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MeCP2 links heterochromatin condensates and neurodevelopmental disease

Abstract

Methyl CpG binding protein 2 (MeCP2) is a key component of constitutive heterochromatin, which is crucial for chromosome maintenance and transcriptional silencing1,2,3. Mutations in the MECP2 gene cause the progressive neurodevelopmental disorder Rett syndrome3,4,5, which is associated with severe mental disability and autism-like symptoms that affect girls during early childhood. Although previously thought to be a dense and relatively static structure1,2, heterochromatin is now understood to exhibit properties consistent with a liquid-like condensate6,7. Here we show that MeCP2 is a dynamic component of heterochromatin condensates in cells, and is stimulated by DNA to form liquid-like condensates. MeCP2 contains several domains that contribute to the formation of condensates, and mutations in MECP2 that lead to Rett syndrome disrupt the ability of MeCP2 to form condensates. Condensates formed by MeCP2 selectively incorporate and concentrate heterochromatin cofactors rather than components of euchromatic transcriptionally active condensates. We propose that MeCP2 enhances the separation of heterochromatin and euchromatin through its condensate partitioning properties, and that disruption of condensates may be a common consequence of mutations in MeCP2 that cause Rett syndrome.

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Fig. 1: MeCP2 forms condensates in vivo and in vitro.
Fig. 2: MeCP2 features that contribute to condensate formation.
Fig. 3: Mutations in patients with Rett syndrome disrupt MeCP2 condensate formation.
Fig. 4: R168X mutant MeCP2 displays reduced partitioning into heterochromatin condensates and causes disease-relevant cellular phenotypes in neurons.

Data availability

Relevant data supporting the findings of this study are available within the paper and its Supplementary Information. RNA-seq datasets generated in this study have been deposited in the Gene Expression Omnibus under accession code GSE139033. Uncropped gel images can be found in Supplementary Fig. 1. Additional data are available from the corresponding author upon reasonable request. The following publicly available data were used in this study: GTEx v. 7 RNA-seq Median Gene TPMs by Tissue (www.gtexportal.org), RettBASE MECP2 Variant List (mecp2.chw.edu.au/mecp2/mecp2_home.php), and UniProt Cluster ID: UniRef50_Q9Z2D6 (www.uniprot.org/uniref/UniRef50_Q9Z2D6). Source data are provided with this paper.

Code availability

Custom code used for analysis of images from in vitro droplet assays is available at www.github.com/jehenninger/in_vitro_droplet_assay. Custom code used for analysis of heterochromatin condensate volumes is available at www.github.com/jehenninger/MECP2_neuron.

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Acknowledgements

We thank A. P. Bird for sharing Mecp2 mutant cell lines; D. Reinberg for sharing purified poly-nucleosomes; P. A. Sharp for discussions; D. Richardson and the Harvard Center for Biological Imaging; W. Salmon and the Whitehead W.M. Keck Microscopy Facility; and R. Flannery, J. Drotar, N. Rosenau and the Whitehead Genetically Engineered Models Center for technical support. The work was supported by NIH grant R01 GM123511 (R.A.Y.), NSF grant PHY1743900 (R.A.Y.), NIH grant 2 R01 MH104610-20 (R.J., R.A.Y.), NIH grant R37 CA084198 (R.J.), Hope Funds for Cancer Research Fellowship (A.D.), NIH grant T32 5T32DK007191-45 (J.M.P.), NSF Graduate Research Fellowship (A.V.Z.), and NIH grant K99/R00 MH113813 (X.S.L.).

Author information

Authors and Affiliations

Authors

Contributions

C.H.L., E.L.C., R.J. and R.A.Y. conceived the project. C.H.L., E.L.C., T.I.L., R.J. and R.A.Y. organized the studies. C.H.L., E.L.C. and R.A.Y wrote the manuscript. E.L.C., J.E.H., O.O., A.V.Z. and J.S. performed in vitro droplet assays. N.M.H. performed protein purification. G.L. performed purification of poly-nucleosomes. C.H.L., E.L.C. and J.E.H. developed and performed computational analyses. E.L.C. and A.D. performed cellular imaging experiments. L.K.A. performed transcriptional reporter assays. C.H.L. generated endogenous-tagged cell lines and gene expression analyses. X.T. and T.L. performed neuronal differentiation. A.D., X.S.L., S.M., D.S.S. and E.W. performed chimeric mouse generation. C.H.L., E.L.C. and J.M.P. generated constructs. R.J. and R.A.Y. supervised the project with help from T.I.L. All authors contributed to editing the manuscript.

Corresponding authors

Correspondence to Rudolf Jaenisch or Richard A. Young.

Ethics declarations

Competing interests

R.A.Y. is a founder and shareholder of Syros Pharmaceuticals, Camp4 Therapeutics, Omega Therapeutics, and Dewpoint Therapeutics. R.J. is an advisor/co-founder of Fate Therapeutics, Fulcrum Therapeutics, Omega Therapeutics, and Dewpoint Therapeutics. T.I.L. is a shareholder of Syros Pharmaceuticals and a consultant to Camp4 Therapeutics. All other authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 MeCP2 and HP1α are dynamic components of heterochromatin condensates.

a, Live-cell images of endogenous-tagged HP1α–mCherry and Hoechst staining in mouse ES cells. b, Live-cell images of endogenous-tagged MeCP2–GFP and HP1α–mCherry in mouse ES cells. c, Live-cell images of FRAP experiments with endogenously tagged HP1α–mCherry mouse ES cells. d, FRAP curves for experiments in c. Photobleaching occurs at t = 0 s. n = 7 cells. e, Half-time of photobleaching recovery for MeCP2–GFP and HP1α–mCherry at heterochromatin condensates in imaging experiments in c and Fig. 1b. n = 7 cells per condition. P = 0.90, t = 0.13, df = 12, two-tailed Student’s t-test. f, Mobile fractions of MeCP2–GFP and HP1α–mCherry within heterochromatin condensates in imaging experiments in c and Fig. 1b, determined by FRAP analysis. n = 7 cells per condition. P = 0.09, t = 1.87, df = 12, two-tailed Student’s t-test. Data are mean ± s.e.m.

Extended Data Fig. 2 Generation of endogenous-tagged MeCP2–GFP chimeric mice.

a, Schematic of MeCP2–GFP chimeric mouse generation using endogenous-tagged MeCP2–GFP mouse ES cells. Endogenous-tagged MeCP2–GFP mouse ES cells derived from V6.5 background were injected into embryos from CD1-IGS mice and multiple embryos were implanted into pseudo-pregnant female mice. Chimeric pups were distinguished from non-chimeric littermates by agouti coat colour. MeCP2–GFP tagged adult chimeric mice were used for experiments. b, Schematic of strategy used to generate endogenous-tagged MeCP2–GFP mouse ES cells. c, PCR genotyping of endogenous-tagged MeCP2–GFP mouse ES cells. For gel source data, see Supplementary Fig. 1. d, MECP2 expression values in transcripts per million (TPM) measured by RNA-seq for various human tissues surveyed by GTEx. Tissues are ordered based on expression level. TPM values greater than 1 are considered to be expressed.

Extended Data Fig. 3 MeCP2 forms phase-separated droplets in vitro.

a, Droplet experiments examining effect of MeCP2 concentration. MeCP2–GFP was added to droplet formation buffers with 100 mM NaCl and 10% PEG-8000. b, Droplet areas for experiments in a. Fields per condition n = 10. c, MeCP2–GFP condensed fraction for experiments in a. Fields per condition n = 10. d, Droplet experiments examining effect of salt concentration. MeCP2–GFP at 10 μM was added to droplet formation buffers with indicated NaCl concentrations and 10% PEG-8000. e, Droplet areas for experiments in d. Fields per condition n = 15. f, MeCP2–GFP condensed fraction for experiments in d. Fields per condition n = 15. g, Phase diagram of MeCP2 droplet formation. MeCP2–GFP was added to droplet formation buffers with indicated NaCl concentrations and 5% PEG-8000. Filled-in circles indicate conditions with droplets. Fields per condition n = 10. h, Droplet experiments showing MeCP2 droplet fusion. MeCP2–GFP at 10 μM was added to droplet formation buffers with 100 mM NaCl and 10% PEG-8000. i, Droplet experiments showing MeCP2 droplet FRAP. Conditions as in h. Photobleaching at t = 0 s. j, Droplet experiments examining HP1α. HP1α–mCherry at 10 μM was added to droplet formation buffers with 100 mM NaCl and 10% PEG-8000. k, DNA-Cy5 partition ratios in MeCP2–GFP droplets for experiments in Fig. 1g. Fields per condition n = 15. l, Expanded schematic of MeCP2 protein (Fig. 2a) with protein sequence conservation, net charge per residue (NCPR), and residue plots. m, Droplet experiments examining MeCP2 deletion mutants. MeCP2–GFP deletion mutants at 10 μM were added to droplet formation buffers with 100 mM NaCl and 10% PEG-8000. n, Droplet areas for experiments in m. Fields per condition n = 5. o, MeCP2–GFP condensed fraction for experiments in m. Fields per condition n = 5. Data are mean ± s.d.

Extended Data Fig. 4 MeCP2 condensates preferentially concentrate HP1α compared to components of transcriptional condensates.

a, Immunofluorescence images of heterochromatin condensates (MeCP2–GFP) and transcriptional condensates (anti-MED1) in mouse ES cells. b, Droplet experiments examining ability of MeCP2 condensates to preferentially concentrate HP1α compared to components of transcriptional condensates. MeCP2–GFP at 7.5 μM was mixed with HP1α–mCherry, MED1-IDR-mCherry, BRD4-IDR-mCherry, or mCherry at 7.5 μM in droplet formation buffers with 150 mM NaCl and 10% PEG-8000. c, mCherry partition ratios in MeCP2–GFP droplets for experiments in b. Fields per condition n = 15. d, Droplet experiments with naked DNA examining ability of MeCP2 condensates to preferentially concentrate HP1α compared to components of transcriptional condensates. Conditions same as in b, but with the addition of 160 nM DNA. e, mCherry partition ratios in MeCP2–GFP droplets for experiments in d. Fields per condition n = 15. f, Droplet experiments with nucleosomal DNA examining ability of MeCP2 condensates to preferentially concentrate HP1α compared to components of transcriptional condensates. MeCP2–GFP at 5 μM was mixed with HP1α–mCherry, MED1-IDR-mCherry, BRD4-IDR-mCherry, or mCherry at 5 μM and 6 nM poly-nucleosomes in droplet formation buffers with 100 mM NaCl and 3 mM MgCl2. g, mCherry partition ratios in MeCP2–GFP droplets for experiments in f. Fields per condition n = 10. h, Brightfield images examining droplet formation with nucleosomal DNA alone and with MeCP2. Poly-nucleosomes at 6 nM were mixed with 5 μM MeCP2–GFP or no MeCP2–GFP in droplet formation buffers with 100 mM NaCl and 3 mM MgCl2. i, Droplet experiments examining MeCP2 droplet formation with nucleosomal DNA. Conditions same as in h. j, Droplet areas for experiments in i. Fields per condition n = 10. k, MeCP2–GFP condensed fraction for experiments in i. Fields per condition n = 10. Data are mean ± s.d.

Extended Data Fig. 5 MeCP2 condensate partitioning of BRD4 domains.

a, Droplet experiments examining ability of MeCP2 condensates to preferentially incorporate and concentrate HP1α compared to BRD4 domains in the presence of naked DNA. BRD4-IDR, bromodomain 1 (BD1), and extra-terminal (ET) domain were examined. MeCP2–GFP at 7.5 μM was mixed with either HP1α–mCherry, BRD4-IDR-mCherry, BRD4-BD1-mCherry, BRD4-ET-mCherry, or mCherry each at 7.5 μM and 160 nM methylated DNA in droplet formation buffers with 150 mM NaCl and 10% PEG-8000. b, mCherry partition ratios in MeCP2–GFP droplets for experiments in a. Fields per condition n = 15.

Extended Data Fig. 6 Rett syndrome mutations disrupt MeCP2 condensate formation.

a, Droplet areas for experiments in Fig. 3b. Fields per condition n = 15. b, Droplet areas for experiments in Fig. 3d. Fields per condition n = 15. c, Droplet areas for experiments in Fig. 3f. Fields per condition n = 15. d, Droplet experiments examining effects of Rett syndrome missense mutations that disrupt IDR-2 on MeCP2 droplet formation. Wild-type MeCP2–GFP and Rett syndrome IDR-2 mutants (P225R and P322L) at indicated concentrations were mixed with 20 nM methylated DNA in droplet formation buffers with 100 mM NaCl. e, MeCP2–GFP condensed fraction as a function of MeCP2–GFP concentration for experiments in d. Data are mean ± s.d. Fields per condition n = 15. f, Droplet areas for experiments in d. Fields per condition n = 15.

Extended Data Fig. 7 TBLR1 partitioning into MeCP2 droplets is disrupted by Rett syndrome mutation R306C.

a, Droplet experiments examining ability of wild-type MeCP2 and R306C mutant condensates to enrich TBLR1-CTD. Wild-type MeCP2–GFP or R306C mutant at 6 μM was mixed with TBLR1-CTD-mCherry at 10 μM in droplet formation buffers with 125 mM NaCl and 10% PEG-8000. b, TBLR1-CTD-mCherry partition ratios in MeCP2–GFP wild-type and R306C mutant droplets for experiments in a. Fields per condition n = 15. c, Droplet experiments examining ability of wild-type MeCP2 and R306C mutant condensates to enrich TBLR1-CTD. Wild-type MeCP2–GFP or R306C mutant at 10 μM was mixed with TBLR1-CTD-mCherry at 4 μM in droplet formation buffers with 125 mM NaCl. d, TBLR1-CTD-mCherry partition ratios in wild-type MeCP2–GFP and R306C mutant droplets for experiments in c. Fields per condition n = 12.

Extended Data Fig. 8 MeCP2 Mini forms droplets in vitro and partitions into heterochromatin condensates in mouse ES cells.

a, Schematic of MeCP2 protein with a minimal MeCP2 protein (Mini)22 that retains the MBD and NID displayed below. b, Droplet experiments examining ability of Mini MeCP2 to form droplets. Wild-type MeCP2–GFP and Mini at 4 μM were added to droplet formation buffers with 125 mM NaCl and 10% PEG-8000. c, Droplet areas for experiments in b. Fields per condition n = 12. d, MeCP2–GFP condensed fraction for experiments in b. Fields per condition n = 12. e, Droplet experiments examining ability of wild-type MeCP2 and Mini to form droplets with HP1α and DNA. Wild-type MeCP2–GFP or Mini at 7.5 μM was mixed with 7.5 μM HP1α–mCherry and 160 nM DNA in droplet formation buffers with 150 mM NaCl and 10% PEG-8000. f, HP1α–mCherry partition ratios in wild-type MeCP2–GFP and Mini droplets for experiments in e. Fields per condition n = 15. g, DNA-Cy5 partition ratios in wild-type MeCP2–GFP and Mini droplets for experiments in e. Fields per condition n = 15. h, Droplet experiments examining ability of MeCP2 wild-type and Mini condensates to enrich TBLR1-CTD. Wild-type MeCP2–GFP or Mini at 4 μM was mixed with TBLR1-CTD-mCherry at 10 μM in droplet formation buffers with 125 mM NaCl and 10% PEG-8000. i, TBLR1-CTD-mCherry partition ratios in wild-type MeCP2–GFP and Mini droplets for experiments in h. Fields per condition n = 12. j, Live-cell microscopy of endogenous-tagged wild-type MeCP2–GFP and Mini proteins with Hoechst staining in mouse ES cells. k, Partition ratios of MeCP2–GFP proteins at heterochromatin condensates for experiments in j. n = 10 cells per condition. P = 0.1161, t = 1.6509, df = 18, two-tailed Student’s t-test. All data are mean ± s.d.

Extended Data Fig. 9 R168X mutant MeCP2 displays reduced partitioning into heterochromatin condensates and causes disease-relevant cellular phenotypes in mouse ES cells.

a, Live-cell images of endogenous-tagged wild-type MeCP2–GFP and R168X mutant proteins with Hoechst staining in mouse ES cells. b, Partition ratios of MeCP2–GFP proteins at heterochromatin condensates for experiments in a. Cells per condition: WT (n = 11), R168X (n = 10). P < 0.0001, t = 12.13, df = 19, two-tailed Student’s t-test. c, MeCP2–GFP signal in endogenous-tagged wild-type MeCP2–GFP and R168X mutant mouse ES cells measured by flow cytometry. n = 3 biologically independent samples per condition. For example flow cytometry gating strategy, see Supplementary Fig. 2. d, Western blot of endogenous-tagged wild-type MeCP2–GFP and R168X mutant mouse ES cells. Anti-H3 was used as a processing control. For gel source data, see Supplementary Fig. 1. e, Number of heterochromatin condensates per cell in endogenous-tagged wild-type MeCP2–GFP and R168X mutant mouse ES cells. n = 16 cells per condition. P = 0.0149, t = 2.5832, df = 30, two-tailed Student’s t-test. f, Heterochromatin condensate volumes in endogenous-tagged wild-type MeCP2–GFP and R168X mutant mouse ES cells. Condensates per condition: WT (n = 206), R168X (n = 273). P < 0.0001, t = 4.2065, df = 477, two-tailed Student’s t-test. g, Live-cell images of endogenous-tagged MeCP2–GFP (wild type or R168X mutant) and HP1α–mCherry in mouse ES cells. h, Partition ratios of HP1α–mCherry at heterochromatin condensates for experiments in g. Cells per condition: WT (n = 6), R168X (n = 20). P < 0.0001, t = 5.7136, df = 24, two-tailed Student’s t-test. i, HP1α–mCherry signal in endogenous-tagged MeCP2–GFP (wild-type or R168X mutant) and HP1α–mCherry mouse ES cells measured by flow cytometry. n = 3 biologically independent samples per condition. For example flow cytometry gating strategy, see Supplementary Fig. 2. j, Normalized major satellite repeat expression in endogenous-tagged wild-type MeCP2–GFP and R168X mutant mouse ES cells. n = 3 biologically independent samples per condition. P = 0.0017, t = 7.5436, df = 4, two-tailed Student’s t-test. k, Total RNA per cell in endogenous-tagged wild-type MeCP2–GFP and R168X mutant mouse ES cells. n = 3 biologically independent samples per condition. P = 0.0324, t = 3.2154, df = 4, two-tailed Student’s t-test. l, RNA-seq comparing endogenous-tagged wild-type MeCP2–GFP and R168X mutant mouse ES cells. Differentially expressed genes (red dots) were determined by two-tailed Wald test with multiple test adjusted P < 0.1. For both conditions, n = 3 biologically independent samples. All data are mean ± s.d.

Extended Data Fig. 10 R168X mutant in mouse ES cells and neurons.

a, Live-cell images of mouse ES cells overexpressing either wild-type or R168X mutant MeCP2–GFP. b, Partition ratios of MeCP2–GFP proteins at heterochromatin condensates relative to the nucleoplasm for experiments in a. Cells per condition: WT (n = 3), R168X (n = 5). P = 0.0008, t = 6.1529, df = 6, two-tailed Student’s t-test. c, Western blot of mouse ES cells overexpressing either wild-type or R168X mutant MeCP2–GFP. Anti-H3 was used as a processing control. For gel source data, see Supplementary Fig. 1. d, Schematic of generation of mouse ES-cell-derived neurons. Endogenous-tagged wild-type MeCP2–GFP and R168X mutant mouse ES cells were modified for Dox-inducible NGN2 expression using the PiggyBac system. Prior to neuronal differentiation, mouse ES cells were seeded on astrocytes. Neuronal differentiation was induced by adding doxycycline to drive NGN2 expression. Five days after induction of NGN2 expression, neurons were analysed. e, Fixed-cell immunofluorescence images of neurons derived from wild-type MeCP2–GFP and R168X mutant mouse ES cells. Anti-TuJ1 staining was used to distinguish neurons. f, Western blot of endogenous-tagged wild-type MeCP2–GFP and R168X mutant neurons. Anti-H3 was used as a loading control. For gel source data, see Supplementary Fig. 1. g, Normalized major satellite repeat expression in endogenous-tagged wild-type MeCP2–GFP and R168X mutant neurons. n = 3 biologically independent samples per condition. P = 0.0061, t = 5.3004, df = 4, two-tailed Student’s t-test. h, Total RNA per cell in endogenous-tagged wild-type MeCP2–GFP and R168X neurons. n = 3 biologically independent samples per condition. P = 0.0141, t = 4.1676, df = 4, two-tailed Student’s t-test. i, RNA-seq comparing endogenous-tagged wild-type MeCP2–GFP and R168X mutant neurons. Differentially expressed genes (red dots) were identified using a two-tailed Wald test with multiple test adjusted P < 0.1. For both conditions, n = 3 biologically independent samples. Data are mean ± s.d.

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This file contains Supplementary Figures 1 and 2. Supplementary Figure 1: Gel source data with size markers. Regions used for display in the indicated figures are denoted with dashed boxes. Supplementary Figure 2: Example flow cytometry gating strategy. Related to Extended Data Fig. 9c, i.

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Li, C.H., Coffey, E.L., Dall’Agnese, A. et al. MeCP2 links heterochromatin condensates and neurodevelopmental disease. Nature 586, 440–444 (2020). https://doi.org/10.1038/s41586-020-2574-4

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