Differential role of p300 and CBP acetyltransferase during myogenesis: p300 acts upstream of MyoD and Myf5 - PubMed (original) (raw)

Differential role of p300 and CBP acetyltransferase during myogenesis: p300 acts upstream of MyoD and Myf5

Jeanne-Françoise Roth et al. EMBO J. 2003.

Abstract

Studies in tissue culture cells have implicated p300 and CBP acetyltransferases in myogenic regulatory factor (MRF) mediated transcription and terminal differentiation of skeletal muscle cells. However, in vivo data placing p300 and CBP on myogenic differentiation pathways are not yet available. In this report we provide genetic evidence that p300 but not CBP acetyltransferase (AT) activity is required for myogenesis in the mouse and in embryonic stem (ES) cells. A fraction of embryos carrying a single p300 AT- deficient allele exhibit impaired MRF expression, delayed terminal differentiation and a reduced muscle mass. In mouse embryos lacking p300 protein, Myf-5 induction is severely attenuated. Similarly, ES cells homozygous for a p300 AT or a p300 null mutation fail to activate Myf5 and MyoD transcription efficiently, while Pax3, acting genetically upstream of these MRFs, is expressed. In contrast, ES cells lacking CBP AT activity express MyoD and Myf5 and undergo myogenic differentiation. These data reveal a specific requirement for p300 and its AT activity in the induction of MRF gene expression and myogenic cell fate determination in vivo.

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Figures

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Fig. 1. Targeting strategy to generate p300 AT mutant ES cells and characterization of p300 or CBP AT-mutant ES cells. (A) Overall organization of the p300 protein, the wild-type p300 genomic locus, the targeting vector and the recombined loci is shown. The asterisks indicate the site of the mutation, which harbors a _de novo Nhe_I restriction site. (B) Southern blot with probe 1 analysing genomic DNA isolated from wild-type, heterozygous (p300+/AS-neo) and homozygous p300 KO (_p300_AS-neo/AS-neo) and p300 AT– (_p300_AS/AS) ES cells. The probes and the size of the genomic DNA fragments following _Nhe_I restriction digest are indicated in (A). (C and F) Western blot analysis showing p300 and CBP protein levels in (C) p300 KO and p300 AT– cells and (F) CBP KO (_cbp_AS-neo/AS-neo) and CBP AT– (_cbp_AS/AS) cells. (E) E1A binding assay: western blot analysis following E1A immunoprecipitation from ES cells infected with adenovirus using, from top to bottom, E1A, p300 or CBP antibodies. (D and G) HAT assay with (D) p300 KO and three independent p300 AT– cell lines and (G) CBP KO and two independent CBP AT– cell lines.

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Fig. 2. Histological analysis of E18.5 wild-type and p300+/AS mouse embryos. HE staining of sagittal (A–D) or transverse (E and F) mouse sections. An overview of the embryos is shown in (A) and (B); black arrows point to the body wall. Enlarged views of the trapezius muscle (C and D), the tongue (E and F), the diaphragm (G and H) and the intercostal muscles (I and J). Scale bars: (A and B), 2 mm; (C–H), 100 µm; (I and J) 500 µm. White arrowheads mark muscle fibers in the trapezius (C and D) and loose muscle fibers in the tongue (F). Black arrowheads indicate the intercostal muscle located between the second and third sternebra (I and J).

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Fig. 3. Skeletal muscle structural protein expression in mouse embryos. (A) Transverse sections of wild-type (wt) and p300+/AS littermates aged E14.5 stained with HE (a and b) and with a sacromeric actin antibody (c–f). Enlarged view (20×) of the paravertebral area: arrowheads indicate the edema (a and b) and the back muscle mass (c and d). Enlarged view (40×) of the tongue (e and f); arrowheads highlight actin staining. (B) Transverse section of wild-type and cbp+/AS mice aged E14.5 stained with HE (a and b) and enlarged view (20×) of the paravertebral region following sarcomeric actin staining (c and d). (C) Myosin heavy-chain whole-mount immunostaining of E10.5 wild-type and p300+/AS littermates using MF20. Scale bar, 500 µm. Filled arrowheads indicate staining in the posterior region of the wild-type embryo which is absent in the p300+/AS littermate.

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Fig. 4. MRF expression in mouse embryos. (A) MRF expression in the paravertebral region of E14.5 wild-type and p300+/AS embryos. MyoD (a and b) and myogenin (c and d) immunostaining. S, spinal chord; arrowheads indicate the muscle groups. (B–D) Whole-mount in situ hybridization of wild-type and p300+/AS littermate embryos aged between E9.5 and E11.5 showing (B) Myf5, (C) myogenin and (D) MyoD expression. The arrowhead in (B) highlights a disruption of the Myf5 expression pattern. Scale bars, 500 µm.

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Fig. 5. Formation and proliferation of embryoid bodies. Wild-type E14, CBP KO, CBP AT–, p300 KO and p300 AT– ES cell lines were aggregated as hanging drops for 2 days to form EBs and transferred to bacterial dishes. (A) Microscope analysis of EBs at D2 and D4. The bar represents 400 µm. (B) Histogram representing the diameter of EBs derived from all five cell lines at D2, D4 and D6 of differentiation. The standard deviation represents the average of three independent experiments. Fifty EBs were measured per cell line at each time point.

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Fig. 6. Skeletal muscle formation during in vitro ES cell differentiation. The ES cell lines used were E14 wild type, CBP KO, CBP AT–, p300 KO and p300 AT–. (A) MHC expression at D44 or D66 of differentiation was determined by immunofluorescence. (B) The signal was quantified at D44 using the Adobe quantification toolpack™. The standard deviation is derived from the results of three independent experiments. (C) RT–PCR showing embryonic myosin heavy chain (eMHC) and skeletal actin (skactin) expression at D44. Hprt was used as an internal control. Lane 1, positive control with total RNA from an E11.5 embryo.

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Fig. 7. In vitro osteogenesis. The ES cell lines used were E14, CBP KO, CBP AT–, p300 KO and p300 AT–. (A) Bone mineral deposition in EBs is indicative of osteogenesis and was visualized by alizarin red staining at D20. (B) Bar diagram illustrating the percentage of EBs containing calcium deposits. (C) Osteocalcin and Hprt expression analysed by RT–PCR of 150 EBs at D20 of osteogenesis.

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Fig. 8. Expression of myogenic regulatory factors in differentiated EBs. RNA expression levels of the indicated genes were determined by RT–PCR at D23 (lanes 2–6) and D44 (lanes 7–11). Lane 1, positive control using RNA from E10.5–E13.5 embryos. The ES cell lines used were E14 wild type (lanes 2 and 7), CBP KO (lanes 3 and 8), CBP AT– (lanes 4 and 9), p300 KO (lanes 5 and 10) and p300 AT– (lanes 6 and 11).

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Fig. 9. Myf5 expression in E8.5 p300+/– and _p300_–/– littermates detected by whole-mount in situ hybridization. Arrowheads indicate the somites expressing Myf5. Scale bars, 500 µm.

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Fig. 10. Model placing p300, CBP and their respective AT activities on genetic pathways controlling skeletal myogenesis. Arrows represent an established link. Dashed arrows represent a suggested effect. Note that arrows do not necessarily reflect a direct effect.

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