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Pluripotency transcription factors and Tet1/2 maintain Brd4-independent stem cell identity

Nature Cell Biology volume 20, pages 565–574 (2018)Cite this article

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Abstract

A robust network of transcription factors and an open chromatin landscape are hallmarks of the naive pluripotent state. Recently, the acetyllysine reader Brd4 has been implicated in stem cell maintenance, but the relative contribution of Brd4 to pluripotency remains unclear. Here, we show that Brd4 is dispensable for self-renewal and pluripotency of embryonic stem cells (ESCs). When maintained in their ground state, ESCs retain transcription factor binding and chromatin accessibility independent of Brd4 function or expression. In metastable ESCs, Brd4 independence can be achieved by increased expression of pluripotency transcription factors, including STAT3, Nanog or Klf4, so long as the DNA methylcytosine oxidases Tet1 and Tet2 are present. These data reveal that Brd4 is not essential for ESC self-renewal. Rather, the levels of pluripotency transcription factor abundance and Tet1/2 function determine the extent to which bromodomain recognition of protein acetylation contributes to the maintenance of gene expression and cell identity.

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Fig. 1: 2i increases acetylation at key pluripotency loci.
Fig. 2: Naive ESCs are resistant to Brd4 inhibitors.
Fig. 3: Brd4 is dispensable in naive ESCs.
Fig. 4: Naive ESCs maintain transcriptional networks in the presence of BET inhibition.
Fig. 5: Increased transcription factor activity provides resistance to BET inhibition in the absence of 2i.
Fig. 6: JQ1 affects Nanog and Tet target genes.
Fig. 7: Tet1/2 are required for resistance to BET inhibitors.

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Acknowledgements

We thank the Thompson and Allis labs for discussion. We thank R. Jaenisch for the induced pluripotent stem cells and A. Smith for the cDNA encoding the chimaeric LIF receptor. L.W.S.F. is the Jack Sorrell Fellow of the Damon Runyon Cancer Research Foundation (DRG-2144-13). B.W.C. received support from the HHMI and the Jane Coffin Childs Memorial Research Fund. D.A.-C. is a recipient of a postdoctoral fellowship from the Ramón Areces Foundation. This work was supported by a grant from the Tri-Institutional Stem Cell Initiative (2014-034 to C.B.T., C.D.A and L.W.S.F.) and the Memorial Sloan Kettering Cancer Center support grant P30 CA008748. C.D.A. acknowledges support from the National Institutes of Health (R01CA204639) and The Rockefeller University.

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  1. These authors contributed equally: Lydia W. S. Finley, Santosha A. Vardhana, and Bryce W. Carey.

Authors and Affiliations

  1. Cell Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Lydia W. S. Finley & Yanyang Chen

  2. Center for Epigenetics Research, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Lydia W. S. Finley, Santosha A. Vardhana, Richard Koche, Yanyang Chen, Bryan King, Megan R. Radler & Craig B. Thompson

  3. Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Santosha A. Vardhana, Direna Alonso-Curbelo, Bryan King, Megan R. Radler, Scott W. Lowe & Craig B. Thompson

  4. Laboratory of Chromatin Biology and Epigenetics, The Rockefeller University, New York, NY, USA

    Bryce W. Carey & C. David Allis

  5. Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medical College, New York, NY, USA

    Duancheng Wen

  6. Ansary Stem Cell Institute and Department of Medicine, Weill Cornell Medical College, New York, NY, USA

    Shahin Rafii

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Contributions

L.W.S.F., S.A.V. and B.W.C. designed, performed and analysed all experiments under the guidance of C.D.A. and C.B.T. R.K. analysed the ChIP–seq and ATAC-seq data. B.K. assisted with the RNA-seq analyses. D.A.-C. and S.W.L. assisted with the generation of Brd4 shRNA and CRISPR-edited cell lines. D.W. and S.R. performed the chimaera experiments. Y.C. and M.R.R. provided technical assistance. L.W.S.F., S.A.V., B.W.C., C.D.A. and C.B.T. wrote the manuscript.

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Correspondence to C. David Allis or Craig B. Thompson.

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Competing interests

C.B.T. is a founder of Agios Pharmaceuticals and a member of its scientific advisory board. He also serves on the board of directors of Merck and Charles River Laboratories.

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Integrated supplementary information

Supplementary Figure 1 Brd4 binding in ESCs.

(a) Density plots of Brd4, H3K9ac and H3K27ac ChIP-seq datasets. Data are centered on Brd4 peaks. Rows represent 10 kb interval around a single Brd4-occupied peak. (b) Scatter plot comparing normalized Brd4 ChIP-seq peak read density in cells cultured in S/L or S/L+2i. 31,132 peaks are shown. Sites changed at least two-fold are colored in red and blue. (c) Gene set enrichment plot showing genes ranked by amount of Brd4 binding. Data are derived from a single ChIP-Seq experiment. P values are calculated based on 1000 permutations by the GSEA algorithm and was not adjusted for multiple comparisons.

Supplementary Figure 2 2i promotes resistance to Brd4 inhibitors.

(a) Volume of ESCs treated for 48 h with DMSO (vehicle) or 500 nM JQ1. (b) Left, population doublings of ESCs cultured for 48 h in indicated doses of JQ1 relative to cells grown in DMSO. Right, volume of cells after 24 h with JQ1. (c) Volume of ESCs grown in S/L or S/L+2i and cultured with 500 nM JQ1. (d,e) Population doublings (d) and volume (e) of ESCs treated for 48 h with 500 nM iBET relative to vehicle (DMSO)-treated controls. (f) Volume of induced pluripotent stem cells (iPSCs) cultured in S/L or S/L+2i with DMSO or 500 nM JQ1. (g) Alkaline phosphatase staining of iPSCs grown in S/L or S/L+2i and treated with 500 nM JQ1 for 48 h. One representative well of a six-well plate is shown. (h) Population doublings of ESCs cultured in indicated medium and 500 nM JQ1. Note that S/L and S/L+2i samples are also shown in Fig. 1a. n=3 independent samples are shown and the connecting line joins the mean values of each time point. (i) GFP intensity of Nanog-GFP reporter ESCs cultured in indicated medium and treated with DMSO or 500 nM JQ1 for 24 h as measured by FACS. Light grey shaded peak represents negative control. Experiment was performed two independent times with similar results. (j) Gating strategy for Nanog-GFP ESCs. Singlets were identified by forward scatter (left) and side scatter (middle). Viable cells were selected based on DAPI exclusion (right). (k) Alkaline phosphatase staining of colonies formed from single ESCs grown in S/L or S/L+2i and treated continuously with DMSO (vehicle) or 500 nM JQ1. Top, representative well of a six-well plate; bottom, close up of boxed region. One representative well of a six-well plate is shown. All bars represent mean ±SD of n=3 independent samples. Statistics calculated by 2-way ANOVA with Sidak’s multiple comparisons post test.

Supplementary Figure 3 Manipulation of Brd4 expression in ESCs.

(a) Western blot depicting clonal ESC lines generated by transfection with Cas9 and sgRNA against a nongenic region of chromosome 8 (ch8) or exon 3 of Brd4. Wild-type Brd4 is indicated with arrowhead; clones with truncated Brd4 are indicated with red asterisk. Top two panels represent samples run on a 6% gel; bottom two panels represent samples run on a 10% gel. Nucleolin is used as a loading control for each set of blots. Unprocessed original scans are shown in Supplementary Fig. 8. (b) Sequencing of Brd4 locus in clonal ESC lines edited with sgRNA against chromosome 8 (ch8) or Brd4 exon 3; the PAM of the Brd4 sgRNA is shown in red. Clone 1 and clone 2 of ch8-edited ESCs have wild-type (WT) Brd4. Brd4-edited clones result in mutation in Brd4 exon 3. Clone 1 has a large rearrangement (shown in blue) that results in insertion of 578 bp from a downstream Brd4 intron and a 149 bp deletion of Brd4 exon 3. The resulting rearrangement results in a stop at codon 32. Clone 2 has a 1 bp insertion (shown in green) that produces a stop at codon 96. Clone 5 has a 6 bp deletion that produces an in-frame deletion of two amino acids in exon 3. Protospacer-adjacent motif (PAM) shown in red. (c) Representative images of clones described in (a,b) grown in S/L+2i. Scale bar, 100 μm. (d) Relative growth of clones. All clones were maintained in S/L+2i and then seeded for growth curve in S/L or S/L+2i. n=3 independent samples were counted daily. Data are presented as population doublings from day 2 to day 3 (thus, after 48h of conversion to S/L) relative to ch8 clone 1 S/L+2i control. Bars, mean ± SD. Statistics calculated by 2-way ANOVA with Sidak’s multiple comparisons post test.

Supplementary Figure 4 2i maintains Brd4-independent Med1 binding and chromatin accessibility at key pluripotency genes.

(a) Heat map illustrating relative Brd4 binding as measured by ChIP-seq in S/L-cultured ESCs treated with vehicle or JQ1. (b-e) Gene set enrichment analyses of genes identified as direct targets of Oct4, Sox2 and Nanog (OSN targets) (b, d) or target genes of pluripotency transcription factors identified by ChIP studies (ChIP targets) (c, e). Genes are ranked as described based on expression determined by RNA-Seq experiments of three replicate samples. For GSEA, P values are calculated based on 1000 permutations by the GSEA algorithm and was not adjusted for multiple comparisons. (f) Scatter plot comparing the effect of JQ1 on 67,053 ATAC-seq peaks. Peaks that are significantly downregulated by JQ1 in S/L but not S/L+2i (blue) or both S/L and S/L+2i (red) cultured ESCs are shown. (g) Scatter plot comparing normalized Med1 ChIP-seq peak read density in cells cultured in S/L or S/L+2i. 3,171 peaks are shown. Sites changed at least two-fold are colored in red and blue. (h, i) Heat maps illustrating relative Med1 binding assessed by ChIP-seq (h) or chromatin accessibility measured by ATAC-seq (i) under the indicated conditions. For a, h, i, all peaks associated with each gene were summed and then normalized relative to the mean of each group. (j, k) ESCs grown in S/L or S/L+2i were treated with 100 nM Dinaciclib (CDK9 inhibitor) or THZ1 (CDK7 inhibitor) for 48h and total population doublings (j) or Nanog-GFP fluorescence relative to vehicle control (k) were quantified. Bars, mean ± SD of n=3 independent samples. See also Supplementary Table 5.

Supplementary Figure 5 Increased transcription factor activity enables resistance to BET inhibitors in the absence of 2i.

(a) Western blot depicting activation of STAT3 and ERK in ESCs expressing GCSF-activated LIFR transgene (GY118F) grown with or without GCSF and then cultured in the presence or absence of LIF for 24 h. Unprocessed original scans of blots are shown in Supplementary Fig. 9. Western blot to verify transgene was performed once. (b) Volume of ESCs expressing GCSF-activated LIFR transgene (GY118F) treated with DMSO or 500 nM JQ1 for 48 h. (c) Density plot of Brd4 ChIP-seq (from S/L-cultured ESCs) and Nanog/Klf4/Oct4/Sox2/Esrrb ChIP-seq datasets (ref. 42). Data are centered on Brd4 peaks. Rows represent 10 kb interval around a single Brd4-occupied peak. (d) 17,696 Brd4 ChIP-seq peaks (as in c) are clustered based on their relative binding of indicated transcription factors. The percent overlap of each factor with Brd4-bound peaks is shown at bottom. (e) Nanog expression in ESCs expressing empty vector or Nanog transgene. (f) Volume of ESCs expressing empty vector or Nanog transgene treated with DMSO, 500 nM JQ1 or 500 nM iBET for 48 h. (g) Alkaline phosphatase staining of ESCs expressing empty vector or Nanog transgene treated with DMSO, 500 nM JQ1 or 500 nM iBET for 48 h. Scale bar, 100 □m. Experiment was performed two independent times. (h) Klf4 expression in ESCs expressing empty vector or Klf4 transgene. (i) Volume of ESCs expressing empty vector or Klf4 transgene treated with DMSO or 500 nM JQ1 for 48 h. (j) Image of representative well of alkaline phosphatase staining of colony formation assay quantified in Fig. 5h. Bars represent mean ± SD (b, f, i) or mean ± SEM (e, h) of n=3 independent samples.

Supplementary Figure 6 Tet1/2 and Brd4 bind similar genomic regions.

(a) Density plots of Brd4 (from S/L cultured ESCs) and Tet1 ChIP-seq datasets (ref. 46). Data are centered on Brd4 peaks. Rows represent 10 kb interval around a single Brd4-occupied peak. (b) Overlap of Brd4 and Tet1 binding sites. (c) Colony formation assay of control ESCs cultured in the presence of 200 nM JQ1 and vehicle or 100 μg/mL Vitamin C for 6 days. Data are presented as mean ± SEM of n=3 independent samples.

Supplementary Figure 7 Tet1/2 determine sensitivity to BET inhibitors.

(a) Sequencing of Tet1 and Tet2 in control and Tet1/2 DKO ESCs. Protospacer-adjacent motif (PAM) shown in red; mutations shown in green. (b) Heat map depicting relative accessibility at all ATAC-seq peaks in control or Tet1/2 DKO ESCs. 638 sites gain accessibility, 693 sites lose accessibility, 27,310 sites exhibit no change in accessibility with Tet1/2 mutation. (c) Brd4 expression in control (ctrl) or Tet1/2 DKO ESCs expressing empty vector or Nanog transgene. (d) Growth curve of Tet1/2 DKO ESCs cultured in S/L. Triplicate wells are shown as individual data points. n=3 independent samples are shown and the connecting line joins the mean values of each time point. (e) Population doublings of control (Ctrl) or Tet1/2 double mutant (DKO) ESCs cultured in S/L and treated for 48 h with 1 μM JQ1 relative to vehicle (DMSO)-treated controls. P values relative to Ctrl lines calculated using 1-way ANOVA with Tukey’s multiple comparison post-test. (f) Nanog expression in control (ctrl) or Tet1/2 DKO ESCs expressing empty vector or Nanog transgene. (g) Image of representative well of alkaline phosphatase staining of colony formation assay quantified in Fig. 7b. (h) Relative accessibility at all n=178 peaks associated with pluripotency genes (using genes shown in Fig. 7g) in Nanog vs Vector expressing cells. Box, 25-75th percentile; bar, median; whiskers, 5-95th percentile. Significance assessed by two-tailed paired Student’s t test relative to Ctrl. Data was generated from ATAC-Seq experiments with 2 samples in each condition. (i) Heat map depicting relative chromatin accessibility as measured by ATAC-seq. Peaks whose accessibility decreased with JQ1 treatment in control cells and whose accessibility was maintained and/or increased with Nanog overexpression were grouped into three clusters. Peaks in Clusters I and II remained open despite JQ1 treatment if Nanog was overexpressed. Tet1/2 were not required for Nanog to preserve accessibility of peaks in cluster I in response to JQ1 treatment; peaks in Cluster II did require Tet1/2 in order for Nanog to preserve accessibility in response to JQ1 treatment. Number of peaks in each cluster is shown in grey. The top motifs enriched in each cluster are shown to the right. %, percent of peaks in each cluster with a given motif. ELKF, Erythrocyte-Klf1; BORIS, K562-CTCFL. (j) Brd4 expression in ESCs cultured as shown in Fig. 7g. Bars are presented as mean ± SEM of n=3 independent samples.

Supplementary Figure 8

Unprocessed original scans of blots.

Supplementary information

Supplementary Information

Supplementary Figures 1–8 and Supplementary Table legends

Reporting Summary

Supplementary Table 1

Sequences for Brd4 shRNA and sgRNA.

Supplementary Table 2

Genotyping primers for D34 ESCs.

Supplementary Table 3

Primers used for real-time PCR.

Supplementary Table 4

Sequencing information.

Supplementary Table 5

Statistics source data.

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Finley, L.W.S., Vardhana, S.A., Carey, B.W. et al. Pluripotency transcription factors and Tet1/2 maintain Brd4-independent stem cell identity. Nat Cell Biol 20, 565–574 (2018). https://doi.org/10.1038/s41556-018-0086-3

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