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Visualization of multivalent histone modification in a single cell reveals highly concerted epigenetic changes on differentiation of embryonic stem cells.

Hattori N, Niwa T, Kimura K, Helin K, Ushijima T - Nucleic Acids Res. (2013)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Epigenomics, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan, Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen, Denmark and Centre for Epigenetics, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen, Denmark.

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.

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Application of iChmo to visualization of bivalent modification. (A) Mouse ESCs were stained by immunofluorescence with antibodies against H3K4me3 and H3K27me3 (scale bar: 10 µm). (B) Coexistence of H3K4me3 and H3K27me3 in WT ESCs was detected by iChmo, but hardly in Suz12 KO ESCs and MEFs (scale bar: 10 µm). (C) The mean number of fluorescence spots was significantly larger in WT ESCs (15.2; n = 58) than in Suz12 KO ESCs (0.9; n = 69) and MEFs (4.1; n = 60) (Mann–Whitney U-test; *P < 0.001).
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gkt528-F2: Application of iChmo to visualization of bivalent modification. (A) Mouse ESCs were stained by immunofluorescence with antibodies against H3K4me3 and H3K27me3 (scale bar: 10 µm). (B) Coexistence of H3K4me3 and H3K27me3 in WT ESCs was detected by iChmo, but hardly in Suz12 KO ESCs and MEFs (scale bar: 10 µm). (C) The mean number of fluorescence spots was significantly larger in WT ESCs (15.2; n = 58) than in Suz12 KO ESCs (0.9; n = 69) and MEFs (4.1; n = 60) (Mann–Whitney U-test; *P < 0.001).

Mentions: We focused on bivalent modification because of its biological significance and applied iChmo to visualize it using mouse ESCs and MEFs that have a lot of and few, respectively, bivalent modifications (3,5). By immunofluorescence staining of ESCs, H3K4me3 signals were observed as interspersed small dots, whereas H3K27me3 signals were enriched at the periphery of the nuclear membrane (Figure 2A). Some signals were merged in the nucleus, appearing to reflect the presence of bivalent modification. However, the same staining pattern was observed in MEFs (Supplementary Figure S1), and it was shown that immunofluorescence was not capable of distinguishing whether H3K4me3 and H3K27me3 were in close proximity. However, notably, by iChmo, a large number of fluorescence spots were observed in ESCs, whereas only a small number of spots were in MEFs (Figure 2B). This demonstrated that iChmo can distinguish whether two modifications coexist in the vicinity at the single cell level.Figure 2.


Visualization of multivalent histone modification in a single cell reveals highly concerted epigenetic changes on differentiation of embryonic stem cells.

Hattori N, Niwa T, Kimura K, Helin K, Ushijima T - Nucleic Acids Res. (2013)

Application of iChmo to visualization of bivalent modification. (A) Mouse ESCs were stained by immunofluorescence with antibodies against H3K4me3 and H3K27me3 (scale bar: 10 µm). (B) Coexistence of H3K4me3 and H3K27me3 in WT ESCs was detected by iChmo, but hardly in Suz12 KO ESCs and MEFs (scale bar: 10 µm). (C) The mean number of fluorescence spots was significantly larger in WT ESCs (15.2; n = 58) than in Suz12 KO ESCs (0.9; n = 69) and MEFs (4.1; n = 60) (Mann–Whitney U-test; *P < 0.001).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3753646&req=5

gkt528-F2: Application of iChmo to visualization of bivalent modification. (A) Mouse ESCs were stained by immunofluorescence with antibodies against H3K4me3 and H3K27me3 (scale bar: 10 µm). (B) Coexistence of H3K4me3 and H3K27me3 in WT ESCs was detected by iChmo, but hardly in Suz12 KO ESCs and MEFs (scale bar: 10 µm). (C) The mean number of fluorescence spots was significantly larger in WT ESCs (15.2; n = 58) than in Suz12 KO ESCs (0.9; n = 69) and MEFs (4.1; n = 60) (Mann–Whitney U-test; *P < 0.001).
Mentions: We focused on bivalent modification because of its biological significance and applied iChmo to visualize it using mouse ESCs and MEFs that have a lot of and few, respectively, bivalent modifications (3,5). By immunofluorescence staining of ESCs, H3K4me3 signals were observed as interspersed small dots, whereas H3K27me3 signals were enriched at the periphery of the nuclear membrane (Figure 2A). Some signals were merged in the nucleus, appearing to reflect the presence of bivalent modification. However, the same staining pattern was observed in MEFs (Supplementary Figure S1), and it was shown that immunofluorescence was not capable of distinguishing whether H3K4me3 and H3K27me3 were in close proximity. However, notably, by iChmo, a large number of fluorescence spots were observed in ESCs, whereas only a small number of spots were in MEFs (Figure 2B). This demonstrated that iChmo can distinguish whether two modifications coexist in the vicinity at the single cell level.Figure 2.

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Epigenomics, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan, Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen, Denmark and Centre for Epigenetics, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen, Denmark.

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.

Show MeSH
Related in: MedlinePlus