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Mitotic bookmarking by SWI/SNF subunits

Nature volume 618, pages 180–187 (2023)Cite this article

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Abstract

For cells to initiate and sustain a differentiated state, it is necessary that a ‘memory’ of this state is transmitted through mitosis to the daughter cells1,2,3. Mammalian switch/sucrose non-fermentable (SWI/SNF) complexes (also known as Brg1/Brg-associated factors, or BAF) control cell identity by modulating chromatin architecture to regulate gene expression4,5,6,7, but whether they participate in cell fate memory is unclear. Here we provide evidence that subunits of SWI/SNF act as mitotic bookmarks to safeguard cell identity during cell division. The SWI/SNF core subunits SMARCE1 and SMARCB1 are displaced from enhancers but are bound to promoters during mitosis, and we show that this binding is required for appropriate reactivation of bound genes after mitotic exit. Ablation of SMARCE1 during a single mitosis in mouse embryonic stem cells is sufficient to disrupt gene expression, impair the occupancy of several established bookmarks at a subset of their targets and cause aberrant neural differentiation. Thus, SWI/SNF subunit SMARCE1 has a mitotic bookmarking role and is essential for heritable epigenetic fidelity during transcriptional reprogramming.

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Fig. 1: Select SWI/SNF subunits bound on mitotic chromatin.
Fig. 2: SWI/SNF subunits preferentially bind at promoters in mitosis.
Fig. 3: SMARCE1 reactivates bookmarked genes.
Fig. 4: Mitotic SMARCE1 and neural differentiation.

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Data availability

The ChIP–seq, Cut&Run, nascent RNA-seq, RNA-seq and ATAC–seq data supporting the findings of this study have been deposited at the GEO database under accession number GSE189563. Source data are provided with this paper.

Code availability

The code for analysing the data has been deposited at GitHub (https://github.com/xiaolongchen0627/SWISNF.mitotic.bookmarking.code).

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Acknowledgements

We thank all of the members of the Roberts laboratory for discussions and G. Riddihough for help with the manuscript; the staff at the Hartwell Center Core for sequencing; the staff at St Jude Center for Applied Bioinformatics (CAB) for help with alignment of RNA-seq, ChIP–seq and CUT&RUN–seq reads; the members of the St Jude Flow Cytometry and Cell Sorting Core for cell sorting. The St Jude core facilities are supported by National Cancer Institute Cancer Center Support Grant (NCI CCSG 2 P30 CA021765) and by American Lebanese Syrian Associated Charities (ALSAC) of St Jude Children’s Research Hospital. The Center for Applied Bioinformatics is supported by the National Cancer Institute, Cancer Center Support Grant P30 CA21765 and ALSAC. This study was supported by the National Cancer Institute (NCI) R01 CA113794 and R01 CA172152 to C.W.M.R.; National Cancer Institute Cancer Center support grant (NCI CCSG 2 P30 CA021765), ALSAC of St Jude Children’s Research Hospital, CURE AT/RT Now to C.W.M.R.; the Garrett B. Smith Foundation to C.W.M.R.; St Jude Children’s Research Hospital Collaborative Research Consortium on Chromatin Regulation in Pediatric Cancer to C.W.M.R.; R01 CA216391 to J.Z.; and National Institutes of Health (NIH) grant R01-AI123322 to D.R.G. S.R.-J. is supported by the St Jude Graduate School of Biomedical Sciences and the Ruth L. Kirschstein National Research Service Award (F31 CA261150). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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  1. These authors contributed equally: Zhexin Zhu, Xiaolong Chen

Authors and Affiliations

  1. Division of Molecular Oncology, Department of Oncology, St Jude Children’s Research Hospital, Memphis, TN, USA

    Zhexin Zhu, Trishabelle Manzano, Patrick J. Walsh, Kendall M. Wills, Rebecca Halliburton, Sandi Radko-Juettner, Raymond D. Carter, Janet F. Partridge & Charles W. M. Roberts

  2. Department of Computational Biology, St Jude Children’s Research Hospital, Memphis, TN, USA

    Xiaolong Chen & Jinghui Zhang

  3. Department of Immunology, St Jude Children’s Research Hospital, Memphis, TN, USA

    Ao Guo & Douglas R. Green

  4. St Jude Graduate School of Biomedical Sciences, St Jude Children’s Research Hospital, Memphis, TN, USA

    Sandi Radko-Juettner

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Contributions

Z.Z. conceived the project, designed and performed most experiments, interpreted results and co-wrote the manuscript. X.C. analysed all of the sequencing data, interpreted results with Z.Z. and co-wrote the manuscript. Z.Z. and P.J.W. initiated and developed the project. A.G. performed flow cytometry, immunohistochemistry and sequencing sample preparation. Z.Z. and T.M. derived the Smarce1-AID cell lines. K.M.W. performed density sedimentation gradients. T.M., R.D.C., R.H. and S.R.-J. performed cell synchronization and western blotting. J.F.P., S.R.-J. and R.H. commented on and edited the manuscript with input from all of the authors. C.W.M.R., J.Z. and D.R.G. conceived the project, supervised experimental designs, interpreted results and co-wrote the manuscript.

Corresponding authors

Correspondence to Zhexin Zhu or Charles W. M. Roberts.

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C.W.M.R. is a scientific advisory board member of Exo Therapeutics, unrelated to this manuscript.

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Extended data figures and tables

Extended Data Fig. 1 Cell cycle analysis and subcellular SWI/SNF interactions in asynchronous and mitotic mouse ES cells.

a, Representative flow cytometry analysis showing DNA contents of asynchronous mouse ES cells and cells synchronized at S, G2, and mitotic phase. b, Schematic for extraction of chromatin fraction from asynchronous mouse ES cells and populations synchronized at S, G2, and mitosis. c, Representative flow cytometry showing the distribution of phosphorylated Serine 10 of histone H3 between asynchronous and synchronized mitotic mouse ES cells. d, Representative flow cytometry analysis showing DNA contents and purity of synchronized mitotic mouse ES cells. e, Representative DAPI staining confirming the high purity of synchronized mitotic cells. Scale bar: 10 μm. f, Schematic showing the isolation of cytoplasm, soluble chromatin, and chromatin fractions from mouse ES cells. g, Immunoprecipitation (IP) of SMARCA4 and SMARCB1 in whole cell lysate from asynchronous (top) and mitotic (bottom) mouse ES cells. h, IP of SMARCB1 and SMARCA4 in the cytoplasmic+ soluble chromatin fractions (top), and chromatin fraction (bottom) isolated from asynchronous and mitotic mouse ES cells. i, Immunoblots in whole cell lysate of asynchronous (lane 1) and mitotic (other lanes) mouse ES cells treated with TBB (CK2 inhibitor) and PD0325901 (ERK1 inhibitor) for 6 h, as indicated. Samples were separated either in NuPAGETM 4 to 12% Bis-Tris gels or in the case of the labelled SMARCA4 sample in Phos-tagTM (50 μmol/L) precast gels. j, Immunoblot in whole cell lysate of asynchronous (lane 1) or mitotic (other lanes) mouse ES cells. Lysates were treated with increasing doses of lambda protein phosphatase as indicated. Samples were separated in NuPAGETM 4 to 12% Bis-Tris gels or in the case of the labelled SMARCA4 sample in Phos-tagTM (50 μmol/L) precast gels. k, IP of SMARCB1 and SMARCA4 in the lambda protein phosphatase treated cytoplasmic+ soluble chromatin fractions (top), and chromatin fraction (bottom) isolated from asynchronous and mitotic mouse ES cells. Data are representative of two (a,c,d,e,h,i,j,k) and three (g) independent biological experiments.

Extended Data Fig. 2 Generation and live-cell imaging of endogenous C-terminal EGFP-tagged SOX2, SMARCA4, SMARCB1, and SMARCE1 mouse ES cells.

a, Images showing mouse ES cells stably expressing H2B-mCherry. b,c,d,e, PCR-based genotyping assay validating the generation of the Sox2-Egfp (b), Smarca4-Egfp (c), Smarcb1-Egfp (d), and Smarce1-Egfp (e) homozygous knock-in mouse ES cells. f,g, Western blot validating SOX2-EGFP and SMARCA4-EGFP (f), and SMARCB1-EGFP and SMARCE1-EGFP (g) protein expression, with H3 or GAPDH as loading control. h,i,j, Glycerol gradient and immunoblot performed on H2B-mCherry parental cells and Smarca4-Egfp (h), Smarcb1-Egfp (i), and Smarce1-Egfp (j) clones of mouse ES cells which were used for live-cell imaging. k, Representative live-cell imaging of asynchronous mouse ES cells stably expressing H2B-mCherry with endogenously tagged EGFP at the C-terminal of SMARCA4, SMARCB1, and SMARCE1, respectively. Scale bar: 10 μm. Data are representative of single experiments that assess several independent clones for genotype (b,c,d,e,f,g) and live-cell imaging (k). Data are representative of two independent biological replicates (h,i,j).

Extended Data Fig. 3 Binding of SOX2, ESRRB and SWI/SNF subunits in asynchronous and mitotic cells.

a, (top) Average binding profile of DSG+ FA cross-linked ChIP-seq signal at the indicated binding regions (±3000-bp peak summit) identified in asynchronous (asyn) and mitotic (mit) AB2.2 and E14TG2a (as indicated) mouse ES cells. (bottom) Scatter plots of DSG+ FA ChIP-seq signal in reads per million at the designated regions (±250-bp peak summit) for asynchronous and mitotic mouse ES cells. Linear regression of RPM value of asynchronous cells divided by the value for mitotic cells were estimated and regression slopes are shown in red. Grey dashed lines indicate the random background. b, Venn diagrams showing the overlap of DSG+ FA ChIP-seq peaks in asynchronous (blue) and mitotic (red) AB2.2 and E14TG2a mouse ES cells as indicated. c, SOX2: POU5F1 co-binding motifs and SOX2 binding motifs identified from SOX2 DSG+ FA ChIP-seq in asynchronous and mitotic AB2.2 and E14TG2a mouse ES cells as indicated. P-value was calculated by two-sided Fisher’s exact test. d, (top) Average binding profile of Cut&Run signal at SOX2 binding regions (±3000-bp peak summit) identified in asynchronous and mitotic AB2.2 or E14TG2a cells. (bottom) Scatter plots of Cut&Run signal in reads per million at the designated regions (±500-bp peak summit) for asynchronous and mitotic mouse ES cells. Linear regression of RPM value of asynchronous cells divided by the value for mitotic cells were estimated and regression slopes are shown in red. Grey dashed lines indicate the random background. e, Venn diagrams showing the overlap of Cut&Run peaks of designated factors in asynchronous (blue) and mitotic (red) in AB2.2 or E14TG2a mouse ES cells as indicated. f, Venn diagram showing the overlaps of SOX2 peaks (left) and ESRRB peaks (right) identified from Cut&Run (blue) and DSG+ FA ChIP-seq (red) in asynchronous mouse ES cells. g, SOX2: POU5F1 co-binding motifs and SOX2 binding motifs identified from SOX2 Cut&Run in asynchronous and mitotic AB2.2 and E14TG2a mouse ES cells as indicated. P-value was calculated by two-sided Fisher’s exact test. h, SOX2: POU5F1 co-binding motifs and SOX2 binding motifs identified from SOX2 Cut&Run peaks subtracting SOX2 DSG+ FA ChIP-seq peaks in mitotic AB2.2 and E14TG2a cells as indicated. P-value was calculated by two-sided Fisher’s exact test. i, Violin plots depicting the FIMO-called best motif score per SOX2 or ESRRB peak in sites losing binding in mitosis (lost) or retaining binding (retained), performed by Cut&Run in the indicated mouse ES cell lines. j, Cut&Run-qPCR validating the lost (asyn-specific), bookmarked (asyn- and mit-shared), and gained (mit-specific) SMARCE1 peaks in asynchronous and mitotic mouse ES cells. k, Venn diagram showing the overlap of SMARCA4, SMARCB1, and SMARCE1 Cut&Run peaks in asynchronous cells (top) and the overlap between SMARCE1 and SMARCB1 Cut&Run peaks in mitotic mouse ES cells (bottom). l, SMARCE1 (left) and SMARCB1 (right) peak length for asyn-specific, mit-specific, and asyn- and mit-shared peaks in asynchronous and mitotic mouse ES cells. m, Average SMARCE1 (left) and SMARCB1 (right) binding profiles at asyn-specific, mit-specific, and asyn- and mit-shared regions. n, To confirm localization of SMARCE1 and SMARCB1 as detected by Cut&Run, ChIP-seq was utilized. The distributions of SMARCE1 (left) and SMARCB1 (right) via ChIP-seq (using DSG+ FA fixation) at promoter and enhancer regions in asynchronous and mitotic mouse ES cells. o, The distributions of ESRRB peaks from DSG+ FA ChIP-seq and Cut&Run in asynchronous and mitotic AB2.2 mouse ES cells. Bookmarked ESRRB and CTCF peaks are as previously reported. All data are compiled from two biological replicates. Statistical analysis was performed using two-sided Fisher’s exact test (c,g,h). Center lines denote medians; box limits 25th-75th percentile; whiskers 5th-95th percentile (i,l).

Source Data

Extended Data Fig. 4 The comprehensive profiles of SMARCE1 and SMARCB1 bindings in asynchronous and mitotic ES cells.

a, The distributions of asyn-specific, mit-specific, asyn- and mit-shared SMARCE1 (left) and SMARCB1 (right) peaks identified from Cut&Run at enhancer and promoter regions in asynchronous and mitotic AB2.2 cells. b, Relative enrichment or depletion of the lost (asynchronous-specific), retained (gained+ bookmarked), gained (mitosis-specific), and bookmarked (present in both asynchronous and mitosis) peaks for ESRRB, SOX2, SMARCB1 and SMARCE1 Cut&Run at the lost, retained, bookmarked, or coating regions of published ATAC signal and indicated factors. Colour indicates ratio of observed (Obs) to expected (Exp) frequency, and p-value (two-sided Fisher’s exact test) is indicated if significant (p < 0.01, 0.0e00 means p-value < e−350). Comparison using <100 overlapping peaks are denoted with a hash mark (#). c, Average binding profile of Cut&Run and DSG+FA ChIP-seq signal at the indicated binding regions (±3000-bp peak summit) identified in asynchronous and mitotic mouse ES cells. Average binding profiles represent reads per million (RPM); the y-axis is scaled by median asynchronous binding. d, Relative enrichment or depletion of the lost (asynchronous-specific), retained (gained+ bookmarked), gained (mitosis-specific), and bookmarked (present in both asynchronous and mitosis) peaks for ESRRB, SOX2, SMARCB1 and SMARCE1 Cut&Run at early, middle, late, and transient reactivated enhancer transcriptional regulatory elements (eTREs), super enhancers and promoters. Colour indicates ratio of observed (Obs) to expected (Exp) frequency, and p-value (two-sided Fisher’s exact test) is indicated if significant (p < 0.01, 0.0e00 means p-value < e−350). Comparison using <100 overlapping peaks are denoted with a hash mark (#). e, Boxplot showing the relative expression levels in asynchronous cells of genes based upon the indicating binding status of SMARCE1. Center lines denote medians; box limits 25th-75th percentile; whiskers 5th-95th percentile. Data are compiled from two (a,b,c,d) and eight (e) biological replicates. Statistical analysis was performed using two-tailed unpaired Student’s t-Test, *** p-value < 0.001 (e).

Extended Data Fig. 5 Generation of endogenous MD and MD (R42A) degron fused SMARCE1 mouse ES cells.

a, Schematic showing the strategy for MD and MD (R42A) knock-in at the C-terminal end of SMARCE1 in mouse ES cells by CRISPR-Cas9. b,c, PCR-based genotyping assay validating the generation of Smarce1-MD (b) and Smarce1-MD (R42A) (c) mouse ES cell lines. d, Sanger-sequencing validating the mutant MD degron (R42A: AGA to GCA) and the wild-type MD degron (R: AGA) sequences in Smarce1-MD(R42A) #A04, #A10 and Smarce1-MD#09, #30 mouse ES cells, respectively. e, Representative immunoblot showing the protein levels of SMARCE1, SMARCB1, SOX2, OCT4 in parental, Smarce1-MD(R42A) #A01, #A02, #A04, #A10, and Smarce1-MD#09, #30, #45, #47 mouse ES cells. GAPDH and H3 were used as loading controls. f, Alkaline phosphatase staining in Smarce1-MD(R42A) and Smarce1-MD mouse ES cells. g, IHC staining of core pluripotency factors (OCT4, NANOG, SOX2, ESRRB) in Smarce1-MD(R42A) and Smarce1-MD mouse ES cells. DAPI was used to indicate nuclei. h, Cell proliferation of Smarce1-MD(R42A) and Smarce1-MD mouse ES cells measuring by BrdU and 7-AAD. i, Statistics of cell proliferation from (h). j, Cell morphologies of Smarce1-MD(R42A) and Smarce1-MD mouse ES cells. Data are representative from one (b,c,d,f,g,) or two biological replicates (e,); or compiled from three biological replicates (h,i). Data are shown as mean ± s.e.m, n = 3 biologically independent experiments. Differences are not statistically significant (n.s.) determined by one-way ANOVA (i). Scale bars: 400 μm (f,g,j).

Source Data

Extended Data Fig. 6 Characterization of the MD degron cells and development of the custom biotin-RNA spike-in for nascent RNA-seq.

a, Schematic for EU-pulse-labelling of newly synthesized transcripts during mitosis and mitotic release in the MD degron system. b, Representative immunoblot measuring SMARCE1 levels in mouse ES cells expressing endogenous SMARCE1-MD(R42A) (left) or SMARCE1-MD (right) in asynchronous cells, mitotic cells and after release from mitotic arrest, with GAPDH as loading control. c, Representative immunoblot measuring SMARCE1 levels in parental AB2.2 mouse ES cells in asynchronous and after release from mitotic arrest, with GAPDH as loading control. d, PCA plot showing RNA-seq data from Smarce1-MD(R42A) and Smarce1-MD mouse ES cells. e, Volcano plot showing differentially expressed genes in Smarce1-MD(R42A) and Smarce1-MD mouse ES cells. P-value in differentially expressed genes test was calculated using Wald test from DESeq2. f, Box plot showing stem cell associated (GO: 0019827) gene expression levels in Smarce1-MD(R42A) and Smarce1-MD mouse ES cells. Center lines denote medians; box limits 25th-75th percentile; whiskers 5th-95th percentile. g, GO analysis of differentially expressed genes in Smarce1-MD(R42A) and Smarce1-MD mouse ES cells. Over-representation test was used to calculated GO term enrichment with FDR for multiple test correction. h, Bar plot showing the expression levels of Sox2 (top) and Bmp4 (bottom) in Smarce1-MD(R42A) and Smarce1-MD mouse ES cells. i, Agarose gel showing the highly purified Chr16 spike-in and ChrX spike-in for nascent RNA-seq experiments. j, Red lines indicating the locations and reads of custom designed nascent RNA spike-ins on chromosome 16 (chr16: 10188072-10188427) (left) and chromosome X (ChrX: 7506352-7506647) (right) in mouse ES RNA-seq, respectively. k, Representative examples of nascent RNA-seq reads indicating high enrichment of custom designed RNA spike-in reads on chromosome 16 (left) and chromosome X (right) in the nascent RNA-seq samples at 90 min after mitotic release. l, Representative SMARCE1 peaks and associated nascent RNA profiles in Smarce1-MD (R42A) and Smarce1-MD cells at 90 min after mitotic release. m, Venn diagram showing the overlap between mitotic SMARCE1 bound genes and down-regulated genes identified at 90 min in Smarce1-MD cells by nascent RNA-seq. Data were processed with ClusterProfiler with default parameters (g). Data are representative of one (i), two (b,c,j,k,l,m) or three (d,e,f,g,h) independent biological replicates. Not significant: n.s., with a two-tailed unpaired Student’s t-Test (f). Data are presented as the mean expression (rlog) ± SD, n = 3 biologically independent experiments; p-value was calculated using two-tailed unpaired Student’s t-Test (h). P-value determined by two-sided Fisher’s exact test is indicated (m).

Source Data

Extended Data Fig. 7 Generation and characterization of endogenous auxin-inducible degradation (AID) degron fused to SMARCE1 in mouse ES cells.

a, PCR-base genotyping assay validating the generation of Smarce1-AID homozygous knock-in mouse ES cell lines (green). b, Representative immunoblot showing the protein levels in the homozygous knock-in clones from (a); clone 13 is a heterozygous one (#13het), clone 7 and clone 85 are off-target edited. c, Representative immunoblot in Smarce1-AID clones #6, #23, and #13het treated with 1 μM 5-Ph-IAA for the indicated time following release from mitotic arrest. GAPDH was used as a loading control. d, Average binding profiles of SMARCE1 Cut&Run signal in asynchronous Smarce1-AID cells (clones #6 and #23) and mitotic cells treated with DMSO or 1 μM 5-Ph-IAA for 1 h. e, Representative immunoblot in mitotic Smarce1-AID cells (clones #6 and #23) treated with DMSO or 1 μM 5-Ph-IAA for 1 h (lanes 1 and 2) and in cells that underwent washout and release from mitotic arrest for the indicated time periods. f, Schematic for EU-pulse-labelling of newly synthesized transcripts and 5-Ph-IAA treatment during mitosis and mitotic release in the AID degron system. g, Representative flow cytometry plot showing DNA contents of asynchronous cells, synchronized mitotic cells, and cells released from mitotic arrest for the indicated time. h, Representative SMARCE1 peaks and associated nascent RNA profiles in DMSO and 1 μM 5-Ph-IAA treated Smarce1-AID (clones 6 and 23) mouse ES cells at 30 min, 60 min and 90 min after mitotic release. i, Violin plot showing the nascent transcriptional levels of genes bound by mitosis-specific (gained) SMARCE1 in asynchronous and mitotic cells and cells released from mitotic arrest in the AID system (left) and in the MD system (right). Center lines denote medians; box limits 25th-75th percentile; whiskers 5th-95th percentile. Data are representative or compiled from one (a) or two biological replicates (b,c,d,e,g,h,i).

Extended Data Fig. 8 The timing and degree of nascent transcription changes in mitotic SMARCE1 depleted mouse ES cells.

a, Cell morphologies of DMSO treated Smarce1-AID cells (clones #6 and #23) and the cells treated with 1 μM 5-Ph-IAA for the indicated days. b, Representative immunoblot of Smarce1-AID cells (clones #6 and #23) treated with DMSO or 1 μM 5-Ph-IAA. c, Bar plot showing the numbers of differentially expressed genes (DEGs) identified from Smarce1-AID cells (clones #6 and #23) treated with 1 μM 5-Ph-IAA for the indicated time. d, (left) Heatmap showing DEGs identified from Smarce1-AID cells (clones #6 and #23) treated with 1 μM 5-Ph-IAA for the indicated time. The DEGs were classified into three clusters by k-means clustering. Colour bar indicates scaled z score of gene expression. (right) GO analysis indicating pathways enriched for the three clusters. Size of the circle represents ratio of observed (Obs) versus expected (Exp) frequency, and p-value was calculated by two-sided Fisher’s exact test. e, Bar plot showing the contributing number of genes identified from down-regulated DEGs (bulk RNA-seq) in asynchronous Smarce1-MD cells (in which SMARCE1 is solely missing in mitotic cells) to the down-regulated genes identified from Smarce1-AID cells treated with 5-Ph-IAA (in which SMARCE1 is missing throughout the cell cycle). f, Bar plot showing the contributing number of genes identified from down-regulated DEGs (bulk RNA-seq) in asynchronous Smarce1-MD cells to the down-regulated genes identified from Smarca4 knockout (KO) mouse ES cells (King HW et al., 2017). g, Bar plot showing the contributing number of genes identified from down-regulated DEGs (bulk RNA-seq) in asynchronous Smarce1-MD cells to the down-regulated genes identified from Brd9 shRNA transfected mouse ES cells (J Gatchalian et al., 2018). h, GO analysis of relative enrichment or depletion of upregulated genes identified from bulk RNA-seq in the scenario of nascent RNA-seq at 240 min after mitotic release and in asynchronous MD cells. Size of circles indicates ratio of observed (Obs) versus expected (Exp) frequency, and colour presents p-value calculated by two-sided Fisher’s exact test. i, The downregulated genes that reached the lowest expression levels in 5-Ph-IAA treated Smarce1-AID mouse ES cells at the indicated time point were defined as “30 min timing” and “60 min timing” genes, respectively; “low expressed” refers to the genes that were consistently lowly expressed (rlog< 4) at all the time points. Genes within the “30 min timing” and “60 min timing” groups from the nascent RNA-seq of 5-Ph-IAA treated Smarce1-AID cells that overlapped with the genes in “90 min timing” group from the nascent transcripts of Smarce1-MD at 90 min are shown in blue. Gene numbers are shown within the bar plot. j, Box plot showing the nascent transcript levels of “30 min timing”, “60 min timing”, and “low expressed” genes at all time points examined in Smarce1-AID cells treated with DMSO or 5-Ph-IAA. k, Heatmap of the DEGs identified from nascent RNA-seq in both the AID system (left) and the MD system (right). Colour bar indicates z scaled rlog value. l, GO analysis of “30 min timing” and “60 min timing” genes. Size of the circle represents ratio of observed (Obs) versus expected (Exp) frequency, and p-value was calculated by two-sided Fisher’s exact test. m, Box plot of “30 min timing”, “60 min timing”, and “low expressed” genes separated into 5 tiers based upon nascent gene expression levels. Box plots depict the median transcriptional activity across the time course; in each cluster, the total number of genes is listed in blue and the number of genes that differ significantly between 5-Ph-IAA and DMSO conditions is listed in yellow. n, Relative enrichment or depletion of the lost, retained, bookmarked, and mitotically bound peaks for chromatin accessibility and the indicated factors at “30 min timing”, “60 min timing”, and “low expressed” genes (y-axis). Colour indicates ratio of observed (Obs) versus expected (Exp) frequency, and p-value (two-sided Fisher’s extract test) is indicated. Comparisons using <100 overlapping peaks are denoted with a hash mark (#). o, (left) Average binding profiles of SMARCA4 Cut&Run signal on SMARCE1 binding sites in Smarce1-MD(R42A) and Smarce1-MD mouse ES cells at 90 min after mitotic release. (right) Heatmaps of SMARCA4 Cut&Run at SMARCE1 binding sites in Smarce1-MD(R42A) and Smarce1-MD mouse ES cells at 90 min after mitotic release. p, Venn diagram showing the overlap between lost SMARCE1 peaks and lost SMARCA4 peaks identified from Smarce1-MD versus Smarce1-MD(R42A) cells at 90 min after mitotic release. Data are compiled from two biological replicates for each clone (Smarce1-AID#06, #23) (c,d,e,f,g,i, j,m). Center lines denote medians; box limits 25th-75th percentile; whiskers 5th-95th percentile (j, m). Statistical analysis was performed using two-sided Fisher exact test (e,f,g). The ratio of observed (Obs) versus expected (Exp) frequency is shown, and p-value determined by two-sided Fisher’s exact test is indicated (p).

Extended Data Fig. 9 Subsequent Activation of Genes Bookmarked by SMARCE1 is ATPase Dependent.

a, (top) Average binding profile of ATAC-seq signal identified in Smarce1-MD (R42A) and Smarce1-MD cells at 90 min following release from mitotic arrest. Cells were treated with either DMSO or 1 μM BRM014 (SMARCA4 ATPase inhibitor). (middle) Heatmaps of ATAC-seq signal. (bottom) Scatter plots of ATAC-seq signal in reads per million in the indicated samples. Dots indicate significant loss (red) or gain (blue) of ATAC-signal. b, Volcano plots showing genes with the significantly decreased nascent RNA transcriptional levels under the treatment of BRM014 versus DMSO in Smarce1-MD(R42A) (left) and Smarce1-MD (right) cells at 90 min after releasing from mitotic arrest. P-value in differentially expressed genes test was calculated using Wald test from DESeq2. c, Venn diagram showing the overlap of decreased ATAC-seq sites affected by ATPase inhibitor BRM014 to those affected by the loss of SMARCE1 in mitosis. d, Scatter plots of SOX2 (left), ESRRB (middle), and EZH2 (right) peak changes between Smarce1-MD (R42A) and Smarce1-MD cells at 90 min after mitotic release. Dots indicate significant binding loss (red) or gain (blue) following mitotic loss of SMARCE1. e, Representative immunoblot of Smarce1-MD (R42A) clones A04 and A10 and Smarce1-MD clones 09 and 30 at 90 min after mitotic release. f, Scatter plots of SOX2 (left), ESRRB (middle), and EZH2 (right) peak changes in mitotic Smarce1-AID mouse ES cells (clones 06 and 23) treated with DMSO or 5-Ph-IAA for 1 h. Dots indicate significant binding loss (red) or gain (blue) following mitotic loss of SMARCE1. g, Venn diagram showing the overlap between decreased/depleted SOX2 (left)/ESRRB (right) peaks and retained SMARCE1 peaks identified from Smarce1-MD versus Smarce1-MD (R42A) cells at 90 min after mitotic release. h, Overlap of genes with lost/decreased SOX2 peaks or gained EZH2 peaks and genes upregulated or downregulated in Smarce1-MD mouse ES cells. i, (top) Average binding profile of SOX2 Cut&Run signal identified in Smarce1-MD (R42A) and Smarce1-MD cells at 90 min following release from mitotic arrest. Cells were treated with either DMSO or 1 μM BRM014. (middle) Heatmaps of SOX2 signal. (bottom) Scatter plots of SOX2 signal in reads per million in the indicated samples. Dots indicate significant loss (red) or gain (blue) of SOX2 signal. j, Venn diagram showing the overlap of depleted/decreased SOX2 peaks identified from Smarce1-MD (R42A) cells treated with BRM014 versus DMSO and Smarce1-MD versus Smarce1-MD (R42A) cells at 90 min after mitotic release. All data are compiled from two biological replicates. Correction values were obtained from Pearson’s product moment correlation (d,f). The ratio of observed (Obs) versus expected (Exp) frequency is shown, and p-value determined by two-sided Fisher’s exact test is indicated (c,g,j).

Extended Data Fig. 10 Mitotic SMARCE1 is required for the appropriate neural differentiation.

a, Bright field images presenting cell morphologies of Smarce1-MD(R42A) and Smarce1-MD derived neural cells at day six after induction. b,c, (b) IHC staining of DAPI and GFAP (top), SOX1 (middle), and NES (bottom) in Smarce1-MD (R42A) and Smarce1-MD derived neural cells at day six after induction. (c) Statistical analysis of (b) (p value for GFAP= 2.72e−10, p value for SOX1 = 3.01e−11, p value for NES = 4.62e−9). d, GO analysis of differentially expressed genes between Smarce1-MD (R42A) and Smarce1-MD cultures at day six after neural induction. Over-representation test was used to calculated GO term enrichment with FDR for multiple test correction. e, Representative morphologies of embryoid bodies derived from Smarce1-MD(R42A) and Smarce1-MD mouse ES cells at day six. f, GO analysis of differentially expressed genes identified in (e). Over-representation test was used to calculated GO term enrichment with FDR for multiple test correction. g, Schematic for direct neural induction and 5-Ph-IAA treatment during mitosis and mitotic release in the AID degron system. h, Bright field images presenting cell morphologies of DMSO or 5-Ph-AA treated mitotic Smarce1-AID derived cultures at day three after induction. i, Quantitative PCR (qPCR) for neuroectoderm markers in DMSO or 5-Ph-AA treated mitotic Smarce1-AID (clones #6 and #23). j, Representative morphologies of Smarce1-MD(R42A) and Smarce1-MD cultures supplemented with indicated dose of BMP4 at day six after neural induction. k, Heatmap showing the differentially expressed genes in (j). l. GO analysis of (k). Over-representation test was used to calculated GO term enrichment with FDR for multiple test correction. All data are representative and compiled from two independent biological experiments. N = 2 biological replicates for each clone (Smarce1-AID#06, #23) (i). Data are shown as mean ± s.e.m., n = 12 images/sample collected from 2 biologically independent experiments (c). Scale bars: 400 μm (a,b,e,j), 1000 μm (h). P-value was calculated using two-tailed unpaired Student’s t-Test (* p < 0.05, ** p < 0.01, *** p < 0.001) (c,i).

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Supplementary Fig. 1 and Supplementary Table 2.

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Supplementary Table 1

Sample-specific spike-in scalar.

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Zhu, Z., Chen, X., Guo, A. et al. Mitotic bookmarking by SWI/SNF subunits. Nature 618, 180–187 (2023). https://doi.org/10.1038/s41586-023-06085-6

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