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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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Visualization of epigenetic and phenotypic layers of differentiation in an early stage of ESC differentiation. (A) Differentiation of ESCs was induced by treatment of all-trans RA, and Oct-4 mRNA expression was measured before, and 24 and 48 h after the RA treatment. The expression levels in the mouse liver and brain are shown as those in Oct-4-negative tissues. Values show mean + SD of three experiments. (B) Expression of Oct-4 and ßIII-tubulin proteins was analyzed in ESCs before, and 24 and 48 h after the RA treatment by immunofluorescence (scale bar: 20 µm). (C) Images of phase contrast and iChmo for the bivalent modification in ESC colonies before, and 24 and 48 h after the RA treatment (upper panel), and differentiated neuron-like cells at 24 and 48 h (lower panel). Regardless of the phenotypic differentiation statuses, the bivalent modification was absent both at 24 and 48 h, supporting highly concerted regulation of epigenetic changes. Scale bar represents 10 µm. (D) The number of fluorescence spots was counted in ESCs before (n = 33), and 24 (n = 37) and 48 (n = 38) h after the RA treatment (Mann–Whitney U-test; *P < 0.001). Although the decrease of Oct-4 expression and increase of ßIII-tubulin were highly variable among the ESCs treated with RA, the decrease of the bivalent modification was highly coordinated.
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gkt528-F3: Visualization of epigenetic and phenotypic layers of differentiation in an early stage of ESC differentiation. (A) Differentiation of ESCs was induced by treatment of all-trans RA, and Oct-4 mRNA expression was measured before, and 24 and 48 h after the RA treatment. The expression levels in the mouse liver and brain are shown as those in Oct-4-negative tissues. Values show mean + SD of three experiments. (B) Expression of Oct-4 and ßIII-tubulin proteins was analyzed in ESCs before, and 24 and 48 h after the RA treatment by immunofluorescence (scale bar: 20 µm). (C) Images of phase contrast and iChmo for the bivalent modification in ESC colonies before, and 24 and 48 h after the RA treatment (upper panel), and differentiated neuron-like cells at 24 and 48 h (lower panel). Regardless of the phenotypic differentiation statuses, the bivalent modification was absent both at 24 and 48 h, supporting highly concerted regulation of epigenetic changes. Scale bar represents 10 µm. (D) The number of fluorescence spots was counted in ESCs before (n = 33), and 24 (n = 37) and 48 (n = 38) h after the RA treatment (Mann–Whitney U-test; *P < 0.001). Although the decrease of Oct-4 expression and increase of ßIII-tubulin were highly variable among the ESCs treated with RA, the decrease of the bivalent modification was highly coordinated.

Mentions: We took advantage of an imaging method to analyze individual cells in a heterogeneous sample, namely, ESCs at an early stage of differentiation. ESC differentiation was induced by all-trans RA treatment 24 h after removal of LIF and feeder layer cells. The expression of Oct-4 mRNA decreased to the half 24 h after the RA treatment, but was still detectable 48 h after the treatment (Figure 3A). At cellular level, Oct-4 protein was detectable in all ESCs before the treatment but was heterogeneously detectable 24 h later (Figure 3B). At 48 h, in accordance with the residual Oct-4 mRNA expression, a minor fraction of ESCs still had the Oct-4 protein expression (Figure 3B). ßIII-tubulin, a differentiated neuron marker, was also expressed in only a fraction of the ESCs (Figure 3B), showing the presence of large variations in the phenotypic differentiation of the ESCs. In contrast, no or little fluorescence spots of the bivalent modification were observed 24 and 48 h after the RA treatment by iChmo, regardless of whether ESCs formed colonies or were differentiated into neuron-like cells (Figure 3C). The mean number of spots of ESCs at 48 h after the RA treatment was at the same level as that of the Suz12 KO ESCs (Figure 3D). These data indicated that the epigenetic layer of differentiation was completed at 2 days in a highly concerted manner, whereas the phenotypic layer of differentiation proceeded with considerable variation.Figure 3.


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)

Visualization of epigenetic and phenotypic layers of differentiation in an early stage of ESC differentiation. (A) Differentiation of ESCs was induced by treatment of all-trans RA, and Oct-4 mRNA expression was measured before, and 24 and 48 h after the RA treatment. The expression levels in the mouse liver and brain are shown as those in Oct-4-negative tissues. Values show mean + SD of three experiments. (B) Expression of Oct-4 and ßIII-tubulin proteins was analyzed in ESCs before, and 24 and 48 h after the RA treatment by immunofluorescence (scale bar: 20 µm). (C) Images of phase contrast and iChmo for the bivalent modification in ESC colonies before, and 24 and 48 h after the RA treatment (upper panel), and differentiated neuron-like cells at 24 and 48 h (lower panel). Regardless of the phenotypic differentiation statuses, the bivalent modification was absent both at 24 and 48 h, supporting highly concerted regulation of epigenetic changes. Scale bar represents 10 µm. (D) The number of fluorescence spots was counted in ESCs before (n = 33), and 24 (n = 37) and 48 (n = 38) h after the RA treatment (Mann–Whitney U-test; *P < 0.001). Although the decrease of Oct-4 expression and increase of ßIII-tubulin were highly variable among the ESCs treated with RA, the decrease of the bivalent modification was highly coordinated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3753646&req=5

gkt528-F3: Visualization of epigenetic and phenotypic layers of differentiation in an early stage of ESC differentiation. (A) Differentiation of ESCs was induced by treatment of all-trans RA, and Oct-4 mRNA expression was measured before, and 24 and 48 h after the RA treatment. The expression levels in the mouse liver and brain are shown as those in Oct-4-negative tissues. Values show mean + SD of three experiments. (B) Expression of Oct-4 and ßIII-tubulin proteins was analyzed in ESCs before, and 24 and 48 h after the RA treatment by immunofluorescence (scale bar: 20 µm). (C) Images of phase contrast and iChmo for the bivalent modification in ESC colonies before, and 24 and 48 h after the RA treatment (upper panel), and differentiated neuron-like cells at 24 and 48 h (lower panel). Regardless of the phenotypic differentiation statuses, the bivalent modification was absent both at 24 and 48 h, supporting highly concerted regulation of epigenetic changes. Scale bar represents 10 µm. (D) The number of fluorescence spots was counted in ESCs before (n = 33), and 24 (n = 37) and 48 (n = 38) h after the RA treatment (Mann–Whitney U-test; *P < 0.001). Although the decrease of Oct-4 expression and increase of ßIII-tubulin were highly variable among the ESCs treated with RA, the decrease of the bivalent modification was highly coordinated.
Mentions: We took advantage of an imaging method to analyze individual cells in a heterogeneous sample, namely, ESCs at an early stage of differentiation. ESC differentiation was induced by all-trans RA treatment 24 h after removal of LIF and feeder layer cells. The expression of Oct-4 mRNA decreased to the half 24 h after the RA treatment, but was still detectable 48 h after the treatment (Figure 3A). At cellular level, Oct-4 protein was detectable in all ESCs before the treatment but was heterogeneously detectable 24 h later (Figure 3B). At 48 h, in accordance with the residual Oct-4 mRNA expression, a minor fraction of ESCs still had the Oct-4 protein expression (Figure 3B). ßIII-tubulin, a differentiated neuron marker, was also expressed in only a fraction of the ESCs (Figure 3B), showing the presence of large variations in the phenotypic differentiation of the ESCs. In contrast, no or little fluorescence spots of the bivalent modification were observed 24 and 48 h after the RA treatment by iChmo, regardless of whether ESCs formed colonies or were differentiated into neuron-like cells (Figure 3C). The mean number of spots of ESCs at 48 h after the RA treatment was at the same level as that of the Suz12 KO ESCs (Figure 3D). These data indicated that the epigenetic layer of differentiation was completed at 2 days in a highly concerted manner, whereas the phenotypic layer of differentiation proceeded with considerable variation.Figure 3.

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