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Mesp1 controls the chromatin and enhancer landscapes essential for spatiotemporal patterning of early cardiovascular progenitors

Abstract

The mammalian heart arises from various populations of Mesp1-expressing cardiovascular progenitors (CPs) that are specified during the early stages of gastrulation. Mesp1 is a transcription factor that acts as a master regulator of CP specification and differentiation. However, how Mesp1 regulates the chromatin landscape of nascent mesodermal cells to define the temporal and spatial patterning of the distinct populations of CPs remains unknown. Here, by combining ChIP–seq, RNA-seq and ATAC-seq during mouse pluripotent stem cell differentiation, we defined the dynamic remodelling of the chromatin landscape mediated by Mesp1. We identified different enhancers that are temporally regulated to erase the pluripotent state and specify the pools of CPs that mediate heart development. We identified Zic2 and Zic3 as essential cofactors that act with Mesp1 to regulate its transcription-factor activity at key mesodermal enhancers, thereby regulating the chromatin remodelling and gene expression associated with the specification of the different populations of CPs in vivo. Our study identifies the dynamics of the chromatin landscape and enhancer remodelling associated with temporal patterning of early mesodermal cells into the distinct populations of CPs that mediate heart development.

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Fig. 1: Dynamics of gene expression regulated by Mesp1.
Fig. 2: Temporal dynamic of chromatin remodelling regulated by Mesp1.
Fig. 3: Characterization of Mesp1-bound enhancers and prediction of putative transcriptional cofactors.
Fig. 4: Validation of Mesp1 target genes and enhancer remodelling in the presence of endogenous Mesp1.
Fig. 5: Zic2 and Zic3 bind to a fraction of Mesp1-bound enhancers.
Fig. 6: Mesp1 and Zic3 co-regulate gene expression during mouse gastrulation.
Fig. 7: Zic3 and Zic2 are essential regulators of Mesp1 activity.

Data availability

The next-generation-sequencing data (ChIP–seq, ATAC-seq and RNA-seq) generated during this study has been deposited in Gene Expression Omnibus (GEO) and is accessible through GEO Series accession number GSE165107. Previously published next-generation-sequencing data and microarray data that were re-analysed here are available under the accession codes GSE41361, GSE44288 and GSE59033. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding authors on reasonable request.

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Acknowledgements

We thank the ULB animal facility and ULB genomic core facility (F. Libert and A. Lefort). We thank Y. Song for bioinformatic assistance. F.L., X.L. and B.S. were supported by an FNRS aspirant fellowship (B.S.), an EMBO long-term fellowship and the Leducq Foundation. S.G. was funded by a Royal Society Newton International Fellowship (grant no. NIF\R1\181950). C.G. is funded by the Swedish Research Council (grant no. 2017-06278). F.L. thanks the MMG imaging and the animal phenotyping core platforms. We thank R. Kelly for sharing antibodies. Work in the Göttgens laboratory is supported by grants from the Wellcome Trust, Blood Cancer UK, Cancer Research UK, NIDDK and core support grants from the Wellcome Trust to the Wellcome–MRC Cambridge Stem Cell Institute. Work in F.L.’s laboratory was supported by the INSERM ATIP-Avenir programme. C.B. is an investigator of WELBIO. Work in C.B.’s laboratory was supported by the FNRS, ULB foundation, European Research Council (ERC), foundation Bettencourt Schueller (C.B. and F.L.) and Leducq Foundation as part of the network ‘22q11.2 deletion syndrome: novel approaches to understand cardiopharyngeal pathogenesis’. C.B. and B.G. acknowledge support from the Fondation Philippe Wiener–Maurice Anspach.

Author information

Authors and Affiliations

Authors

Contributions

X.L., B.S., F.L. and C.B. designed the experiments, performed data analyses and wrote the manuscript. X.L. and B.S. performed most of the biological experiments. B.S. performed bioinformatic analyses of all sequencing data. C.D. performed FACS. E.P. and F.L. performed the immunofluorescence and RNAscope experiments on mouse embryos. Y.A. generated the Zic3-KO mouse lines. B.S., S.Z., E.B. and F.L. described and analysed the Zic3-mutant phenotypes. Chimeric embryos were generated by C.G., M.-L.N.T. and W.M., and processed and analysed by F.L. and B.G. Knockout cell lines for the chimeric embryos were generated by B.S. and C.G. C.P. provided technical support. E.C. and F.F. provided help with some of the next-generation sequencing. S.G. and J.C.M. performed the scRNA-seq analyses for the initial submission. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Fabienne Lescroart or Cédric Blanpain.

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The authors declare no competing interests.

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Nature Cell Biology thanks José Luis de la Pompa and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Temporal regulation of gene expression mediated by homogeneous Mesp1 induction within embryoid bodies.

a, RNA-FISH on sections of EBs from Mesp1 Dox-inducible PSC lines in control conditions (NO DOX) or 24 h after doxycycline induction (+DOX) showing Mesp1 expression in red. (representative image of 6 independent embryonic bodies). b, Percentage of cells that are positive for Mesp1 using RNA in situ hybridization, in control (NO DOX) or upon Mesp1 overexpression (+DOX) (n = 6 for NO DOX and n = 7 for +DOX independent embryoid bodies). Error bars indicate SEM. Statistical analyses were performed by two-tail unpaired student t tests. p = 2.53×10−6 c, Level of Mesp1 expression in control (NO DOX) or doxycycline condition (+DOX) as measured by the signal intensity from the smRNA-FISH. Error bars indicate SEM. Statistical analyses were performed by two-tail unpaired student t tests. n = 30 representative Mesp1+ cells per condition. p = 6.87×10−10 d, Principal component analysis of RNA-seq samples performed at day 2.5 (0 h), day 3 (12 h following Mesp1 overexpression) and day 3.5 (24 h following Mesp1 overexpression) in control (no dox) and Mesp1 overexpression (dox) conditions during PSC differentiation. Note the excellent concordance between biological duplicates. e, Representative examples of genes that undergo early, constant or late downregulation mediated by Mesp1. Examples were chosen to represent the diversity of kinetics we could find in genes repressed by Mesp1. f, Plots representing all genes classified as early, constant or late downregulated, as well as their respective average profile (thick lines).

Source data

Extended Data Fig. 2 Temporal analysis of Mesp1 TF activity.

a, Pie chart representing the distribution of the position of Mesp1 ChIP-seq peaks relative to protein-coding genes (data shown represent two biologically independent replicates. b-c, Graph representing the dynamics of ATAC-seq (b) and H3K27Ac ChIP-seq (c) signal within early, constant and late Mesp1 ChIP-seq peaks. Average profiles are shown by the think red (dox) and blue (no dox) lines. d, Dot plot illustrating the correlation between temporality of Mesp1 binding and surrounding H3K27Ac deposition (left), or Mesp1 binding and ATAC-seq opening of the chromatin (right). For each individual Mesp1 ChIP-seq peak, the average signal was measured for all three types of experiments at 12 h and 24 h dox, in order to extract a measure of fold-change between 12 and 24 h. The log2 value of this fold-change for each peak was compared between each type of experiment. Data shown represent two biologically independent replicates; p-values were calculated through a two-tailored t-test.

Extended Data Fig. 3 Motif discovery of enhancers activated by Mesp1.

a, Motif discovery performed separately in Mesp1 ChIP-seq peaks classified as early, constant or late peaks. p-values are calculated through a binomial test. b, Same pipeline as panel a but separating peaks as de novo versus primed Mesp1 peaks. Stars represent the enrichment of the CAAATGG motif in pioneer peaks in comparison to non-pioneer peaks through a two-tailored Z-test. Data shown represent two biologically independent replicates. c, Quantification of the occurrence of all forms of bHLH motifs in Mesp1 ChIP-seq peaks, showing the prevalence of CAAATG motif, most often with an extra G. *** The top 2 bHLH motifs were notably over-represented (p < 0.00001) through a two-tailored Z-test, with z = 17.5 for CAAATG and z = 5.9 for CAGATG. d, Representative genomic locus where ATAC-seq and H3K27Ac ChIP-seq peaks are upregulated 24 h following Mesp1 expression but which are not directly bound by Mesp1 (red boxes), suggesting that their regulation is mediated by other TFs, whose expression is induced directly or indirectly by Mesp1. Gata4 ChIP-seq data31 (green) shows a strong overlap between these de novo opened peaks not bound but induced by Mesp1. e, Motif discovery searching for known TF binding sites within ATAC-seq peaks that get opened by Mesp1 but are not directly bound by Mesp1. p-values are calculated through a binomial test. f, Quantification of peaks containing a Gata4 motif, both in Mesp1 ChIP-seq peaks and ATAC-seq peaks UP without Mesp1 binding, separated as peaks bound by Gata4 or not bound by Gata4 using a published ChIP-seq dataset31. *** Gata4 motifs are statistically notably enriched in Gata4-binding peaks in Mesp1 ChIP-seq (z = 8.34, p < 0.00001) and in ATAC-seq peaks UP (z = 13.6, p < 0.00001). Data shown represent two biologically independent replicates; n = 1 for previously published Gata4 ChIP-seq31. These values were calculated through a two-tailored Z-test.

Extended Data Fig. 4 Repression of the core pluripotency network by Mesp1.

a, Representative genomic locus (Cdh1) where ATAC-seq and H3K27Ac ChIP peaks found in control (no dox) conditions are absent or smaller in dox conditions, without presenting any Mesp1 binding (blue boxes), suggesting indirect repression of chromatin opening by other factors. b, Motif discovery within peaks that are closed in dox conditions, including enrichment of a compound OCT-SOX-TCF-NANOG motif. p-values are calculated through a binomial test. c, Heatmap showing signal of ATAC-seq, H3K27Ac ChIP-seq and published Nanog, Oct4 and Sox2 ChIP-seq data within peaks that were closed after dox-induced Mesp1 overexpression32.

Extended Data Fig. 5 Context-dependency of Mesp1 activator potential.

a, Heatmap of the expression values of Mesp1 upregulated genes in undifferentiated PSCs (2i conditions), with or without Mesp1 induction (dox). RNA-seq samples in 2i were performed twice. b, Overlap between genes directly and indirectly activated by Mesp1 during PSC differentiation or in 2i, illustrating the paucity of Mesp1-mediated gene activation in pluripotency. c, Heatmap illustrating Mesp1 binding affinity to its enhancers in 2i by Mesp1 ChIP-seq and the subsequent lack of chromatin opening by ATAC-seq in 2i conditions. Each row represents a Mesp1 binding site detected during differentiation at 24 h dox. Peaks were ordered by unsupervised k-means clustering. 1, de novo peaks where Mesp1 binding and subsequent chromatin opening is lost in 2i; 2 and 2’, primed peaks with conserved Mesp1 binding and chromatin opening in 2i; 3, de novo ATAC-seq peaks where Mesp1 binding and chromatin opening is conserved in 2i. All samples collected in 2i were performed twice. d, Representative examples of Mesp1 binding loci in 2i conditions. e-f, Classification of Mesp1 ChIP-seq peaks found in 2i into previously detailed chromatin opening (e) or kinetic (f) groups. *** Late peaks were notably depleted in 2i conditions (z = -7.86, p < 0.00001). Data shown represent two biologically independent replicates. These values were calculated through a two-tailored Z-test. g, Quantification of the correlation between Mesp1 binding strength measured by Mesp1 ChIP-seq and chromatin opening in ATAC-seq in 2i (24 h dox), demonstrating a linear correlation between these two variables. Data shown represent two biologically independent replicates.

Extended Data Fig. 6 Zic2 and Zic3 cooperate with Mesp1 and potentially with other mesoderm-inducing TFs.

a, Western blot illustrating the expression of Zic2 and Zic3 with and without Mesp1 induction, at day 3.5 (24 h) of PSC differentiation, as well as the lack of Zic2 and Zic3 protein expression in Zic2/3 double KO cell lines. (Data shown represent 2 independent experiments) b, Illustration of the two most enriched motifs in all Zic2 and Zic3 ChIP-seq detected peaks. p-values are calculated through a binomial test. c, Venn diagram illustrating the number of overlapping peaks between Mesp1, Zic2 and Zic3 ChIP-seq datasets. P-value was calculated using a hypergeometric test, using bedtools fisher. d, Illustration of the temporality of Zic3 binding within primed and de novo Mesp1-bound peaks, with associated ATAC-seq signal. 1, peaks already bound by Zic3 at day 2.5 (0 h); 2, peaks bound by Zic3 at 24 h no dox; 3, peaks bound by Zic3 at 24 h dox.

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Extended Data Fig. 7 Zic2 and Zic3 regulate Mesp1-induced CP specification and differentiation.

a, mRNA expression of the core pluripotency associated TFs in Mesp1-inducible WT, Zic2 and Zic3 KO PSCs in Lif/2i pluripotency conditions, as measured by RT-qPCR. (n = 4 biologically independent replicates covering two independent KO clones with each assessed by two independent experiments. Error bars indicate mean + /-SEM, statical analysis was performed by 2-way ANOVA. b, Representative immunofluorescence for Troponin T in Mesp1-inducible WT, Zic2 and Zic3 KO cell lines at day 10 of differentiation, illustrating the ability of Mesp1 overexpression to overcome cardiac differentiation defects in Zic2 and Zic3 KO cell lines. (Data shown represent 6 independent experiments. Scale bars=100 µm. c, Illustrative examples of genes that are notably upregulated in Zic3 KO cells in comparison to WT cells, including known important factors of pluripotency and endoderm differentiation.

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Extended Data Fig. 8 Heart defects observed in the newly generated Zic3-KO line.

a, Zygote injection strategy used to generate Zic3 KO mice. b, Pictures of Wild type (WT) and homozygous null (Zic3KO) E14.5 embryos, showing severe neural tube closure defects and exencephaly found in a subset of Zic3 KO embryos (n = 10/32). Scale bars= 1 mm. c, Range of cardiac morphological abnormalities found in Zic3 KO embryos at E14.5. We observe outflow tract defects with persistent truncus arteriosus (PTA bottom left panel – n = 1/32), hypoplasia of the right ventricle (RV) (upper right panel – n = 5/32) and mutants with a situs inversus phenotype (bottom right panel – n = 2/32). Scale bars= 1 mm. d, ratio of the surface area of the right ventricle compared to the surface area of the left ventricle in wild type (black – n = 26) and Zic3 KO (red – n = 44). Error bars indicate mean + /-SEM. Unpaired, two-tailed t-test showed a p-value=0.0004. e, Mean thickness of the compact myocardial (CM) layer of the ventricles in wild type (black – n = 6) and Zic3 KO embryos (red – n = 12). Error bars indicate mean + /-SEM. Unpaired, two-tailed t-test showed a p-value=0.0074. f-f’, Immunofluorescence on E14.5 wild type (f) and Zic3 KO (f’) hearts using an anti-cardiac Troponin T (cTnT) antibody to label the cardiomyocytes and isolectinB4 to label the endocardium (representative pictures from 4 independent hearts of each genotype). No endocardial defect was observed in Zic3 KO embryos while the cTnT+ layer was thinner in f’. Scale bars= 200 µm. RV, right ventricle; LV, left ventricle; RA, right atrium; LA, left atrium; pt, pulmonary trunk; ao, aorta.

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Extended Data Fig. 9 Zic2 and Zic3 redundantly regulate Mesp1 activity.

a, Expression of three core pluripotency genes in Mesp1 WT and two independent Zic2/3 KO PSC cell lines cultured in Lif/2i medium. Data from two independent experiments, b, FACS profiles of EBs at day 4 of differentiation from Mesp1 WT and Zic2/3 dKO cell lines, illustrating the decrease in Flk1 and PDGFRa expression in Zic2/3 dKO cell lines both in no dox and dox conditions. c, Table shows the distribution of genes that were downregulated in Zic3 KO and Zic2/3 dKO cells within the temporal categories of Mesp1 direct upregulated target genes. There was no particular enrichment for early, constant and late genes within the Zic2/3-dependent fraction of Mesp1 target. d, Barplot illustrating the proportion of early, constant and late Mesp1 binding sites within Mesp1 ChIP-seq peaks conserved in Zic2/3 dKO cell lines. *** for late genes, z = -5.845, p < 0.00001. These values were calculated through a two-tailored Z-test. Data shown represent two biologically independent replicates. e, Representation of the proportion of Mesp1 ChIP-seq peaks as well as ATAC-seq peaks that are opened (UP) or closed (DOWN- upon Mesp1 induction in WT cells which are preferentially closed in Zic2/3 dKO cells after Mesp1 induction. n = 2 independent experiments for Mesp1 ChIP-seq and ATAC-seq in WT cells; n = 3 independent experiments for ATAC-seq in Zic2/3 dKO cell lines. *** all three comparisons were significant with p < 0.00001 and respectively z = 34.9 (Mesp1 ChIP-seq), z = 54.2 (ATAC-seq UP) and z = -20.1 (ATAC-seq DOWN). These values were calculated through a two-tailored Z-test. f, Motif enrichment analysis of ATAC-seq peaks that were preferentially closed in Zic2/3 dKO cells in comparison to WT cells. p-values are calculated through a binomial test.

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Supplementary information

Reporting Summary

Supplementary Table 1

Supplementary Table 1. Guide RNA sequences used for CRISPR–Cas9n gene and enhancer KO as well as enhancer-motif editing. Supplementary Table 2. PCR primers used for screening of the KO cell lines. Supplementary Table 3. Sequences of qPCR primers used. Supplementary Table 4. List of primary antibodies used. Supplementary Table 5. Probes used for smRNA-FISH.

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Source Data Fig. 3

Statistical source data for Fig. 3b,e,g.

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Unprocessed western blots for Fig. 5f.

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Statistical source data for Fig. 5a,b.

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Statistical source data for Fig. 6a,b.

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Statistical source data for Fig. 7a,b,k.

Source Data Extended Data Fig. 1

Statistical source data for Extended Data Fig. 1b,c.

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Unprocessed western blots for Extended Data Fig. 6a.

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Statistical source data for Extended Data Fig. 7a.

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Statistical source data for Extended Data Fig. 8d,e.

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Statistical source data for Extended Data Fig. 9a.

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Lin, X., Swedlund, B., Ton, ML.N. et al. Mesp1 controls the chromatin and enhancer landscapes essential for spatiotemporal patterning of early cardiovascular progenitors. Nat Cell Biol 24, 1114–1128 (2022). https://doi.org/10.1038/s41556-022-00947-3

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