Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Biotin tagging of MeCP2 in mice reveals contextual insights into the Rett syndrome transcriptome

Abstract

Mutations in MECP2 cause Rett syndrome (RTT), an X-linked neurological disorder characterized by regressive loss of neurodevelopmental milestones and acquired psychomotor deficits. However, the cellular heterogeneity of the brain impedes an understanding of how MECP2 mutations contribute to RTT. Here we developed a Cre-inducible method for cell-type-specific biotin tagging of MeCP2 in mice. Combining this approach with an allelic series of knock-in mice carrying frequent RTT-associated mutations (encoding T158M and R106W) enabled the selective profiling of RTT-associated nuclear transcriptomes in excitatory and inhibitory cortical neurons. We found that most gene-expression changes were largely specific to each RTT-associated mutation and cell type. Lowly expressed cell-type-enriched genes were preferentially disrupted by MeCP2 mutations, with upregulated and downregulated genes reflecting distinct functional categories. Subcellular RNA analysis in MeCP2-mutant neurons further revealed reductions in the nascent transcription of long genes and uncovered widespread post-transcriptional compensation at the cellular level. Finally, we overcame X-linked cellular mosaicism in female RTT models and identified distinct gene-expression changes between neighboring wild-type and mutant neurons, providing contextual insights into RTT etiology that support personalized therapeutic interventions.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Utilization and characterization of Mecp2Tavi mice and associated RTT variants.
Figure 2: Cell-type-specific transcriptional profiling of neuronal nuclei.
Figure 3: Analysis of T158M and R106W DEGs.
Figure 4: Genome-wide length-dependent transcriptional changes in mutant mice.
Figure 5: T158M and R106W DEGs in mosaic female mice.

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. Chahrour, M. & Zoghbi, H.Y. The story of Rett syndrome: from clinic to neurobiology. Neuron 56, 422–437 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. Amir, R.E. et al. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat. Genet. 23, 185–188 (1999).

    Article  CAS  PubMed  Google Scholar 

  3. Shahbazian, M.D., Antalffy, B., Armstrong, D.L. & Zoghbi, H.Y. Insight into Rett syndrome: MeCP2 levels display tissue- and cell-specific differences and correlate with neuronal maturation. Hum. Mol. Genet. 11, 115–124 (2002).

    Article  CAS  PubMed  Google Scholar 

  4. Lewis, J.D. et al. Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA. Cell 69, 905–914 (1992).

    Article  CAS  PubMed  Google Scholar 

  5. Jones, P.L. et al. Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nat. Genet. 19, 187–191 (1998).

    Article  CAS  PubMed  Google Scholar 

  6. Lyst, M.J. et al. Rett syndrome mutations abolish the interaction of MeCP2 with the NCoR/SMRT co-repressor. Nat. Neurosci. 16, 898–902 (2013).

    Article  CAS  PubMed  Google Scholar 

  7. Nan, X. et al. Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature 393, 386–389 (1998).

    Article  CAS  PubMed  Google Scholar 

  8. Skene, P.J. et al. Neuronal MeCP2 is expressed at near histone-octamer levels and globally alters the chromatin state. Mol. Cell 37, 457–468 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Chahrour, M. et al. MeCP2, a key contributor to neurological disease, activates and represses transcription. Science 320, 1224–1229 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Chen, L. et al. MeCP2 binds to non-CG methylated DNA as neurons mature, influencing transcription and the timing of onset for Rett syndrome. Proc. Natl. Acad. Sci. USA 112, 5509–5514 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Li, Y. et al. Global transcriptional and translational repression in human-embryonic-stem-cell-derived Rett syndrome neurons. Cell Stem Cell 13, 446–458 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Cuddapah, V.A. et al. Methyl-CpG-binding protein 2 (MECP2) mutation type is associated with disease severity in Rett syndrome. J. Med. Genet. 51, 152–158 (2014).

    Article  CAS  PubMed  Google Scholar 

  13. Ghosh, R.P., Horowitz-Scherer, R.A., Nikitina, T., Gierasch, L.M. & Woodcock, C.L. Rett syndrome–causing mutations in human MeCP2 result in diverse structural changes that impact folding and DNA interactions. J. Biol. Chem. 283, 20523–20534 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Ho, K.L. et al. MeCP2 binding to DNA depends upon hydration at methyl-CpG. Mol. Cell 29, 525–531 (2008).

    Article  CAS  PubMed  Google Scholar 

  15. Ballestar, E., Yusufzai, T.M. & Wolffe, A.P. Effects of Rett syndrome mutations of the methyl-CpG binding domain of the transcriptional repressor MeCP2 on selectivity for association with methylated DNA. Biochemistry 39, 7100–7106 (2000).

    Article  CAS  PubMed  Google Scholar 

  16. Brown, K. et al. The molecular basis of variable phenotypic severity among common missense mutations causing Rett syndrome. Hum. Mol. Genet. 25, 558–570 2016).

    Article  CAS  PubMed  Google Scholar 

  17. Goffin, D. et al. Rett syndrome mutation MeCP2 T158A disrupts DNA binding, protein stability and ERP responses. Nat. Neurosci. 15, 274–283 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Baker, S.A. et al. An AT-hook domain in MeCP2 determines the clinical course of Rett syndrome and related disorders. Cell 152, 984–996 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Katz, D.M. et al. Preclinical research in Rett syndrome: setting the foundation for translational success. Dis. Model. Mech. 5, 733–745 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Lamonica, J.M. et al. Elevating expression of MeCP2 T158M rescues DNA binding and Rett syndrome–like phenotypes. J. Clin. Invest. 127, 1889–1904 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Lyst, M.J. & Bird, A. Rett syndrome: a complex disorder with simple roots. Nat. Rev. Genet. 16, 261–275 (2015).

    Article  CAS  PubMed  Google Scholar 

  22. Fishell, G. & Heintz, N. The neuron identity problem: form meets function. Neuron 80, 602–612 (2013).

    Article  CAS  PubMed  Google Scholar 

  23. Molyneaux, B.J. et al. DeCoN: genome-wide analysis of in vivo transcriptional dynamics during pyramidal neuron fate selection in neocortex. Neuron 85, 275–288 (2015).

    Article  CAS  PubMed  Google Scholar 

  24. Mo, A. et al. Epigenomic signatures of neuronal diversity in the mammalian brain. Neuron 86, 1369–1384 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Zhao, Y.-T., Goffin, D., Johnson, B.S. & Zhou, Z. Loss of MeCP2 function is associated with distinct gene expression changes in the striatum. Neurobiol. Dis. 59, 257–266 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Gabel, H.W. et al. Disruption of DNA-methylation-dependent long gene repression in Rett syndrome. Nature 522, 89–93 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Guo, J.U. et al. Distribution, recognition and regulation of non-CpG methylation in the adult mammalian brain. Nat. Neurosci. 17, 215–222 (2014).

    Article  CAS  PubMed  Google Scholar 

  28. Rube, H.T. et al. Sequence features accurately predict genome-wide MeCP2 binding in vivo. Nat. Commun. 7, 11025 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Deal, R.B. & Henikoff, S. A simple method for gene expression and chromatin profiling of individual cell types within a tissue. Dev. Cell 18, 1030–1040 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Lakso, M. et al. Efficient in vivo manipulation of mouse genomic sequences at the zygote stage. Proc. Natl. Acad. Sci. USA 93, 5860–5865 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Samaco, R.C. et al. A partial loss of function allele of methyl-CpG-binding protein 2 predicts a human neurodevelopmental syndrome. Hum. Mol. Genet. 17, 1718–1727 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Guy, J., Gan, J., Selfridge, J., Cobb, S. & Bird, A. Reversal of neurological defects in a mouse model of Rett syndrome. Science 315, 1143–1147 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Kumar, A. et al. Analysis of protein domains and Rett syndrome mutations indicate that multiple regions influence chromatin-binding dynamics of the chromatin-associated protein MECP2 in vivo. J. Cell Sci. 121, 1128–1137 (2008).

    Article  CAS  PubMed  Google Scholar 

  34. Goebbels, S. et al. Genetic targeting of principal neurons in neocortex and hippocampus of NEX-Cre mice. Genes 44, 611–621 (2006).

    Article  CAS  Google Scholar 

  35. Monory, K. et al. The endocannabinoid system controls key epileptogenic circuits in the hippocampus. Neuron 51, 455–466 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Bhatt, D.M. et al. Transcript dynamics of proinflammatory genes revealed by sequence analysis of subcellular RNA fractions. Cell 150, 279–290 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Ameur, A. et al. Total RNA sequencing reveals nascent transcription and widespread co-transcriptional splicing in the human brain. Nat. Struct. Mol. Biol. 18, 1435–1440 (2011).

    Article  CAS  PubMed  Google Scholar 

  38. Sugino, K. et al. Cell-type-specific repression by methyl-CpG-binding protein 2 is biased toward long genes. J. Neurosci. 34, 12877–12883 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Maniatis, T. & Reed, R. An extensive network of coupling among gene expression machines. Nature 416, 499–506 (2002).

    Article  CAS  PubMed  Google Scholar 

  40. Core, L.J., Waterfall, J.J. & Lis, J.T. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322, 1845–1848 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Brennan, C.M. & Steitz, J.A. HuR and mRNA stability. Cell. Mol. Life Sci. 58, 266–277 (2001).

    Article  CAS  PubMed  Google Scholar 

  42. Höck, J. & Meister, G. The Argonaute protein family. Genome Biol. 9, 210 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Flavell, S.W. & Greenberg, M.E. Signaling mechanisms linking neuronal activity to gene expression and plasticity of the nervous system. Annu. Rev. Neurosci. 31, 563–590 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Yang, H. et al. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154, 1370–1379 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Müller, M. & Can, K. Aberrant redox homoeostasis and mitochondrial dysfunction in Rett syndrome. Biochem. Soc. Trans. 42, 959–964 (2014).

    Article  CAS  PubMed  Google Scholar 

  46. Zylka, M.J., Simon, J.M. & Philpot, B.D. Gene length matters in neurons. Neuron 86, 353–355 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Linhoff, M.W., Garg, S.K. & Mandel, G. A high-resolution imaging approach to investigate chromatin architecture in complex tissues. Cell 163, 246–255 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. King, I.F. et al. Topoisomerases facilitate transcription of long genes linked to autism. Nature 501, 58–62 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Nott, A. et al. Histone deacetylase 3 associates with MeCP2 to regulate FOXO and social behavior. Nat. Neurosci. 19, 1497–1505; advance online publication (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Djebali, S. et al. Landscape of transcription in human cells. Nature 489, 101–108 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Buxbaum, A.R., Yoon, Y.J., Singer, R.H. & Park, H.Y. Single-molecule insights into mRNA dynamics in neurons. Trends Cell Biol. 25, 468–475 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Mauger, O., Lemoine, F. & Scheiffele, P. Targeted intron retention and excision for rapid gene regulation in response to neuronal activity. Neuron 92, 1266–1278 (2016).

    Article  CAS  PubMed  Google Scholar 

  53. Khwaja, O.S. et al. Safety, pharmacokinetics, and preliminary assessment of efficacy of mecasermin (recombinant human IGF-1) for the treatment of Rett syndrome. Proc. Natl. Acad. Sci. USA 111, 4596–4601 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Lombardi, L.M., Baker, S.A. & Zoghbi, H.Y. MECP2 disorders: from the clinic to mice and back. J. Clin. Invest. 125, 2914–2923 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  55. Xiao, C. et al. MiR-150 controls B cell differentiation by targeting the transcription factor c-Myb. Cell 131, 146–159 (2007).

    Article  CAS  PubMed  Google Scholar 

  56. de Boer, E. et al. Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice. Proc. Natl. Acad. Sci. USA 100, 7480–7485 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Driegen, S. et al. A generic tool for biotinylation of tagged proteins in transgenic mice. Transgenic Res. 14, 477–482 (2005).

    Article  CAS  PubMed  Google Scholar 

  58. Greer, C.B. et al. Histone deacetylases positively regulate transcription through the elongation machinery. Cell Rep. 13, 1444–1455 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

    Article  CAS  PubMed  Google Scholar 

  60. Robinson, M.D., McCarthy, D.J. & Smyth, G.K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

    Article  CAS  PubMed  Google Scholar 

  61. Love, M.I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Merico, D., Isserlin, R., Stueker, O., Emili, A. & Bader, G.D. Enrichment map: a network-based method for gene-set enrichment visualization and interpretation. PLoS One 5, e13984 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Smoot, M.E., Ono, K., Ruscheinski, J., Wang, P.-L. & Ideker, T. Cytoscape 2.8: new features for data integration and network visualization. Bioinformatics 27, 431–432 (2011).

    CAS  PubMed  Google Scholar 

  64. Hart, T., Komori, H.K., LaMere, S., Podshivalova, K. & Salomon, D.R. Finding the active genes in deep RNA-seq gene expression studies. BMC Genomics 14, 778 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. R Core Team. R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, 2014).

Download references

Acknowledgements

We would like to thank the IDDRC Mouse Gene Manipulation Core at Children's Hospital Boston (U54HD090255, M. Thompson), the Gene Targeting Core (P01DK049210, K. Kaestner) and the Transgenic and Chimeric Mouse Facility (J. Richa) at University of Pennsylvania for help in generating transgenic mice, the Flow Cytometry and Cell Sorting Resource Laboratory (H. Pletcher, W. DeMuth), and the Next Generation Sequencing Core (J. Schug) for technical assistance. B.S.J. is supported by a Cell and Molecular Biology Training Grant (TG32GM072290) and the UNCF/Merck Graduate Research Dissertation Fellowship. This work is supported by NIH grants K22AI112570 (G.V.), R21AI107067 and R01CA140485 (T.H.K.), R01MH091850 and R01NS081054 (Z.Z.), and a basic research grant from Rettsyndrome.org (Z.Z.). Z.Z. is a Pew Scholar in the Biomedical Sciences.

Author information

Authors and Affiliations

Authors

Contributions

Conceptualization, B.S.J. and Z.Z.; methodology, B.S.J., J.M.L., D.G. and Z.Z.; investigation, B.S.J., Y.-T.Z., M.F., J.M.L., K.H.W., Y.J.K. and D.B.; formal analyses, B.S.J., Y.-T.Z., G.G. and T.H.K.; validation, B.S.J., M.F., J.M.L. and G.V.; resources, B.S.J., Y.-T.Z. and Y.C.; data curation, Y.-T.Z.; writing manuscript, B.S.J.; review and editing, B.S.J., Y.-T.Z., M.F. and Z.Z.; visualization, B.S.J.; project administration and funding acquisition, Z.Z.

Corresponding author

Correspondence to Zhaolan Zhou.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–13 (PDF 29209 kb)

Life Sciences Reporting Summary (PDF 171 kb)

Supplementary Table 1

Summary of RNA-seq experimental conditions used in this study (XLSX 14 kb)

Supplementary Table 2

RT-PCR primers used in this study (XLSX 65 kb)

Supplementary Table 3

List of HITS-CLIP data sets used for RBP analysis (XLSX 55 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Johnson, B., Zhao, YT., Fasolino, M. et al. Biotin tagging of MeCP2 in mice reveals contextual insights into the Rett syndrome transcriptome. Nat Med 23, 1203–1214 (2017). https://doi.org/10.1038/nm.4406

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.4406

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing