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Detection of histone modifications at specific gene loci in single cells in histological sections

Nature Methods volume 10, pages 171–177 (2013)Cite this article

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Abstract

Chromatin immunoprecipitation assays have contributed greatly to our understanding of the role of histone modifications in gene regulation. However, they do not permit analysis with single-cell resolution, thus confounding analyses of heterogeneous cell populations. Here we present a method that permits visualization of histone modifications of single genomic loci with single-cell resolution in formaldehyde-fixed paraffin-embedded tissue sections based on combined use of in situ hybridization and proximity ligation assays. We show that dimethylation of lysine 4 of histone H3 (H3K4me2) at the MYH11 locus is restricted to the smooth muscle cell (SMC) lineage in human and mouse tissue sections and that the mark persists even in phenotypically modulated SMC in atherosclerotic lesions that show no detectable expression of SMC marker genes. This methodology has promise for broad applications in the study of epigenetic mechanisms in complex multicellular tissues in development and disease.

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Figure 1: ISH-PLA: a new method of detection of histone modifications at a single genomic locus in tissue sections.
Figure 2: Validation of ISH-PLA using an SMC lineage–tracing mouse model.
Figure 3: Visualization of H3K4me2 on the MYH11 promoter in SMCs in situ in histological sections of human carotid arteries.
Figure 4: H3K4me2 on the MYH11 promoter persists during phenotypic switching in vivo in SMC lineage–tracing mice developing atherosclerosis.
Figure 5: Identification of epigenetic regulation of phenotypically modulated SMCs in human coronary atherosclerotic lesions by ISH-PLA.

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Acknowledgements

We thank M.E. McCanna and R.S. Tripathi for their knowledge and technical expertise, J.W. Mandell (University of Virginia) for providing human brain sections and S. Offermanns (Max Planck Institute) for Myh11-CreERT2 mice. This work was supported by US National Institutes of Health grants R01 HL57353, R01 HL098538 and R01 HL087867 (to G.K.O.). D.G. is supported by the American Heart Association Postdoctoral Fellowship 11POST7760009. L.S.S. is funded by a predoctoral American Heart Association Fellowship 11PRE17008. A.T.N. is funded by a postdoctoral American Heart Association Fellowship 12POST11630032.

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  1. Delphine Gomez and Laura S Shankman: These authors contributed equally to this work.

Authors and Affiliations

  1. Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, Virginia, USA

    Delphine Gomez, Laura S Shankman, Anh T Nguyen & Gary K Owens

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  1. Delphine Gomez
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Contributions

G.K.O. supervised this study; D.G. and G.K.O. conceived of the ISH-PLA strategies, designed studies and wrote the paper; D.G. generated labeled DNA probes, performed immunostaining and all ISH-PLA experiments and analyzed data; D.G. performed in vitro experiments, ChIP and quantitative PCR; L.S.S. generated Myh11-CreERT2 ROSA STOP-flox EYFP+/+ mice; L.S.S. and A.T.N. performed immunostaining on mouse sections; and D.G., L.S.S. and A.T.N. performed image acquisition and analysis.

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Correspondence to Gary K Owens.

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Gomez, D., Shankman, L., Nguyen, A. et al. Detection of histone modifications at specific gene loci in single cells in histological sections. Nat Methods 10, 171–177 (2013). https://doi.org/10.1038/nmeth.2332

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