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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.

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Differential recruitment of MMR proteins at nascent DNA.(A) ESCs were incubated with EdU as indicated. The iPOND samples were analysed by western blot using the indicated antibodies. Functional clusters according to Figure 2 are indicated. (B) ESCs were differentiated in N2B27 media and stained with anti-Nanog and anti-Nestin antibodies. The cells in S-phase were labelled by EdU and visualized using the Click reaction. (C) ESCs and differentiated cells were incubated with EdU as indicated. The input and the iPOND samples were analysed by western blot using the indicated antibodies. The amount of EdU-labelled DNA was analysed by dot-blot.
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Figure 3: Differential recruitment of MMR proteins at nascent DNA.(A) ESCs were incubated with EdU as indicated. The iPOND samples were analysed by western blot using the indicated antibodies. Functional clusters according to Figure 2 are indicated. (B) ESCs were differentiated in N2B27 media and stained with anti-Nanog and anti-Nestin antibodies. The cells in S-phase were labelled by EdU and visualized using the Click reaction. (C) ESCs and differentiated cells were incubated with EdU as indicated. The input and the iPOND samples were analysed by western blot using the indicated antibodies. The amount of EdU-labelled DNA was analysed by dot-blot.

Mentions: In addition to the DNA replication cluster, the protein network at nascent DNA in ESCs encompasses 10 functional clusters (Figure 2A). The DNA repair cluster included proteins such as members of the mismatch repair system (MMR: MHS2, MSH6, MLH1) and the double-strand break repair protein MRE11A. These proteins were confirmed to be enriched at nascent DNA in ESCs by western blot with specific antibodies (Figure 3A). The enrichment on MMR proteins at nascent DNA concurs with previous studies in yeast (26) and the more recent iPOND mass-spectrometry data on HEK-293T cells (10,11). These results suggest that DNA repair systems could be an integral part of the replication machinery across different cell types, although analysis of the mutation frequency (12) and total proteins levels between fibroblasts and ESCs (Supplementary Figure S5) indicate quantitative differences in MMR system activity in ESCs and somatic cells.


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)

Differential recruitment of MMR proteins at nascent DNA.(A) ESCs were incubated with EdU as indicated. The iPOND samples were analysed by western blot using the indicated antibodies. Functional clusters according to Figure 2 are indicated. (B) ESCs were differentiated in N2B27 media and stained with anti-Nanog and anti-Nestin antibodies. The cells in S-phase were labelled by EdU and visualized using the Click reaction. (C) ESCs and differentiated cells were incubated with EdU as indicated. The input and the iPOND samples were analysed by western blot using the indicated antibodies. The amount of EdU-labelled DNA was analysed by dot-blot.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: Differential recruitment of MMR proteins at nascent DNA.(A) ESCs were incubated with EdU as indicated. The iPOND samples were analysed by western blot using the indicated antibodies. Functional clusters according to Figure 2 are indicated. (B) ESCs were differentiated in N2B27 media and stained with anti-Nanog and anti-Nestin antibodies. The cells in S-phase were labelled by EdU and visualized using the Click reaction. (C) ESCs and differentiated cells were incubated with EdU as indicated. The input and the iPOND samples were analysed by western blot using the indicated antibodies. The amount of EdU-labelled DNA was analysed by dot-blot.
Mentions: In addition to the DNA replication cluster, the protein network at nascent DNA in ESCs encompasses 10 functional clusters (Figure 2A). The DNA repair cluster included proteins such as members of the mismatch repair system (MMR: MHS2, MSH6, MLH1) and the double-strand break repair protein MRE11A. These proteins were confirmed to be enriched at nascent DNA in ESCs by western blot with specific antibodies (Figure 3A). The enrichment on MMR proteins at nascent DNA concurs with previous studies in yeast (26) and the more recent iPOND mass-spectrometry data on HEK-293T cells (10,11). These results suggest that DNA repair systems could be an integral part of the replication machinery across different cell types, although analysis of the mutation frequency (12) and total proteins levels between fibroblasts and ESCs (Supplementary Figure S5) indicate quantitative differences in MMR system activity in ESCs and somatic cells.

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