Hypersaline archaeal histone share a mosaic of functions with DNA binding proteins across domains of life
Creators: name: Schmid, Amy
name: Darnell, Cynthia
name: Vreugdenhil, Angie
name: Martinez-Pastor, Mar
name: Hackley, Rylee
name: Sakrikar, Saaz
Publisher: name: figshare
Year: 2022
Subjects: subject: 60199 Biochemistry and Cell Biology not elsewhere classified
subject: FOS: Biological sciences
subject: 60102 Bioinformatics
subject: FOS: Computer and information sciences
subject: Computational Biology
subject: 60408 Genomics
subject: Microbiology

description: Supplementary material accompanying manuscript. <br> <strong>File S1</strong>: Details of methods and results obtained when searching for a DNA sequence motif for HstA and HpyA binding. <strong>Figure S1: ∆</strong><em><strong>hstA</strong></em><strong> phenotype in response to diverse stress conditions</strong>. (<strong>A</strong>) Raw growth curves of multiple biological replicate cultures under standard growth conditions (YPC medium, 2.5M NaCl, 0.3 M Mg2+, 42ºC). (<strong>B</strong>-<strong>E</strong>) Each graph depicts the area under the log-transformed growth curve (AUC) under the conditions indicated at the top of each panel. Each point represents AUC for one growth curve, and the horizontal lines depict the median of the AUC distribution for each strain under each growth condition. (<strong>B</strong>) Growth in increased sodium (4M NaCl); (<strong>C</strong>) growth in reduced sodium (1.5M NaCl). (<strong>D</strong>) Oxidative stress (0.05M H2O2). (<strong>E</strong>) gluconeogenic conditions (no sugar added to HvCa medium). HvCa medium contains identical components to YPC18 described above, except without the addition of peptone or yeast extract (69). (<strong>F</strong>) Growth curves in high MgCl2 (overall Mg2+ 0.48M), low MgCl2 (overall Mg2+ 0.19M); visual analysis of the data confirmed that growth under Mg stress conditions was identical to non-stress conditions. Δ<em>hstA</em>: parent growth ratio in all cases was 0.84 or higher, indicating <em>hstA</em> is not required for stress response. <strong>Figure S2:</strong> <strong>HstA deletion can be complemented </strong><em><strong>in trans</strong></em>; <strong>HA tag does not interfere with HstA function.</strong> Dot plot representing growth measured by area under the curve (AUC) of ∆<em>hstA</em> containing plasmids as calculated by the R package grofit; each dot represents AUC for one technical replicate growth curve, central line represents median value. Samples from left to right: <em>∆hstA </em>expressing wild type <em>hstA in trans; ∆hstA </em>expressing wild type <em>hstA </em>fused to the HA tag; ∆<em>hstA</em> strain transformed with empty vector control. <strong>Figure S3</strong>: <strong>HpyA and HstA bind in few, discrete peaks, like TFs, and contrasting with histones and NAPs.</strong> (<strong>A</strong>) HpyA (dark blue) and HstA (light blue) binding is observed as discrete reproducible peaks (marked with arrow); shown here are 2 representative replicates each. (<strong>B</strong>) Halophilic TF TrmB (pink) binds in discrete reproducible peaks; 2 replicates shown (<strong>C</strong>) Bacterial TF FNR (purple) binds in discrete reproducible peaks (2 replicates shown). (<strong>D</strong>) Bacterial NAPs cover a large part of the genome, either with many peaks: IHF (green), FIS (olive); and/or broad peaks: HNS (black). (<strong>E</strong>) Yeast histone ChIP-seq (red) shows depletions in promoter regions of some genes (representative regions marked with black arrows). Broad peak locations of nucleosomes positioned in the gene body are indicated with grey dots. Shown here are data from histone H2B, H4, H3 in quiescent phase (H3_Q), and H3 in logarithmic growth phase (H3_log). <strong>Figure S4</strong>: <strong>Binding occupancy at start sites of bacterial NAPs. </strong>Average binding occupancy across all genes for: (<strong>A</strong>) <em>E. coli</em> IHF (green, 2 subunits), H-NS (black), FIS (olive); (<strong>B</strong>) Lrp in minimal medium in stationary phase (orange, mean TSS occupancy for two biological replicate experiments are shown). <strong>Figure S5</strong>: <strong>Heatmap of binding occupancy for yeast histone and halophilic histone</strong>. (<strong>A</strong>) Heatmap of yeast histone H3, with each row representing the start site of one gene. Color scale at right represents average normalized occupancy. (<strong>B</strong>) Line graph of histone H3 showing the average across all genes. (<strong>C</strong>) HpyA heatmap with color scale as in A. (<strong>D</strong>) HpyA average line graph. <strong>Figure S6</strong>: (<strong>A</strong>) AA/TT/TA dinucleotide periodicity of archaeal species with characterized histones (red; <em>M. stadtmanae, M. thermoautotrophicus, M. jannaschii</em>) and with non-histone chromatin (blue; <em>T. acidophilum, P. calidifontis</em>). Dotted red line represents 10 bp periodicity peak location. (<strong>B</strong>) Periodicity of GC dinucleotides in species with &gt;60%G+C genomic content: histone-containing <em>M. kandleri</em> (red), model halophile species <em>Hbt. salinarum, Hfx. volcanii, Hfx. mediterranei, Hca. hispanica</em> (black). Red dotted line indicates 10bp, black dotted line indicates 11bp. Within halophiles, we also examined the periodicity of the GC dinucleotide, because, unlike the genomes of archaea known to use histones to package their genomes, halophilic genomes are &gt; 60% G+C (80). Therefore, the histone binding signal might be revealed by GC dinucleotides, which are also known to play a role in histone binding (81). <em>Methanopyrus kandleri</em>, a GC-rich species (82) whose histone protein has been shown to be capable of nucleosome formation (83), was also included in this comparison. For the four model halophiles, we detected relatively weak periodicity that skewed away from 10bp (~10.5-11bp <strong>Fig. S6B</strong>). One peak exhibited a periodicity close to 11bp, which, as noted above, is more likely linked to supercoiling than histone binding. On the other hand, a periodicity closer to ~10bp was indeed observed for GC dinucleotides in the <em>M. kandleri</em> genome. (<strong>C</strong>) 10 bp periodicity of species with known histones and MNase-seq. MNase protected regions (nucleosomes) of <em>S. cerevisiae </em>(green), <em>T. kodakarensis </em>(red), <em>Hfx. volcanii </em>(black). Dotted black line indicates the ~10bp periodicity peak (not detected for <em>Hfx. volcanii</em>). Normalized spectra are plotted in this panel to facilitate clarity in the visualization. (<strong>D</strong>) Genome-wide average of micrococcal nuclease digestion (MNase-seq) pattern for <em>Hfx. volcanii. </em>We note that, to our knowledge, no ChIP-seq data are available in the literature for archaeal histones other than HpyA or HstA. However, genome-wide mapping of nucleosomes using MNase-seq has been carried out in <em>T. kodakarensis </em>(9), <em>Methanothermobacter thermoautotrophicus (13)</em>, and <em>Hfx. volcanii </em>(54). In all these studies, a depletion in occupancy just upstream of the start site, similar to the nucleosome-free region of eukaryotic promoters, was reported. We could reproduce this TSS occupancy depletion using our analysis pipeline on the publicly available <em>Hfx. volcanii</em> MNase data (<strong>Fig. S4C</strong>); however, this MNase profile is different from the HstA ChIP-seq occupancy profile (light blue lines, <strong>Fig. 4D</strong>). By contrast, in the case of yeast histones, the histone ChIP-seq occupancy (<strong>Fig. 4A</strong>) correlates with known MNase digestion patterns (3). Hence, the dissimilarity between <em>Hfx. volcanii</em> MNase-seq and HstA ChIP-seq occupancy suggest that HstA is not the major chromatin protein for <em>Hfx volcanii</em>, in contrast with the original interpretation of this MNase-seq dataset (54). <strong>Table S1: </strong>Two tabs in the supplementary excel file: (a) Growth data (OD600) for ∆<em>hstA</em> and ∆<em>pyrE</em> parent strain in optimal conditions; (b) whole genome resequencing data for ∆<em>hstA</em> strain. <strong>Table S2: </strong>Manually curated list of ChIP-seq peaks for HstA. <strong>Table S3:</strong> Details of characteristics (number of peaks, average width, total area covered by peaks) of ChIP-seq peaks for HpyA, HstA, and other DNA binding proteins (shown graphically in main text figures). <strong>Table S4: </strong>Results of hypergeometric test to check over-representation of ChIP-seq peaks in intergenic regions of the genome. <strong>Table S5:</strong> List of ChIP-seq datasets used (with SRA trace where available), and genomes analysed for periodicity (with link to the relevant NCBI assembly). <strong>Table S6: </strong>List of primers, strains, and plasmids used in this study. <br>