this library, monitoring heterochromatin gene silencing and
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genome stability, to identify residues which could compromise normal function
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when mutated. Many distinctive regions within the nucleosome were revealed.
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Furthermore, we used the bar-seq method to profile the mutant composition of
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many libraries in one high-throughput sequencing experiment, greatly reducing
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the labor and increasing the capacity. This study not only demonstrates the
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applications of the versatile histone library, but also revealed many previously
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unknown functions of histone H2A and H2B.
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INTRODUCTION
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In eukaryotes, genomic DNA is compacted into chromatin to fit into the nucleus.
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As the functional unit of chromatin, a nucleosome consists of histone octamers,
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including two copies each of the four core histones H2A, H2B, H3 and H4, and
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about 147bp DNA (LUGER et al. 1997). Histones are known to be modified at
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multiple positions and numerous studies have been carried out in recent years
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to understand the function and regulation of these post-translational
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modifications (PTMs) (MORRISON et al. 2004; KEOGH et al. 2005; MASUMOTO et
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al. 2005; SHILATIFARD 2006; KOUZARIDES 2007; CHANG AND PILLUS 2009; DANG
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et al. 2009; MOSAMMAPARAST
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WURTELE et al. 2012; PATEL AND WANG 2013; WATANABE et al. 2013; ZENTNER
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AND
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Currently, about 20 types of PTMs, which are distributed among more than 100
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residues of core histones, have been identified (TAN et al. 2011; HUANG et al.
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2014). Although several “popular” modifications have been well studied, the
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functions of many newly discovered histone marks remain poorly understood.
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In addition, critical functions of residues without any PTMs have also been
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reported (LUO et al. 2010; YU et al. 2011), underscoring the importance of each
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histone residue.
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Compared to histone H3 and H4, histone H2A and H2B are less conserved
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from yeast to human, especially in the N terminal tails. In addition, H3-H4
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tetramers form the core of the nucleosomes while H2A and H2B are more
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HENIKOFF 2013; TESSARZ
SHI 2010; ZHOU et al. 2011; RANDO 2012;
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KOUZARIDES 2014; SEN et al. 2015).
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readily displaced (KIMURA AND COOK 2001; KIREEVA et al. 2002). Perhaps for
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these reasons, fewer studies have focused on histones H2A and H2B than for
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H3 and H4. Although modifications at more than 50 residues of H2A and H2B
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were characterized (TAN et al. 2011; HUANG et al. 2014), only a few modified
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residues have been well characterized. For example, ubiquitination of histone
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H2B K123, mediated by Rad6, stimulates the methylation of H3K4 and H3K79
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and plays important roles in transcription regulation and DNA damage
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checkpoint signaling (ROBZYK et al. 2000; NG et al. 2002; SUN AND ALLIS 2002;
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GIANNATTASIO et al. 2005; PAVRI et al. 2006; FLEMING et al. 2008; MOYAL et al.
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2011; HUNG et al. 2017). The functions and regulation of most modifications
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remain unknown. Until now, only two versions of the histone H2A and H2B
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mutant library were available for S. cerevisiae (MATSUBARA et al. 2007;
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NAKANISHI et al. 2008; SAKAMOTO et al. 2009), and both of these libraries
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consist of exclusively alanine substitutions and can only be used in an
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episomal plasmid format.
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In this study, we used a comprehensive new library of histone H2A/H2B
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mutants to systematically probe the functions of histone H2A/H2B residues in
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different biological processes, including DNA damage repair, temperature
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tolerance, genome stability and heterochromatic gene silencing, revealing
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many interesting features of these less well-known histones. In addition, the
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presence of two unique barcodes on every mutant allowed adoption of a
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barcode sequencing method to dissect complex phenotypic assays which
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otherwise would be labor intensive and time-consuming. Other applications,
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such as construction of an interaction network among the core histone
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surfaces, can be envisioned.
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MATERIALS AND METHODS
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Yeast strains, plasmids and media
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Strains in BY-H2ML1&2 library were used for high-throughput phenotype
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analysis under different stress conditions with serial dilution. Linearized
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plasmids of H2ML1 by BciVI were transformed into JDY187 (a derivative of
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strain GRF167) and SHY15 (a derivative of W303) to screen the mutants
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which affect heterochromatin silencing and genome stability, respectively.
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Strains in BY-H2ML1 library were used for MMS assay by bar-seq or serial
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dilution. Genotype of all strains are listed in Table S1.
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Synthetic complete (SC) medium contained 0.17% (w/v) yeast nitrogen base
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without amino acids and ammonium sulfate (YNB, USBio), 0.5% (w/v)
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ammonium sulfate, 2% (w/v) glucose, and 2% (w/v) bacto-agar, supplemented
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with 0.2% (w/v) amino acids drop-out powder. The concentration of 5-FOA in
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synthetic media was 1 g/l. Drug-containing media were prepared by
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supplementing YPD with 200 mM HU, 0.03% (v/v) MMS, 10 μg/ml benomyl, 8
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μg/ml CPT, 2 μg/ml nocodazole, 25 nM rapamycin, or 0.3% (v/v) acetic acid,
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respectively.
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Chromosome loss assay
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The reporter strain (SHY015) was derived from SBY8054, which contains an
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artificial chromosome III fragment with SUP11 and HIS3 markers (NG et al.
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2013). The HTA2-HTB2 locus was knocked out. ura3-1 and leu2-3 were
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corrected to ura3∆0 and leu2∆0 to generate the final strain. Individual histone
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mutations were integrated at the endogenous HTA1-HTB1 locus and
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confirmed by colony PCR as described above. Chromosome loss assay was
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done as described (NG et al. 2013). At least two independent isolates were
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tested for each mutant.
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Heterochromatin silencing assay
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The reporter strain (JDY187) was derived from YNB12, containing two makers,
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MET15 and ADE2, within the rDNA repeat and the subtelomeric region on
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chromosome V respectively (SMITH et al. 1999). Genomic HTA1-HTB1 and
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HTA2-HTB2 were knocked out and the cells were supplied with pJD78 to
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support viability, which was removed after a histone mutant was introduced. At
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least two independent isolates were tested for each mutant. For rDNA
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silencing, diluted fresh cells were plated on lead plates (1 g/l) for one week at
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30° before we scored the colony color (DAI et al. 2010). For telomeric silencing,
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cells were spotted on SC–Leu plates and incubated at 30° for 3 days. Pictures
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of the red colony color were taken after storing the cells at 4° for 4 days.
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Identify MMS-sensitive alleles with barcode sequencing
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Viable H2A/H2B mutants were pooled together and stored in 15% glycerol at –
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80°. For the MMS assay, cells of H2A/H2B pool were inoculated in 50 ml of
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YPD for 4 hours at 30°. 10 ml of cell cultures were transferred into two new
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tubes: one with MMS (final concentration at 0.05%) and the other without. The
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cells were cultured at 30° for 2 hours, precipitated by centrifugation and
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washed with ddH2O twice before inoculating into 50 ml YPD for overnight
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growth at 30°. The cell density of the overnight cultures was measured and
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2.5X108 cells from both cultures were harvested for genomic DNA preparation.
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For barcode sequencing, genomic DNA was isolated and used as the
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templates for two rounds of PCR to construct the sequencing libraries. At first,
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the TAG regions of each histone mutant were amplified using the primers with
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same annealing sequences (Table S6) (DAI et al. 2008). To sequence different
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samples, a five-digit index sequence, among which at least two nucleotides
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were different from each other, were introduced. Next, the Illumina sequencing
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adaptors (P5/P7) were added by second round of PCR using diluted first PCR
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products as templates. These PCR products were mixed, gel purified and
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subjected to Illumina GAII pair-end sequencing. We got over 95k pairs of reads
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per sample. Statistics of log2 and p-value with FDR/FWER false positive
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control were used to compare the sequencing data for different mutants in
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MMS treated and YPD group. The mutants with log2 ratio
