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Genome-wide ATAC-see screening identifies TFDP1 as a modulator of global chromatin accessibility

Nature Genetics volume 56, pages 473–482 (2024)Cite this article

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Abstract

Chromatin accessibility is a hallmark of active regulatory regions and is functionally linked to transcriptional networks and cell identity. However, the molecular mechanisms and networks that govern chromatin accessibility have not been thoroughly studied. Here we conducted a genome-wide CRISPR screening combined with an optimized ATAC-see protocol to identify genes that modulate global chromatin accessibility. In addition to known chromatin regulators like CREBBP and EP400, we discovered a number of previously unrecognized proteins that modulate chromatin accessibility, including TFDP1, HNRNPU, EIF3D and THAP11 belonging to diverse biological pathways. ATAC-seq analysis upon their knockouts revealed their distinct and specific effects on chromatin accessibility. Remarkably, we found that TFDP1, a transcription factor, modulates global chromatin accessibility through transcriptional regulation of canonical histones. In addition, our findings highlight the manipulation of chromatin accessibility as an approach to enhance various cell engineering applications, including genome editing and induced pluripotent stem cell reprogramming.

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Fig. 1: Optimization of ATAC-see assay.
Fig. 2: CRISPR screening for identifying effectors of chromatin accessibility.
Fig. 3: ATAC-seq profiling on knockout of effector genes in eHAP cells.
Fig. 4: The TFDP1/E2F heterodimer plays a role in regulating chromatin accessibility.
Fig. 5: TFDP1 modulates global chromatin accessibility through direct regulation of histones.
Fig. 6: TFDP1 depletion boosts DNA-related biological applications.

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Data availability

The RNA-seq, ChIP–seq, ATAC-seq, MNase-seq and CRISPR-screening datasets in this research are available from the NCBI GEO repository: GSE144454. Data used in, but not generated in, this study include ChIP–seq datasets: GSE72800 (POLR2A), GSE94992 (SMC1A and CTCF), GSE108390 (H3K36me3, H3K4me1, H3K9me3 and H2AK119ub), PRJEB8671 (TFDP1 in LoVo cells), GSE105217 (TFDP1 in K562 cells), GSE80661 (TFDP1 in U266 cells) and GSE80661 (TFDP1 in MM1.S cells). Source data are provided with this paper.

Code availability

The publicly available softwares used are indicated in the Reporting Summary. The custom codes to analyze ATAC-seq data in Fig. 3 and Extended Data Fig. 7 are available in GitHub (https://github.com/Park-Sung-Joon/ATACprofWS, https://doi.org/10.5281/zenodo.10417228)76.

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Acknowledgements

We appreciate all members of Miyanari lab for their kind discussion, M. Hirabayashi and T. Kobayashi (National Institute for Physiological Sciences) for help with FACS analysis, T. Hiyama and M. Noda (National Institute for Basic Biology, NIBB) for providing HEK293T cells, T. Nishiyama (Nagoya University) and T. Kuraishi (Kanazawa University) for providing S2 cells, E. V. Sheval (Moscow State University) for providing pEGFP–H2B (21–35), K. Yamaguchi, A. Akita, S. Shigenobu and M. Matsumoto (NIBB) for technical support for deep sequencing, K. Naruse and A. Kato (NIBB) for Sanger sequencing, M. E. Torres-Padilla (Helmholtz Munich), T. Ishiuchi (Yamanashi University), M. A. Koyanagi, T. Aoi (Kobe University) and K. Nishimura (Tsukuba University) for comments on the manuscript, the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley, NYU Genome Technology Center for their deep sequencing service, NIBB Data Integration and Analysis Facility, the NIG supercomputer at ROIS National Institute of Genetics for the computational resources, and NIBB Bioimaging Facility for their imaging supports. S.I. and T.K. are supported by JSPS Grant-in-Aid for Young Scientists. S.-J.P. is supported by JSPS KAKENHI (18H04710 and 20H05940). Y.O. is supported by JSPS KAKENHI (JP23H00372, JP22H04676 and JP22K19275), AMED BINDS (JP22ama121017j0001), the Medical Research Center Initiative for High Depth Omics and the Cooperative Research Project Program of the Medical Institute of Bioregulation at Kyushu University.　Y.M. is supported by JSPS KAKENHI (16H06279 (PAGS), 22H04925 (PAGS), 18H04722, 22H04688 and 21H04765), World Premier International Research Center Initiative (WPI, MEXT), Okazaki Orion Project, Astellas Foundation, JST/PRESTO, Takeda Science Foundation, the Cell Science Research Foundation, Mochida Memorial Foundation for Medical and Pharmaceutical Research, the Naito Foundation, the Cannon Foundation and Ohsumi Frontier Science Foundation.

Author information

Authors and Affiliations

  1. The Graduate University for Advanced Studies, SOKENDAI, Okazaki, Japan

    Satoko Ishii

  2. National Institute for Basic Biology, National Institutes of Natural Sciences, Okazaki, Japan

    Satoko Ishii & Chiaki Sanbo

  3. Exploratory Research Center on Life and Living Systems, National Institutes of Natural Sciences, Okazaki, Japan

    Satoko Ishii & Chiaki Sanbo

  4. Graduate School of Frontier Biosciences, Osaka University, Suita, Japan

    Taishi Kakizuka

  5. Human Genome Center, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

    Sung-Joon Park & Kenta Nakai

  6. WPI Nano Life Science Institute, Kanazawa University, Kanazawa, Japan

    Ayako Tagawa & Yusuke Miyanari

  7. Research Center for Integrative Evolutionary Science, SOKENDAI, Hayama, Japan

    Hideyuki Tanabe

  8. Division of Transcriptomics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan

    Yasuyuki Ohkawa

  9. TOKIWA-Bio Inc., Tsukuba, Japan

    Mahito Nakanishi

  10. Cancer Research Institute, Kanazawa University, Kanazawa, Japan

    Yusuke Miyanari

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Contributions

S.I. and Y.M. designed and performed experiments and wrote the paper. T.K. set up the optimized ATAC-see. A.T. supported cell sorting. C.S. performed the construction of plasmids. S.-J.P. and Y.M. conducted bioinformatic analyses. K.N. supported the bioinformatic analyses. H.T. provided a resource and technical advice for DNA FISH analysis. Y.O. contributed to the initial setup for deep sequencing. M.N. provided a resource for iPS cell reprogramming.

Corresponding author

Correspondence to Yusuke Miyanari.

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Competing interests

M.N. is the founder and Chief Technology Officer of Tokiwa-Bio, Inc. The other authors declare no competing interests.

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Nature Genetics thanks Andrey Krokhotin and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Characterization of optimized ATAC-see.

a, Mouse genomic DNA (gDNA) were tagmented using Tn5 transposome with Cy3-adaptor DNAs (ME19 or MEA34) with or without MgCl2. Agarose gel images for ethidium bromide (EtBr) and Cy3 channels are displayed. b, Representative images of NIH3T3 cells stained for ATAC-see, DAPI and H3K9me3 under the non-crosslinked condition. scale bar, 2 μm. c, Representative images of NIH3T3 cells stained for ATAC-see and DAPI under indicated crosslink conditions (the left panels). Green dashed circles, mitotic cells. scale bars, 5 μm. d, A bar plot shows intensity of ATAC-see on cells at metaphase relative to that at G2 phase. Data are presented as mean values +/− SD. n, number of cells analyzed. Statistics: a two-tailed unpaired Student’s t-test. e, Representative images of female mouse embryonic fibroblasts (MEFs) stained for ATAC-see, H3K27me3, DAPI, and X chromosome (Chr X, DNA-FISH) without cell-crosslinking (the top panels). Xi and Xa indicate inactive and active Chr X, respectively. Relative fluorescent intensities for indicated marks in Chr X territories are displayed at bottom panels. Box plots (the Bottom panels) are shown as Fig. 1c. n, number of cells analyzed. Scale bars, 5 μm. Statistics: a two-tailed unpaired Student’s t-test. f, Images for ATAC-see and H3K27me3 on female MEFs crosslinked with indicated concentration of PFA. Arrowheads and green dashed circles, inactive Chr X. Scale bars, 5 μm. g, Representative images of NIH3T3 cells stained for ATAC-see, DAPI, phospho histone H3 Ser10 (H3S10P) under the non-crosslinked condition (the upper panels, scale bar, 30 µm). Arrows, H3S10P-positive mitotic cells. Lower panels show higher magnification images of indicated cell-cycle stages. Scale bars, 5 μm. h, Scatter plot and histograms representing intensities for DAPI and ATAC-see in cells at i n, number of cells analyzed. Experiments in a, b, c, and g were reproduced three times.

Source data

Extended Data Fig. 2 Validation of optimized ATAC-see by ATAC-seq analysis.

a, Schematic illustrations displaying Tn5 transposome assembled with indicated adaptor DNAs. b, Representative images of NIH3T3 cells analyzed by ATAC-see using indicated transposomes (the left panels, scale bars, 30 μm). n, number of cells analyzed. A boxplot in the right panel shows relative intensity of ATAC-see. Statistics and definition of the box plot are the same as Fig. 1i. Experiments were reproduced three times. c, Representative genome browser tracks of ATAC-seq analysis with mES cells using indicated transposomes. d, A correlation matrix between ATAC-seq using indicated transposomes. A heat map represents clustering of three independent biological replicates for each experimental condition based on read enrichments in merged ATAC-seq peaks. e, Pairwise scatter plots of signals in the merged peaks. Values of correlation coefficients (Pearson) for each comparison are indicated. f, Insert-size distributions of ATAC-seq using indicated adaptor DNAs. g, Metagene profiles for read enrichments around the transcriptional start sites (TSS). h, Signal extraction scaling (SES) computed by using deeptools plotFingerprint represents cumulative read coverages.

Source data

Extended Data Fig. 3 CRISPR-mediated depletion of chromatin related genes.

eHAP cells expressing Cas9 were transfected with a plasmid encoding two pairs of sgRNAs against indicated genes and analyzed by either western blotting (a) or immunostaining (b) on day 4-5 post-transfection. a, Specific bands using indicated antibodies are shown with arrowheads. Size markers (kDa) are indicated in left side of each panel. These experiments were reproduced twice. b, Immunofluorescence images with indicated antibodies (gray) and DNA staining (green). scale bars, 30 μm. c, Knockout efficiency calculated based on immunostaining data. n; number of cells analyzed. Scale bars, 10 μm.

Source data

Extended Data Fig. 4 Extended results of the CRISPR screening.

a, A gating strategy for flow cytometry to collect cells used for the CRISPR screening. Cells stained for ATAC-see, DAPI, and phospho Histone H3 were analyzed for a DAPI channel to gate cells at G2/M phase. The cells were subsequently gated out based on the signals of phospho Histone H3 to eliminate the mitotic fraction. A cell population displaying the highest or lowest 5% of ATAC-see signals was collected, respectively. Immunostaining images for phospho H3 and DAPI before and after the cell-sorting (the bottom panels) confirm the high purity of G2 phase. Scale bars, 15 μm. b, Pearson correlations of CRISPR screen data between biological replicates from the high and low fractions. Data from day 5 or day 7 after transduction were analyzed by MAGeCK-VISPR. c, The beta score and rank of genes analyzed in Fig. 1i. Only the data from day 7 is shown here. d, A volcano plot for gene hits from the screening result at day 5. Data is shown as in Fig. 2d (the left panel). Lists of chromatin regulating enzymes highlighted in the plot (the right panel). Genes are included in these categories only in the case where they are expressed in eHAP cells (TPM ≧10) and corresponding sgRNAs are not depleted at day 7.

Extended Data Fig. 5 Validation of screen hits.

a, ATAC-see analysis with indicated cell lines as shown in Fig. 2c. b, Distribution of essential scores and proportion of the essential genes (the left and right panels) for all genes and indicated screen hits. c, ATAC-see analysis measured by FACS (the left panel), annexin V staining (the middle panel), and bright field images with 30 µm scale bars (the right panel) under indicated conditions. Ctrl: control, H2O2: Hydrogen peroxide, Rap: Raptinal, Stau: Staurosporine. d, eHAP cells were cultured with or without QVQ-OPh during the knockout, and subsequently assayed for ATAC-see and survival rates (the left and right panels). e, ATAC-see analysis measured by FACS on knockouts for indicated non-essential and essential genes selected from the non-hit group. Essential score for each gene is displayed at bottom. All the samples showed statistically non-significant (P > 0.05). f, eHAP or HeLa cells were treated with 1 µM SAHA, for 8 hrs and subjected to ATAC-see measured by FACS (the top panel) and western blotting with indicated antibodies (the bottom panel). g, Representative genome browser tracks of spike-in calibrated ATAC-seq on control and SMARCA4 knockout eHAP cells. h, Relative mean ATAC-seq signals around merged ATAC-seq peaks between control and SMARCA4 KO. i, The left panel, the total number of ATAC-seq peaks detected in indicated knockouts. The right panels, schematic illustration showing classification of differentially accessible regions (DARs, top) and the number of DARs in SMARCA4 knockout eHAP cells (bottom). j, Gene enrichment analyses with gene hits. Statistics, hypergeometric test computed using g:Profiler. n in a, c-f indicates number of cells analysed. x in c and d indicates number of independent experiments. Statistics in the right panels of c and d: a two-tailed unpaired Student’s t-test. Statistics and the definition of box plots for ATAC-see assays in a, c-f, and i are the same as Fig. 1i. Data in the right panels of c and d are presented as mean values +/− SD. *: P < 0.05.

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Extended Data Fig. 6 Validation of ATAC-seq analyses for knockout of the selected screen hits.

a, Knockouts of indicated genes (colored in red) were confirmed by western blotting. Arrowheads indicate specific bands and asterisks indicate non-specific bands. These experiments were reproduced twice. b, Results of CRISPR screen for the selected screen hits at day 5 and day 7. Left panels for each day show log2 fold changes of 4 sgRNAs designed to the indicated positive (blue) and negative regulators (red) analyzed by ATAC-seq in Fig. 3. Volcano plots for these genes are shown as Fig. 2d (the right panels). c, Schematic illustrations displaying the quantitative ATAC-seq with spike-in control. d, Numbers of total ATAC-seq peaks in the indicated knockout conditions. e, Pearson correlation between two biological replicates. Normalized read enrichments within TSS proximal regions (TSS + /− 1 kb, 22987 RefSeq genes) were analyzed. f, Pairwise Pearson correlation of ATAC-seq samples based on normalized read enrichments in the consensus peak set (n = 256,617). g, PCA clustering analysis of ATAC-seq data. h, ATAC-qPCR analysis for TFDP1 KO (the left panels) and HNRNPU KOs (the right panels) to confirm differential accessibility at DARs identified by ATAC-seq analysis. Genome browser tracks for ATAC-seq of control and the indicated knockout cells at corresponding DARs are shown on the right side. The DAR categories are shown at the bottom of corresponding browser tracks. Statistics: a one-side unpaired Student’s t-test. *: P < 0.05. NS, not significant. n, independent experiments, Data of ATAC-qPCR are presented as mean values +/− SD.

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Extended Data Fig. 7 Extended ATAC-seq analyses for DARs of each knockout.

a, Enrichments of genomic elements (upper half) and chromatin signatures (lower half) at DARs categorized as Fig. 3a in indicated knockouts. b, Proportion of genomic features in conserved-up and conserved-down DARs in indicated knockouts. c, Enrichments of transcription factor motifs at the opened and closed DARs in indicated knockouts. d-f, Representative genome browser tracks of ATAC-seq analysis of the control and the indicated knockouts. The DARs are highlighted in grey. The GC contents of the indicated regions are shown in a violin plot (the bottom of d). Red bars show the medians. The box plots are shown as Fig. 1c. Aggregation plots at the bottom of e and f indicate normalized density of insertion sites at the indicated genomic sites. g, Box plots show proportion of GC content of indicated DARs in each knockout. The width of the boxes indicates a relative number of DARs. The box plots are shown as Fig. 1c. n, number of DARs. h, Motif enrichment analysis of the DARs. Each dot represents significantly enriched motif. x-axis, logFC of ATAC-seq signals at the DARs associated with enriched motif. y-axis, significance of motif enrichment. Motifs with similar sequences are marked with dashed circles.

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Extended Data Fig. 8 Characterization of TFDP1 knockout cells.

a, Growth curves of eHAP cells transfected with either non-targeting (control) or TFDP1 sgRNAs. b, Western blotting with the indicated cell lines transfected with sgRNA (for eHAP, HeLa and 293 T cells) and siRNA (for TIG3, NIH3T3, and mES cells) against non-targeting control (Ctrl), TFDP1 (TFDP1-depletion) or TFDP2 (TFDP2-depletion). Arrowheads indicate specific bands and asterisks indicate non-specific bands. These experiments were reproduced twice. c, Growth curves of eHAP cells transfected with either non-targeting (control) or both TFDP1 and TFDP2 sgRNAs (the left panel). Relative cell number of mES cells 4 days after the transfection with indicated siRNA (the right panel). Statistics: a two-tailed unpaired Student’s t-test. NS: non-significant. d, ChIP-qPCR analysis for the putative E2F4 binding sites of indicated gene loci. eHAP cells were cultured with or without 5 µM HLM006474. (the left panel). Growth curves of eHAP cells treated with the indicated concentration of HLM006474 (the right panel). Statistics: a two-tailed unpaired Student’s t-test. e, Averaged nucleosome occupancy of the control and TFDP1 KO at positions across 300% of coding regions (the left panels) or within 2 kb of TSS of all genes, high-, low-, and non-expressed genes (the right panels). The expression status (High, Low, or no expression) is categorized based on transcripts per kilobase million (TPM). f, Normalized MNase-seq read counts of distances between neighboring nucleosomes (the top panel). Linear fits to the phase peaks calculated from the data in the top are shown with resulting NRLs (the bottom panel). g, Gene enrichment analysis on RNA-seq data for differentially expressed genes upon TFDP1 KO. P-values were computed by representation analysis using Metascape. h, A MA plot for the comparison between TFDP1 KO and control cells with gene spots for up- (red spots), down-regulated genes (blue spots), screen hit genes (green spots), and TFDP1 (an orange spot). Data in a, c-d are presented as mean values +/− SD. n in a, c and d, independent experiments.

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Extended Data Fig. 9 Characterizations of FLAG or mAID knock-in TFDP1 eHAP cells.

a, Schematic diagram showing the generation of FLAG-TFDP1 knock-in eHAP cells. The targeting cassette was knocked-in to TFDP1 locus by CRISPR-Cas9 mediated homologous recombination. Representative clone was further transfected with a plasmid expressing Cre recombinase to flip out Puromycin resistance cassette, resulting in FLAG-TFDP1 knock-in cells. b, Western blotting for indicated proteins in wild-type and FLAG-TFDP1 eHAP cells. c, TFDP1 knockout eHAP cells (TFDP1 KO) were transfected with either an empty or FLAG-TFDP1 expressing plasmid. 3 days after the transfection, cells were analyzed by ATAC-see (the top panel). The statistical test and definition of the box plot are the same as in Fig. 1i. *: P < 0.05. Western blotting for TFDP1 and α-Tubulin are shown in the bottom panels. d, Top 5 transcription factor-binding motifs enriched in TFDP1 binding sites. Statistics, Hypergeometric test computed using Pscan-ChIP. e, Heat maps of the indicated chromatin profiles aligned at ± 5,000 bp of TSSs in 22,987 individual RefSeq genes, sorted by TFDP1 ChIP-seq enrichment levels. Expression levels of corresponding genes analysed by RNA-seq are shown in the right side. f, Pearson correlation between enrichments of indicated factors on ATAC-seq peaks. g, Schematic diagram representing the generation of mAID-TFDP1 knock-in cells (the left panel). Acute depletion of TFDP1 upon adding 5Ph-IAA for 6 hrs was confirmed by western blotting (the right panel). Experiments in b and g were reproduced three times.

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Extended Data Fig. 10 TFDP1 transcriptionally regulates canonical histones.

a, Relative expression levels of canonical histones calculated from total RNA-seq of wild-type and TFDP1 KO eHAP cells. Only expressed histone genes (LogCPM>2) are shown here. b, Quantitative fluorescent western blotting analyses (left) and CBB staining quantification (right) in either whole cell extracts or chromatin fractions of wild-type and TFDP1 KO eHAP cells. Amount of genomic DNA was used as an internal control for calibrating sample variations. Statistical comparison; a two-side unpaired Student’s t-test. *: P < 0.05. n, independent experiments. Data are presented as mean values +/− SD. c, Relative expression levels of mRNA of the indicated non-canonical histone variants in control and TFDP1 KO cells. Statistics: a two-tailed unpaired Student’s t-test. NS: non-significant. *: P < 0.05. n, independent experiments. Data are presented as mean values +/− SD. d, Genome browser track of TFDP1 ChIP-seq at a histone cluster HIST1 locus in the indicated human cell lines. e, Proportion of the indicated canonical histone gene loci occupied by TFDP1. The total numbers of analyzed genes for each canonical histone are shown in the right side.

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Supplementary information

Supplementary Information

Supplementary Tables 1–3, a list of source data, Notes 1–3, Extended Data figure captions, Methods and References.

Reporting Summary

Peer Review File

Supplementary Table 1

Materials used in this study, including oligonucleotides, adapter DNAs, sgRNAs, viruses, siRNAs, plasmid DNAs, antibodies and cell lines.

Supplementary Table 2

Results of CRISPR screening.

Supplementary Table 3

Results of RNA-seq on TFDP1 knockout.

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Ishii, S., Kakizuka, T., Park, SJ. et al. Genome-wide ATAC-see screening identifies TFDP1 as a modulator of global chromatin accessibility. Nat Genet 56, 473–482 (2024). https://doi.org/10.1038/s41588-024-01658-1

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