Article Figures & Data
Figures
- Figure 1. In S. pombe, a deletion of the abo1 gene causes a marked cell proliferation defect suppressed by mutations in HIRA or CAF1 complexes or in histones H3 or H4.
(A) Schematic representation of human ATAD2 and S. pombe Abo1 proteins. The percentage of amino acid sequence identity is indicated for the most conserved domains. ATP1 and ATP2, AAA+ ATPase domain 1 and 2; BRD, Bromo domain; CD, C-terminal domain. (B) Photographs of wild-type (wt, SPV 8) and abo1∆ (SPV 3789) colonies grown on solid medium for 4 d, with magnified views to highlight the co-existence of small size colonies (orange arrowhead) and large size colonies (green arrowhead, which correspond to suppressor clones) on the abo1∆ plate. (C) Growth curves of wt, abo1∆, and abo1∆ suppressor (abo1∆_sup) cells obtained from three biological replicates done with three different clones (SPV 3,789, 3,790, and 3,791) and grown in liquid medium over the indicated times. (D) Diagram of the screen conducted to identify mutations responsible for the suppression of abo1∆ cell growth’s defect by using RNA sequencing. Single colonies of wt, abo1∆ (with severe growth defect) and abo1∆_sup cells were picked to inoculate liquid cultures and total RNA was purified. Massive parallel sequencing from the purified total RNAs depleted from rRNAs was used to detect SNVs (for details see Table S1). Spotting assays of wild-type, abo1∆ and abo1∆_suppressor clones are shown in Fig S1. (E) Circos plot showing the SNVs identified in 11 independent isolates of abo1∆_sup (A to K) along the three S. pombe chromosomes (chr. I, II and III). The positions of all SNVs identified, either in coding or non-coding regions of the genome, are highlighted by dots colored according to the nature of the mutations, as indicated. Blue dashed lines highlight SNVs localizing within the coding sequence (cds) of subunits of histone chaperone complexes (hip3, slm9, and pcf1) or histones (hht2 and hhf1) genes, as well as two SNVs, in the cds of SPCC622.11 or esf1 genes, identified in a majority of abo1∆_sup isolates but that do not contribute to the suppression of abo1∆ cells growth defect. Spotting assays of backcrossed abo1∆_suppressor clones are shown in Fig S3. (F) Position and nature of the amino acid modifications induced by the SNVs found in the 11 abo1∆_sup isolates. The color code of the mutations is the same as in (E). fs: frameshift, dot: non-sense mutation. (G) Scheme showing the functional interplay between Abo1 and HIRA complex allowing correct loading of histones on chromatin in wild-type cells (left part of the panel), and the potential effect of a lack of Abo1 on HIRA’s function and histone loading (right part of the panel) in abo1∆ cells.
- Figure S1. Spotting assay of wild-type, abo1∆, and abo1∆_suppressor clones.
Cells from single colonies of wild-type (wt, SPV 8), abo1∆ (SPV 3789), and abo1∆_sup were picked, grown in liquid medium, and spotted on solid medium at for 4 d. Related to Fig 1D.
- Figure S2. Identification and validation of single-nucleotide variants (SNVs) from RNA-seq data.
(A) Example of a SNV identification by the ArrayStar module of the DNASTAR software corresponding to abo1∆_supA RNA-seq data. Alignment of the reads was done over the reference genome of S. pombe (upper sequence) and are shown here for the fraction of hip3 coding sequence that contains the SNV. The SNV G>A is common to all 718 reads and highlighted in blue. This non-sense mutation introduces a stop codon after the amino acid 1,302 of Hip3. Changes of nucleotides that most likely correspond to sequencing errors were also found and are highlighted in red. (B) For three different abo1∆_suppressors (supA, supB and supG), the SNVs found in the RNA-seq data were checked at the genomic level by DNA sequencing, after PCR amplification of the region containing the SNVs. Sequencing traces show, as expected, that SNVs (underlined in gray) are only detected in abo1∆_sup isolates and not in the genomes of wild-type or parental abo1∆ cells. Note that the sequence shown on the electropherograms for hip3 corresponds to the anti-sense strand. Related to Table 1.
- Figure S3. Spotting assay of backcrossed abo1∆_suppressor clones.
Cells from single colonies of wild-type (wt, SPV 8), abo1∆ (SPV 3789), and abo1∆_sup before backcrossing (O: original) and abo1∆_sup after backcrossing (BC, backcrossed) were picked, grown in liquid medium then spotted on solid medium, and grown for 4 d. Related to Fig 1E.
- Figure 2. A reduction of H3 and H4 genes copy number suppresses the growth defect caused by Abo1 depletion.
(A) Scheme of the conditional knock-down strategy of abo1 using the auxin-inducible degron system. TIR1 F-box proteins from Arabidopsis thaliana and Oryza sativa (rice) are expressed fused to S. pombe Skp1 (green rectangle TIR1), whereas endogenous Abo1 is fused to the 2xHA-IAA degron (blue triangle, IAA17) double tag. Addition of synthetic auxin 1-naphthaleneacetic acid (NAA) to the medium promotes association between Skp1–Cullin–F-box (SCF) E3 ubiquitin ligases complex and the degron tag, leading to subsequent ubiquitination of Abo1 and its degradation by the proteasome. (B) Western blot showing the level of Abo1-HA2-IAA17 fusion protein, 6 h after addition of DMSO (−) or NAA (+) in cells expressing TIR1-Skp1 proteins (right panel, Abo1-HA2-IAA17, SPV 4530). Protein extracts from untagged cells (left panel, untagged, SPV 4451) show the absence of cross-reaction with the α-HA antibody. Molecular weight markers (kD) are shown on the left. Kinetics of the effect of the conditional knock-down of Abo1 on cell growth are shown in Fig S4. (C) Growth curves of wt (SPV 4,451), abo1-aid (SPV 4,530, 4,531, and 4,532), abo1-aid, hht1∆, hhf1∆ (SPV 4,817, 4,818 and 4,819) and abo1-aid, hht2∆, hhf2∆ (SPV 4,822, 4,823 and 4,824) cells obtained from three biological replicates issued from two different clones. Cells were grown in liquid medium containing DMSO (upper graph, − NAA) or 0.5 mM NAA (lower graph, + NAA) for the indicated times before measuring the OD at 600 nm. In the presence of NAA, t test indicates a significant difference in abo1-aid cells’ growth, compared to all three other cell cultures, ***P < 0.001.
- Figure S4. Kinetics of the effect of the conditional knock-down of Abo1 on cell growth.
Growth curves of wt (SPV 4,451) and abo1-aid (SPV 4,530, 4,531 and 4,532) cells were obtained from three biological replicates corresponding to three different clones. Cells were grown overnight at 25°C in liquid EMMC medium up to an optical density at 600 nm (OD600) of 2–3, then diluted at an OD600 of 0.01 in fresh EMMC medium containing 0.5 mM NAA (+NAA) or an equal volume of DMSO (−NAA), and grown for the indicated times. Related to Fig 2.
- Figure 3. Atad2 controls FACT and HIRA interaction with chromatin.
(A) Extracts from human HeLa and HepG2 and mouse embryonic stem cells after ATAD2 KO (by CRISPR/Cas9 system) were probed with the indicated antibodies. Three independent different biological replicates are shown (left panels). SSRP1- and HIRA-encoding mRNAs were also quantified from parallel cultures of the same cell lines (three independent biological replicates) by RT-qPCR. The mean values were calculated from triplicates for each biological replicate (n = 3) and are shown in the bar diagrams for wild-type (red) and ATAD2 KO (green) cells (right panels). Error bars indicate the standard errors of the mean values. (B) Mononucleosomes generated after extensive digestion of chromatin from wild-type and Atad2 KO embryonic stem cells were immunoprecipitated with the indicated antibodies and the immunoprecipitated DNA fragments were sequenced. A heat map of read density over −5 to +5 kb centred on all transcription start sites is shown (the density scale is shown on the right).
Source data are available for this figure.
Source Data for Figure 3[LSA-2021-01151_SdataF3.pdf]
- Figure 4. Atad2-dependent control of histone deposition by FACT and HIRA in embryonic stem cells.
(A) RPKM-normalized read coverage mean values over a region spanning from −500 to 1,500 bp with respect to the gene transcriptional start site (TSS) are shown for the input DNA (black line, wild type; red line, Atad2 KO). DNA co-immunoprecipitated with HIRA (blue line) and Ssrp1 (green line) from Atad2 KO embryonic stem cells are also shown over the input signal. For this analysis, the gene TSSs were grouped into quartiles as a function of gene transcriptional activity calculated from our own RNA-seq data (Morozumi et al, 2016), and the TSS corresponding to the top (fourth quartile) most expressed genes, third quartile (mid-expression) and second quartile (low expression) are shown separately from left to right. The red arrow indicates the peak of read coverage value present at the HIRA-bound nucleosome-free region (NFR) region of highly active genes, which disappears on the NFRs of the less active gene TSSs. (B) Input and ChIP read signals shown in panel (A) for the top 25% active genes are shown at higher resolution to visualize the distance separating neighbouring nucleosomes from dyad to dyad. A schematic representation of the nucleosomal organization over gene TSSs is shown below. (C) Models summarizing the ChIP-seq mapping data of nucleosome distribution and HIRA and FACT localization in wild-type and Atad2 KO active gene TSSs are shown. In wild-type cells, Atad2 ensures a dynamic interaction of HIRA and FACT with chromatin at the gene TSSs, maintaining an equilibrium between histone deposition and removal. In Atad2 KO cells the residence time of HIRA and FACT on nucleosomes is significantly increased, especially on the NFR region.
Tables
Suppressor strains Single-nucleotide variant Amino acid modification Protein name Function (protein complex) abo1∆_supA G>A (3778764) R1303stop Hip3 HIRA complex abo1∆_supB G>A (3780744) Q643stop abo1∆_supC A>C (3779930) L914stop abo1∆_supD T>A (3780930) K581stop abo1∆_supE A>T (3780247) C808stop abo1∆_supF ins T (3782449) N74fs abo1∆_supG G>T (3016457) G307A Slm9 abo1∆_supH C>T (3017455) Q640stop abo1∆_supI ins A (2539317) I213fs Pcf1 CAF1 complex abo1∆_supJ T>C (1365405) L83S Hht2 (H3) Histones abo1∆_supK T>G (4699234) T55P Hhf1 (H4) Detailed description of the single-nucleotide variants in genes encoding subunits of HIRA and CAF1 histone chaperone complexes or histone H3 and H4 found in the 11 abo1∆_sup (A to K) clones (also see Fig S3). The genomic positions of the single-nucleotide variants are indicated in brackets. >, substitution; ins, insertion; fs, frameshift; stop, non-sense.
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