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Transient Downregulation of Nanog and Oct4 Induced by DETA/NO Exposure in Mouse Embryonic Stem Cells Leads to Mesodermal/Endodermal Lineage Differentiation.

Mora-Castilla S, Tejedo JR, Tapia-Limonchi R, Díaz I, Hitos AB, Cahuana GM, Hmadcha A, Martín F, Soria B, Bedoya FJ - Stem Cells Int (2014)

Bottom Line: Simultaneously, mRNA of mesodermal markers Flk1 and Mef2c are also regulated by the treatment.Acetylated histone H3 occupancy at the promoter of Nanog is involved in the process of reexpression.Furthermore, Nanog binding to the promoter of Brachyury leads to repression of this gene, thus disrupting mesendoderm transition.

View Article: PubMed Central - PubMed

Affiliation: Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER), University Pablo de Olavide, Biomedical Research Network (CIBER) of Diabetes and Related Metabolic Diseases, Red-Tercel, Avenida Américo Vespucio S/N, 41092 Seville, Spain ; Sanford Consortium for Regenerative Medicine, University of California San Diego, 2880 Torrey Pines Scenic Drive, La Jolla, CA 92037, USA.

ABSTRACT
The function of pluripotency genes in differentiation is a matter of investigation. We report here that Nanog and Oct4 are reexpressed in two mouse embryonic stem cell (mESC) lines following exposure to the differentiating agent DETA/NO. Both cell lines express a battery of both endoderm and mesoderm markers following induction of differentiation with DETA/NO-based protocols. Confocal analysis of cells undergoing directed differentiation shows that the majority of cells expressing Nanog express also endoderm genes such as Gata4 and FoxA2 (75.4% and 96.2%, resp.). Simultaneously, mRNA of mesodermal markers Flk1 and Mef2c are also regulated by the treatment. Acetylated histone H3 occupancy at the promoter of Nanog is involved in the process of reexpression. Furthermore, Nanog binding to the promoter of Brachyury leads to repression of this gene, thus disrupting mesendoderm transition.

No MeSH data available.


Related in: MedlinePlus

Nanog reexpression during differentiation acts as a regulator of cell fate decisions (a) ChIP analysis shows relative levels of acetylated histone H3 occupancy at Nanog promoter following NO treatment. (b) Real-time PCR analysis of Nanog and (c) Brachyury expression levels before and after NO treatment. (d) ChIP analysis shows Nanog binding to Brachyury promoter following NO treatment. Histogram plots represent cells from control, NO treated (day 4), and 2 days after NO treatment (day 6) in the presence (left bars) or absence (right bars) of LIF, respectively. Data are mean ± SEM from 5 independent biological replicates.  *Significant difference from undifferentiated control condition (+LIF).
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fig2: Nanog reexpression during differentiation acts as a regulator of cell fate decisions (a) ChIP analysis shows relative levels of acetylated histone H3 occupancy at Nanog promoter following NO treatment. (b) Real-time PCR analysis of Nanog and (c) Brachyury expression levels before and after NO treatment. (d) ChIP analysis shows Nanog binding to Brachyury promoter following NO treatment. Histogram plots represent cells from control, NO treated (day 4), and 2 days after NO treatment (day 6) in the presence (left bars) or absence (right bars) of LIF, respectively. Data are mean ± SEM from 5 independent biological replicates.  *Significant difference from undifferentiated control condition (+LIF).

Mentions: Previous reports have shown that NO induces chromatin reorganizations in mESCs [25, 27]. Thus, we studied changes in the levels of acetylated histone occupancy at the Nanog promoter (Figure 2(a)). Chromatin immunoprecipitation studies show occupancy of acetylated histone H3 on Nanog promoter in control cells cultured in the presence of LIF. A significant reduction was observed in cells cultured in the absence of LIF for the same period of time (Figure 2(a)). The DETA/NO treatment led to a strong and significant reduction in occupancy, either in the absence or presence of LIF. Occupancy is fully recovered 2 days after exposure to DETA/NO. This phenomenon is more apparent in the presence of LIF. Occupancy at Nanog promoter by acetylated histone H3 parallels the Nanog reexpression after DETA/NO treatment both at mRNA (Figure 2(b)) and protein (Figure 1(a)) in cells cultured with LIF. The fact that no substantial recovery of Nanog mRNA levels is observed in cells grown in the absence of LIF indicates that LIF signaling is required for the expression of this gene.


Transient Downregulation of Nanog and Oct4 Induced by DETA/NO Exposure in Mouse Embryonic Stem Cells Leads to Mesodermal/Endodermal Lineage Differentiation.

Mora-Castilla S, Tejedo JR, Tapia-Limonchi R, Díaz I, Hitos AB, Cahuana GM, Hmadcha A, Martín F, Soria B, Bedoya FJ - Stem Cells Int (2014)

Nanog reexpression during differentiation acts as a regulator of cell fate decisions (a) ChIP analysis shows relative levels of acetylated histone H3 occupancy at Nanog promoter following NO treatment. (b) Real-time PCR analysis of Nanog and (c) Brachyury expression levels before and after NO treatment. (d) ChIP analysis shows Nanog binding to Brachyury promoter following NO treatment. Histogram plots represent cells from control, NO treated (day 4), and 2 days after NO treatment (day 6) in the presence (left bars) or absence (right bars) of LIF, respectively. Data are mean ± SEM from 5 independent biological replicates.  *Significant difference from undifferentiated control condition (+LIF).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2: Nanog reexpression during differentiation acts as a regulator of cell fate decisions (a) ChIP analysis shows relative levels of acetylated histone H3 occupancy at Nanog promoter following NO treatment. (b) Real-time PCR analysis of Nanog and (c) Brachyury expression levels before and after NO treatment. (d) ChIP analysis shows Nanog binding to Brachyury promoter following NO treatment. Histogram plots represent cells from control, NO treated (day 4), and 2 days after NO treatment (day 6) in the presence (left bars) or absence (right bars) of LIF, respectively. Data are mean ± SEM from 5 independent biological replicates.  *Significant difference from undifferentiated control condition (+LIF).
Mentions: Previous reports have shown that NO induces chromatin reorganizations in mESCs [25, 27]. Thus, we studied changes in the levels of acetylated histone occupancy at the Nanog promoter (Figure 2(a)). Chromatin immunoprecipitation studies show occupancy of acetylated histone H3 on Nanog promoter in control cells cultured in the presence of LIF. A significant reduction was observed in cells cultured in the absence of LIF for the same period of time (Figure 2(a)). The DETA/NO treatment led to a strong and significant reduction in occupancy, either in the absence or presence of LIF. Occupancy is fully recovered 2 days after exposure to DETA/NO. This phenomenon is more apparent in the presence of LIF. Occupancy at Nanog promoter by acetylated histone H3 parallels the Nanog reexpression after DETA/NO treatment both at mRNA (Figure 2(b)) and protein (Figure 1(a)) in cells cultured with LIF. The fact that no substantial recovery of Nanog mRNA levels is observed in cells grown in the absence of LIF indicates that LIF signaling is required for the expression of this gene.

Bottom Line: Simultaneously, mRNA of mesodermal markers Flk1 and Mef2c are also regulated by the treatment.Acetylated histone H3 occupancy at the promoter of Nanog is involved in the process of reexpression.Furthermore, Nanog binding to the promoter of Brachyury leads to repression of this gene, thus disrupting mesendoderm transition.

View Article: PubMed Central - PubMed

Affiliation: Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER), University Pablo de Olavide, Biomedical Research Network (CIBER) of Diabetes and Related Metabolic Diseases, Red-Tercel, Avenida Américo Vespucio S/N, 41092 Seville, Spain ; Sanford Consortium for Regenerative Medicine, University of California San Diego, 2880 Torrey Pines Scenic Drive, La Jolla, CA 92037, USA.

ABSTRACT
The function of pluripotency genes in differentiation is a matter of investigation. We report here that Nanog and Oct4 are reexpressed in two mouse embryonic stem cell (mESC) lines following exposure to the differentiating agent DETA/NO. Both cell lines express a battery of both endoderm and mesoderm markers following induction of differentiation with DETA/NO-based protocols. Confocal analysis of cells undergoing directed differentiation shows that the majority of cells expressing Nanog express also endoderm genes such as Gata4 and FoxA2 (75.4% and 96.2%, resp.). Simultaneously, mRNA of mesodermal markers Flk1 and Mef2c are also regulated by the treatment. Acetylated histone H3 occupancy at the promoter of Nanog is involved in the process of reexpression. Furthermore, Nanog binding to the promoter of Brachyury leads to repression of this gene, thus disrupting mesendoderm transition.

No MeSH data available.


Related in: MedlinePlus