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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

Curcumin inhibits myogenic differentiation. (A) Cells were treated with curcumin (Cur, 10 µM) during EB formation. Endogenous p300 was immunoprecipitated on day 4 and its HAT activity was examined by HAT assay. Quantification of the p300 HAT activity was presented as percentage of the DMSO control. Error bars represent the standard deviation (n = 5, * P < 0.05). (B) Cells were differentiated with DMSO or RA (10 nM) in the presence or absence of curcumin during EB formation and co-stained for myosin heavy chain (MyHC) and Myf5 on day 9, and with Hoechst to visualize nuclei (blue). The cells were also co-stained for myosin heavy chain and MyoD in parallel. (C) Quantifications of myocytes are presented as the fractions of cells stained positively for myosin heavy chain, Myf5 or MyoD (mean ± SD, n = 3) in relation to the total cell populations. Statistical significance is denoted by * to indicate p < 0.05. (D) The expression of myogenin protein on day 4 and day 9 of differentiation was determined by Western blot analysis.
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Figure 2: Curcumin inhibits myogenic differentiation. (A) Cells were treated with curcumin (Cur, 10 µM) during EB formation. Endogenous p300 was immunoprecipitated on day 4 and its HAT activity was examined by HAT assay. Quantification of the p300 HAT activity was presented as percentage of the DMSO control. Error bars represent the standard deviation (n = 5, * P < 0.05). (B) Cells were differentiated with DMSO or RA (10 nM) in the presence or absence of curcumin during EB formation and co-stained for myosin heavy chain (MyHC) and Myf5 on day 9, and with Hoechst to visualize nuclei (blue). The cells were also co-stained for myosin heavy chain and MyoD in parallel. (C) Quantifications of myocytes are presented as the fractions of cells stained positively for myosin heavy chain, Myf5 or MyoD (mean ± SD, n = 3) in relation to the total cell populations. Statistical significance is denoted by * to indicate p < 0.05. (D) The expression of myogenin protein on day 4 and day 9 of differentiation was determined by Western blot analysis.

Mentions: To delineate the interplay of histone acetylation and nuclear HAT activity during myogenic differentiation, we used curcumin, which inhibits the HAT activity of p300 and CBP, but not PCAF [25]. We tested different concentrations of curcumin (1–100 µM) in P19 stem cells, and administered 10 µM in our studies, since it did not exhibit apparent toxicity to the differentiating cells, while still inhibiting about 40% of p300 HAT activity as determined by the HAT assay (Figure 2A). Next, we examined the effects of curcumin on myogenic differentiation, particularly at the stage of lineage specification.


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)

Curcumin inhibits myogenic differentiation. (A) Cells were treated with curcumin (Cur, 10 µM) during EB formation. Endogenous p300 was immunoprecipitated on day 4 and its HAT activity was examined by HAT assay. Quantification of the p300 HAT activity was presented as percentage of the DMSO control. Error bars represent the standard deviation (n = 5, * P < 0.05). (B) Cells were differentiated with DMSO or RA (10 nM) in the presence or absence of curcumin during EB formation and co-stained for myosin heavy chain (MyHC) and Myf5 on day 9, and with Hoechst to visualize nuclei (blue). The cells were also co-stained for myosin heavy chain and MyoD in parallel. (C) Quantifications of myocytes are presented as the fractions of cells stained positively for myosin heavy chain, Myf5 or MyoD (mean ± SD, n = 3) in relation to the total cell populations. Statistical significance is denoted by * to indicate p < 0.05. (D) The expression of myogenin protein on day 4 and day 9 of differentiation was determined by Western blot analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Curcumin inhibits myogenic differentiation. (A) Cells were treated with curcumin (Cur, 10 µM) during EB formation. Endogenous p300 was immunoprecipitated on day 4 and its HAT activity was examined by HAT assay. Quantification of the p300 HAT activity was presented as percentage of the DMSO control. Error bars represent the standard deviation (n = 5, * P < 0.05). (B) Cells were differentiated with DMSO or RA (10 nM) in the presence or absence of curcumin during EB formation and co-stained for myosin heavy chain (MyHC) and Myf5 on day 9, and with Hoechst to visualize nuclei (blue). The cells were also co-stained for myosin heavy chain and MyoD in parallel. (C) Quantifications of myocytes are presented as the fractions of cells stained positively for myosin heavy chain, Myf5 or MyoD (mean ± SD, n = 3) in relation to the total cell populations. Statistical significance is denoted by * to indicate p < 0.05. (D) The expression of myogenin protein on day 4 and day 9 of differentiation was determined by Western blot analysis.
Mentions: To delineate the interplay of histone acetylation and nuclear HAT activity during myogenic differentiation, we used curcumin, which inhibits the HAT activity of p300 and CBP, but not PCAF [25]. We tested different concentrations of curcumin (1–100 µM) in P19 stem cells, and administered 10 µM in our studies, since it did not exhibit apparent toxicity to the differentiating cells, while still inhibiting about 40% of p300 HAT activity as determined by the HAT assay (Figure 2A). Next, we examined the effects of curcumin on myogenic differentiation, particularly at the stage of lineage specification.

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