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Regulation of Myf5 Early Enhancer by Histone Acetyltransferase p300 during Stem Cell Differentiation.

Francetic T, Le May M, Hamed M, Mach H, Meyers D, Cole PA, Chen J, Li Q - Mol. Biol. (N.Y.) (2012)

Bottom Line: Thus, p300 is directly involved in the regulation of the Myf5 early enhancer, and is important for specific histone acetylation and transcription factor recruitment.This connection of p300 HAT activity with H3-K27 acetylation and β-catenin signalling during myogenic differentiation in vitro offers a molecular insight into the enhancer-elements participation observed in embryonic development.In addition, pluripotent stem cell differentiation is a valuable system to dissect the signal-dependent regulation of specific enhancer element during cell fate determinations.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cellular and Molecular, Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON Canada.

ABSTRACT
Skeletal myogenesis is an intricate process coordinated temporally by multiple myogenic regulatory factors (MRF) including Myf5, which is the first MRF expressed and marks the commitment of skeletal muscle lineage. The expression of Myf5 gene during early embryogenesis is controlled by a set of enhancer elements, and requires the histone acetyltransferase (HAT) activity of transcriptional coactivator p300. However, it is unclear as to how different regulatory signals converge at enhancer elements to regulate early Myf5 gene expression, and if p300 is directly involved. We show here that p300 associates with the Myf5 early enhancer at the early stage of stem cell differentiation, and its HAT activity is important for the recruitment of β-catenin to this early enhancer. In addition, histone H3-K27 acetylation, but not H3-K9/14, is intimately connected to the p300 HAT activity. Thus, p300 is directly involved in the regulation of the Myf5 early enhancer, and is important for specific histone acetylation and transcription factor recruitment. This connection of p300 HAT activity with H3-K27 acetylation and β-catenin signalling during myogenic differentiation in vitro offers a molecular insight into the enhancer-elements participation observed in embryonic development. In addition, pluripotent stem cell differentiation is a valuable system to dissect the signal-dependent regulation of specific enhancer element during cell fate determinations.

No MeSH data available.


Related in: MedlinePlus

Histone acetylation and myogenic differentiation. (A) Schematic presentation of the aggregation protocol for P19 cell differentiation. Cells were treated with DMSO in the presence or absence of RA (10 nM) during EB formation and maintained as adhesive culture for additional 5 days without treatments to develop skeletal myocytes. (B) The cells were stained on day 9 with specific antibodies for microscopic analysis of MyoD (red), myosin heavy chain (MyHC, green), and with Hoechst to visualize nuclei (blue). (C) Myogenin gene expression and global histone H3 acetylation (H3-Ac) on day 4 and day 9 of differentiation was examined by Western blot analysis. (D) Quantification of the H3 acetylation blots is presented as fold changes in relation to the undifferentiated control (mean ± SD, n = 3). Statistical significance is denoted by * to indicate p < 0.05 relative to the undifferentiated control.
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Figure 1: Histone acetylation and myogenic differentiation. (A) Schematic presentation of the aggregation protocol for P19 cell differentiation. Cells were treated with DMSO in the presence or absence of RA (10 nM) during EB formation and maintained as adhesive culture for additional 5 days without treatments to develop skeletal myocytes. (B) The cells were stained on day 9 with specific antibodies for microscopic analysis of MyoD (red), myosin heavy chain (MyHC, green), and with Hoechst to visualize nuclei (blue). (C) Myogenin gene expression and global histone H3 acetylation (H3-Ac) on day 4 and day 9 of differentiation was examined by Western blot analysis. (D) Quantification of the H3 acetylation blots is presented as fold changes in relation to the undifferentiated control (mean ± SD, n = 3). Statistical significance is denoted by * to indicate p < 0.05 relative to the undifferentiated control.

Mentions: P19 pluripotent stem cells have been used extensively to study the molecular mechanisms of cellular differentiation [22–24]. In tissue cultures, P19 cells can be induced into myogenic differentiation with an aggregation protocol (Figure 1A) which involves the formation of embryonic bodies (EBs) and the use of small molecule inducers [23,24]. As previously reported, treatment with DMSO during EB formation induced the commitment of P19 cells into skeletal myocytes in a relatively low efficacy, and the elongated bipolar skeletal myocytes developed by day 9 of differentiation (Figure 1B). Cotreatment of the EBs with all-trans retinoic acid (RA) significantly enhanced the development of skeletal myocytes, which also exhibited a more intensive staining of myosin heavy chain as revealed by the immune fluorescence microscopic analysis (Figure 1B). In addition, MyoD protein co-stained with myosin heavy chain in the developing myocytes (Figure 1B) and the myogenin protein, an identity marker of skeletal myocytes, was detected by Western blot analysis by day 9 (Figure 1C).


Regulation of Myf5 Early Enhancer by Histone Acetyltransferase p300 during Stem Cell Differentiation.

Francetic T, Le May M, Hamed M, Mach H, Meyers D, Cole PA, Chen J, Li Q - Mol. Biol. (N.Y.) (2012)

Histone acetylation and myogenic differentiation. (A) Schematic presentation of the aggregation protocol for P19 cell differentiation. Cells were treated with DMSO in the presence or absence of RA (10 nM) during EB formation and maintained as adhesive culture for additional 5 days without treatments to develop skeletal myocytes. (B) The cells were stained on day 9 with specific antibodies for microscopic analysis of MyoD (red), myosin heavy chain (MyHC, green), and with Hoechst to visualize nuclei (blue). (C) Myogenin gene expression and global histone H3 acetylation (H3-Ac) on day 4 and day 9 of differentiation was examined by Western blot analysis. (D) Quantification of the H3 acetylation blots is presented as fold changes in relation to the undifferentiated control (mean ± SD, n = 3). Statistical significance is denoted by * to indicate p < 0.05 relative to the undifferentiated control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222083&req=5

Figure 1: Histone acetylation and myogenic differentiation. (A) Schematic presentation of the aggregation protocol for P19 cell differentiation. Cells were treated with DMSO in the presence or absence of RA (10 nM) during EB formation and maintained as adhesive culture for additional 5 days without treatments to develop skeletal myocytes. (B) The cells were stained on day 9 with specific antibodies for microscopic analysis of MyoD (red), myosin heavy chain (MyHC, green), and with Hoechst to visualize nuclei (blue). (C) Myogenin gene expression and global histone H3 acetylation (H3-Ac) on day 4 and day 9 of differentiation was examined by Western blot analysis. (D) Quantification of the H3 acetylation blots is presented as fold changes in relation to the undifferentiated control (mean ± SD, n = 3). Statistical significance is denoted by * to indicate p < 0.05 relative to the undifferentiated control.
Mentions: P19 pluripotent stem cells have been used extensively to study the molecular mechanisms of cellular differentiation [22–24]. In tissue cultures, P19 cells can be induced into myogenic differentiation with an aggregation protocol (Figure 1A) which involves the formation of embryonic bodies (EBs) and the use of small molecule inducers [23,24]. As previously reported, treatment with DMSO during EB formation induced the commitment of P19 cells into skeletal myocytes in a relatively low efficacy, and the elongated bipolar skeletal myocytes developed by day 9 of differentiation (Figure 1B). Cotreatment of the EBs with all-trans retinoic acid (RA) significantly enhanced the development of skeletal myocytes, which also exhibited a more intensive staining of myosin heavy chain as revealed by the immune fluorescence microscopic analysis (Figure 1B). In addition, MyoD protein co-stained with myosin heavy chain in the developing myocytes (Figure 1B) and the myogenin protein, an identity marker of skeletal myocytes, was detected by Western blot analysis by day 9 (Figure 1C).

Bottom Line: Thus, p300 is directly involved in the regulation of the Myf5 early enhancer, and is important for specific histone acetylation and transcription factor recruitment.This connection of p300 HAT activity with H3-K27 acetylation and β-catenin signalling during myogenic differentiation in vitro offers a molecular insight into the enhancer-elements participation observed in embryonic development.In addition, pluripotent stem cell differentiation is a valuable system to dissect the signal-dependent regulation of specific enhancer element during cell fate determinations.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cellular and Molecular, Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON Canada.

ABSTRACT
Skeletal myogenesis is an intricate process coordinated temporally by multiple myogenic regulatory factors (MRF) including Myf5, which is the first MRF expressed and marks the commitment of skeletal muscle lineage. The expression of Myf5 gene during early embryogenesis is controlled by a set of enhancer elements, and requires the histone acetyltransferase (HAT) activity of transcriptional coactivator p300. However, it is unclear as to how different regulatory signals converge at enhancer elements to regulate early Myf5 gene expression, and if p300 is directly involved. We show here that p300 associates with the Myf5 early enhancer at the early stage of stem cell differentiation, and its HAT activity is important for the recruitment of β-catenin to this early enhancer. In addition, histone H3-K27 acetylation, but not H3-K9/14, is intimately connected to the p300 HAT activity. Thus, p300 is directly involved in the regulation of the Myf5 early enhancer, and is important for specific histone acetylation and transcription factor recruitment. This connection of p300 HAT activity with H3-K27 acetylation and β-catenin signalling during myogenic differentiation in vitro offers a molecular insight into the enhancer-elements participation observed in embryonic development. In addition, pluripotent stem cell differentiation is a valuable system to dissect the signal-dependent regulation of specific enhancer element during cell fate determinations.

No MeSH data available.


Related in: MedlinePlus