Contrasting roles for MyoD in organizing myogenic promoter structures during embryonic skeletal muscle development

Abstract

Background: Among the complexities of skeletal muscle differentiation is a temporal distinction in the onset of expression of different lineage-specific genes. The lineage-determining factor MyoD is bound to myogenic genes at the onset of differentiation whether gene activation is immediate or delayed. How temporal regulation of differentiation-specific genes is established remains unclear.

Results: Using embryonic tissue, we addressed the molecular differences in the organization of the myogenin and muscle creatine kinase (MCK) gene promoters by examining regulatory factor binding as a function of both time and spatial organization during somitogenesis. At the myogenin promoter, binding of the homeodomain factor Pbx1 coincided with H3 hyperacetylation and was followed by binding of co-activators that modulate chromatin structure. MyoD and myogenin binding occurred subsequently, demonstrating that Pbx1 facilitates chromatin remodeling and modification before myogenic regulatory factor binding. At the same time, the MCK promoter was bound by HDAC2 and MyoD, and activating histone marks were largely absent. The association of HDAC2 and MyoD was confirmed by co-immunoprecipitation, proximity ligation assay (PLA), and sequential ChIP.

Conclusions: MyoD differentially promotes activated and repressed chromatin structures at myogenic genes early after the onset of skeletal muscle differentiation in the developing mouse embryo.

Keywords: Pbx; gene expression; gene regulation; histone deacetylase; myogenesis; somite.

Figure 1

Pbx1 and acetylated H3K9K14 are present on the myogenin promoter in embryos containing 5 somites and precede the interaction of the MyoD and myogenin regulators and other co-activators and histone modifications associated with myogenin activation. The binding of (A) MyoD, (B), myogenin, (C) Pbx1, (D) AcH3K9K14, (E) Brg1, (F) Prmt5, (G) H3R8Me2, (H) AcH4 to either the myogenin promoter or to a negative control (NC) sequence in embryos containing 5 or 10 somites (5ss or 10ss), in E9.5 embryos containing 25–40 somites, or in yolk sac was determined by real-time PCR. Data represent the mean of three independent experiments +/− standard deviation. p < 0.05 was obtained for all comparisons between yolk sac values and values for any developmental stage for which signal was obtained as well as for values between different developmental stages, with the following exceptions: MyoD and Pbx1 binding in 10 ss and E9.5 embryos were not statistically different and AcH3K9K14 binding in 5ss, 10ss and E9.5 was not statistically different.

Figure 2

Isolation and characterization of E9.5 somite-enriched tissue. (A) E9.5 embryo hybridized to show myogenin localization showing dissection to divide the trunk region into rostral, medial, or caudal somite-containing tissue. For illustrative purposes, the schematic is overlaid on an image of an E9.5 embryo stained for myogenin protein. (B) Western blot analysis showing the protein levels of the indicated proteins present in extracts from rostral, medial, or caudal somite-containing tissue.

Figure 3

Pbx1 and the Brg1 and Prmt5 cofactors, but not MyoD or myogenin, are present on the myogenin promoter in caudal somite-containing tissue isolated from E9.5 embryos. (A) Rostral or caudal somite-containing tissue was used for ChIP for the indicated factors and was assayed by semi-quantitative PCR followed by native gel electrophoresis. Real-time PCR was subsequently used to analyze ChIP experiments performed for (B) MyoD, (C) myogenin, (D) Pbx1, (E) Brg1, (F) Prmt5. Binding to either the myogenin promoter or to a negative control (NC) sequence in rostral or caudal somite-containing tissue from E9.5 embryos or in yolk sac was determined. Data represent the mean of three independent experiments +/− standard deviation. p < 0.05 was obtained for all comparisons between yolk sac and rostral tissue values and for all comparisons between rostral and caudal tissue values.

Figure 4

MyoD and HDAC2 are associated in mouse embryonic tissue. Extracts were made from mouse embryos at the indicated stage and subjected to co-immunoprecipitation experiments as detailed in the Experimental Procedures.

Figure 5

MyoD and HDAC2 are associated in C2C12 myoblasts and in embryonic tissue containing caudal, but not rostral somatic tissue. (A) PLA assays were performed on C2C12 cells in growth (G) phase, at the onset of differentiation (0 h), or at 24 or 48 h post-differentiation. DAPI staining and the overlay is shown for each image. (B) Sections of E9.5 mouse embryos were used for PLA assay. The yellow boxes indicate the areas magnified in the following panel. (C) Images from the yellow boxed fields of tissue in the caudal or rostral somite regions in (B) showing the results of the PLA assay, Hoechst staining, the overlay between the PLA and Hoechst staining, or differential interference contrast (DIC). Within the PLA image, the inset images show four different areas, each outlined by a dotted white line. Scale bar in the insets, 5 micrometers. Images presented are representative. The experiment in (A) was performed independently three times. The analysis presented in (B–C) was performed independently on three different embryos.

Figure 6

Binding of MyoD, myogenin, co-regulators, and modified histones to the MCK promoter in rostral and caudal somite-containing tissue isolated from E9.5 embryos. Real-time PCR was used to analyze ChIP experiments performed for (A) MyoD, (B) myogenin, (C) Brg1, (D) Prmt5, (E) Mef2, (F) AcH3K9K14, (G) AcH4, (H) HDAC2. Binding to either the MCK promoter or to a negative control (NC) sequence in rostral or caudal somite-containing tissue from E9.5 embryos or in yolk sac was determined. Data represent the mean of three independent experiments +/− standard deviation, except for the HDAC2 experiment, which is the mean of six independent experiments +/− standard deviation. p < 0.05 was obtained for all comparisons between yolk sac and rostral tissue values and for all comparisons other than MyoD and Mef2 between rostral and caudal tissue values.

Figure 7

Sequential, or Re-ChIP experiments show co-localization of MyoD and HDAC2 on the MCK promoter in caudal somite-containing tissue. (A) Semi-quantitative PCR analysis of Re-ChIP experiments. The first antibody (1°) and second antibody (2°) used are indicated. (B) Real-time PCR was used to analyze Re-ChIP experiments. Binding to either the MCK promoter or to a negative control (NC) sequence in rostral or caudal somite-containing tissue from E9.5 embryos or in yolk sac was determined. Data represent the mean of three independent experiments +/− standard deviation. p < 0.05 was obtained for all comparisons between yolk sac and rostral tissue values and for all comparisons between rostral and caudal tissue values.

Figure 8

Proposed model indicating the order of events during activation of the myogenin promoter in mouse embryonic tissue. This schematic diagram is a significantly modified version of the diagram published in Figure 10 of (de la Serna et al., 2005) that was amended with permission from the American Society for Microbiology.

Figure 9

Proposed model illustrating the simultaneous presence of MyoD with identified coactivators at the myogenin locus state and with the corepressor HDAC2 at the MCK locus after the onset of differentiation in the somites but before activation of the MCK gene.