Abstract
Combinations of histone modifications have significant biological roles, such as maintenance of pluripotency and cancer development, but cannot be analyzed at the single cell level. Here, we visualized a combination of histone modifications by applying the in situ proximity ligation assay, which detects two proteins in close vicinity (∼30 nm). The specificity of the method [designated as imaging of a combination of histone modifications (iChmo)] was confirmed by positive signals from H3K4me3/acetylated H3K9, H3K4me3/RNA polymerase II and H3K9me3/H4K20me3, and negative signals from H3K4me3/H3K9me3. Bivalent modification was clearly visualized by iChmo in wild-type embryonic stem cells (ESCs) known to have it, whereas rarely in Suz12 knockout ESCs and mouse embryonic fibroblasts known to have little of it. iChmo was applied to analysis of epigenetic and phenotypic changes of heterogeneous cell population, namely, ESCs at an early stage of differentiation, and this revealed that the bivalent modification disappeared in a highly concerted manner, whereas phenotypic differentiation proceeded with large variations among cells. Also, using this method, we were able to visualize a combination of repressive histone marks in tissue samples. The application of iChmo to samples with heterogeneous cell population and tissue samples is expected to clarify unknown biological and pathological significance of various combinations of epigenetic modifications.
Highlights
Histone modifications are known to play important roles in various biological and pathological processes, such as cell type-specific gene expression and cancer development [1,2]
Bivalent modification was clearly visualized by imaging of a combination of histone modifications (iChmo) in wild-type embryonic stem cells (ESCs) known to have it, whereas rarely in Suz12 knockout ESCs and mouse embryonic fibroblasts known to have little of it. iChmo was applied to analysis of epigenetic and phenotypic changes of heterogeneous cell population, namely, ESCs at an early stage of differentiation, and this revealed that the bivalent modification disappeared in a highly concerted manner, whereas phenotypic differentiation proceeded with large variations among cells
To visualize a combination of histone modifications, we first performed the in situ PLA focusing on two combinations of epigenetic marks known to be present at active loci, one H3K4me3 and H3K9ac and the other H3K4me3 and RNA polymerase II (RNAPII) [18]
Summary
Histone modifications are known to play important roles in various biological and pathological processes, such as cell type-specific gene expression and cancer development [1,2]. Aberrant expression of a protein that binds to a specific combination of histone modifications was associated with poor prognosis in breast cancer, suggesting the importance of a combination in pathological processes [11]. A sequential-ChIP assay has the advantage to identify genomic regions with a specific combination of histone modifications, it suffers from the necessity of a large number of cells, and its application is limited to samples containing >106 cells. In samples with heterogeneous cell populations, it is impossible to identify cells with a specific combination of histone modifications. We cannot identify which cells have a specific multivalent
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