Limits...
Identification of a large protein network involved in epigenetic transmission in replicating DNA of embryonic stem cells.

Aranda S, Rutishauser D, Ernfors P - Nucleic Acids Res. (2014)

Bottom Line: In particular, we show that the chromatin remodeller HDAC1-NuRD complex is enriched at nascent DNA.Consistently, in contrast to what has been described in tumour cell lines, these chromatin marks were found to be stable during cell cycle progression of ESCs.Our results are therefore compatible with a rapid deacetylation-coupled methylation mechanism during the replication of DNA in ESCs that may participate in the preservation of pluripotency of ESCs during replication.

View Article: PubMed Central - PubMed

Affiliation: Unit of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, 17177 Stockholm, Sweden sergi.aranda@crg.eu.

Show MeSH

Related in: MedlinePlus

The acetylation and methylation of H3K9 are functionally linked upon replication fork passage in ESC. (A) ESCs were incubated with 1 mM VPA for 30 min and labelled the last 10 min with EdU. Input and iPOND samples were analysed by western blot using the indicated antibodies. The arrows indicate specific bands for H3K9me3 and H3K27Ac, the asterisks indicate an unspecific cross-reaction of the antibody. (B) Quantification of the average relative enrichment for the repressive histone marks lysine 9 mono- and trimehtylation and lysine 27 trimethylation (K9me1, K9me3 and K27me3, respectively) of histone H3 and its corresponding acetylated forms (K9Ac and K27Ac), normalized by the levels of PCNA (average ± SEM, biological replicates n = 3; *P < 0.05, t-test). (C) Scheme of FUCCI reporter mAG1-hGem transfected in ESCs. (D) Coloured code scheme for the ESCs transiently transfected with the FUCCI reported mAG-hGem. (E) ESCs expressing the FUCCI reporter gene were analysed by flow cytometry. The histogram shows the proportion of cells and fluorescence intensity. The DNA content for each gated population is indicated. Green negative population (blue marked) are cells in G1 phase, low-medium intensity green positive population (orange marked) are cells in S phase, and high intensity green positive population (green marked) are cells in late S/G2/M phase. (F) ESCs expressing the FUCCI reporter gene were isolated by FACS and total cell extracts were analysed by western blot with the indicated antibodies.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4066787&req=5

Figure 6: The acetylation and methylation of H3K9 are functionally linked upon replication fork passage in ESC. (A) ESCs were incubated with 1 mM VPA for 30 min and labelled the last 10 min with EdU. Input and iPOND samples were analysed by western blot using the indicated antibodies. The arrows indicate specific bands for H3K9me3 and H3K27Ac, the asterisks indicate an unspecific cross-reaction of the antibody. (B) Quantification of the average relative enrichment for the repressive histone marks lysine 9 mono- and trimehtylation and lysine 27 trimethylation (K9me1, K9me3 and K27me3, respectively) of histone H3 and its corresponding acetylated forms (K9Ac and K27Ac), normalized by the levels of PCNA (average ± SEM, biological replicates n = 3; *P < 0.05, t-test). (C) Scheme of FUCCI reporter mAG1-hGem transfected in ESCs. (D) Coloured code scheme for the ESCs transiently transfected with the FUCCI reported mAG-hGem. (E) ESCs expressing the FUCCI reporter gene were analysed by flow cytometry. The histogram shows the proportion of cells and fluorescence intensity. The DNA content for each gated population is indicated. Green negative population (blue marked) are cells in G1 phase, low-medium intensity green positive population (orange marked) are cells in S phase, and high intensity green positive population (green marked) are cells in late S/G2/M phase. (F) ESCs expressing the FUCCI reporter gene were isolated by FACS and total cell extracts were analysed by western blot with the indicated antibodies.

Mentions: To investigate whether histone deacetylation and methylation are functionally linked after fork passage in ESCs, we used valproic acid (VPA), a specific inhibitor of class I HDACs with high affinity for HDAC1 (39). VPA holds promise in regenerative medicine, since it has shown to be a potent inducer of pluripotency from somatic cells (40,41). ESCs were treated with a short pulse (30 min) of VPA at low concentration with an EdU pulse during the last 10 min. Nascent chromatin was thereafter purified by iPOND and selected histone modifications were analysed by western blot with specific antibodies (Figure 6A). As expected, acute treatment with VPA led to a marked increase of H3K9Ac levels, both in the input and at nascent DNA (Figure 6A and B). Interestingly, H3K9 mono- and trimethylation was markedly reduced specifically on nascent DNA, in contrast to the input chromatin that remained unaffected. However, the other major repressive histone mark, the methylated lysine 27 at histone H3 and its acetylated form was not significantly affected. These results show that VPA has pronounced effects on the deposition of epigenetic marks during DNA replication, and suggests that in ESCs, HDAC1 could act at nascent DNA by regulating the rapid deacetylation of H3K9 in ESCs, which is necessary for their subsequent methylation during replication.


Identification of a large protein network involved in epigenetic transmission in replicating DNA of embryonic stem cells.

Aranda S, Rutishauser D, Ernfors P - Nucleic Acids Res. (2014)

The acetylation and methylation of H3K9 are functionally linked upon replication fork passage in ESC. (A) ESCs were incubated with 1 mM VPA for 30 min and labelled the last 10 min with EdU. Input and iPOND samples were analysed by western blot using the indicated antibodies. The arrows indicate specific bands for H3K9me3 and H3K27Ac, the asterisks indicate an unspecific cross-reaction of the antibody. (B) Quantification of the average relative enrichment for the repressive histone marks lysine 9 mono- and trimehtylation and lysine 27 trimethylation (K9me1, K9me3 and K27me3, respectively) of histone H3 and its corresponding acetylated forms (K9Ac and K27Ac), normalized by the levels of PCNA (average ± SEM, biological replicates n = 3; *P < 0.05, t-test). (C) Scheme of FUCCI reporter mAG1-hGem transfected in ESCs. (D) Coloured code scheme for the ESCs transiently transfected with the FUCCI reported mAG-hGem. (E) ESCs expressing the FUCCI reporter gene were analysed by flow cytometry. The histogram shows the proportion of cells and fluorescence intensity. The DNA content for each gated population is indicated. Green negative population (blue marked) are cells in G1 phase, low-medium intensity green positive population (orange marked) are cells in S phase, and high intensity green positive population (green marked) are cells in late S/G2/M phase. (F) ESCs expressing the FUCCI reporter gene were isolated by FACS and total cell extracts were analysed by western blot with the indicated antibodies.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4066787&req=5

Figure 6: The acetylation and methylation of H3K9 are functionally linked upon replication fork passage in ESC. (A) ESCs were incubated with 1 mM VPA for 30 min and labelled the last 10 min with EdU. Input and iPOND samples were analysed by western blot using the indicated antibodies. The arrows indicate specific bands for H3K9me3 and H3K27Ac, the asterisks indicate an unspecific cross-reaction of the antibody. (B) Quantification of the average relative enrichment for the repressive histone marks lysine 9 mono- and trimehtylation and lysine 27 trimethylation (K9me1, K9me3 and K27me3, respectively) of histone H3 and its corresponding acetylated forms (K9Ac and K27Ac), normalized by the levels of PCNA (average ± SEM, biological replicates n = 3; *P < 0.05, t-test). (C) Scheme of FUCCI reporter mAG1-hGem transfected in ESCs. (D) Coloured code scheme for the ESCs transiently transfected with the FUCCI reported mAG-hGem. (E) ESCs expressing the FUCCI reporter gene were analysed by flow cytometry. The histogram shows the proportion of cells and fluorescence intensity. The DNA content for each gated population is indicated. Green negative population (blue marked) are cells in G1 phase, low-medium intensity green positive population (orange marked) are cells in S phase, and high intensity green positive population (green marked) are cells in late S/G2/M phase. (F) ESCs expressing the FUCCI reporter gene were isolated by FACS and total cell extracts were analysed by western blot with the indicated antibodies.
Mentions: To investigate whether histone deacetylation and methylation are functionally linked after fork passage in ESCs, we used valproic acid (VPA), a specific inhibitor of class I HDACs with high affinity for HDAC1 (39). VPA holds promise in regenerative medicine, since it has shown to be a potent inducer of pluripotency from somatic cells (40,41). ESCs were treated with a short pulse (30 min) of VPA at low concentration with an EdU pulse during the last 10 min. Nascent chromatin was thereafter purified by iPOND and selected histone modifications were analysed by western blot with specific antibodies (Figure 6A). As expected, acute treatment with VPA led to a marked increase of H3K9Ac levels, both in the input and at nascent DNA (Figure 6A and B). Interestingly, H3K9 mono- and trimethylation was markedly reduced specifically on nascent DNA, in contrast to the input chromatin that remained unaffected. However, the other major repressive histone mark, the methylated lysine 27 at histone H3 and its acetylated form was not significantly affected. These results show that VPA has pronounced effects on the deposition of epigenetic marks during DNA replication, and suggests that in ESCs, HDAC1 could act at nascent DNA by regulating the rapid deacetylation of H3K9 in ESCs, which is necessary for their subsequent methylation during replication.

Bottom Line: In particular, we show that the chromatin remodeller HDAC1-NuRD complex is enriched at nascent DNA.Consistently, in contrast to what has been described in tumour cell lines, these chromatin marks were found to be stable during cell cycle progression of ESCs.Our results are therefore compatible with a rapid deacetylation-coupled methylation mechanism during the replication of DNA in ESCs that may participate in the preservation of pluripotency of ESCs during replication.

View Article: PubMed Central - PubMed

Affiliation: Unit of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, 17177 Stockholm, Sweden sergi.aranda@crg.eu.

Show MeSH
Related in: MedlinePlus