Distinct activities of Myf5 and MyoD indicate sequential roles in skeletal muscle lineage specification and differentiation (RNA-Seq)
Ontology highlight
ABSTRACT: In this work we compare the molecular functions of Myf5 and MyoD, two highly related bHLH transcription factors that regulate skeletal muscle specification and differentiation. We find MyoD and Myf5 bind the same sites genome-wide but have distinct functions: Myf5 induces histone acetylation without Pol II recruitment or robust gene activation, whereas MyoD induces histone acetylation, recruits PolII and robustly activates gene transcription. RNA-Seq profiling of MyoD and Myf5
Project description:In this work we compare the molecular functions of Myf5 and MyoD, two highly related bHLH transcription factors that regulate skeletal muscle specification and differentiation. We find MyoD and Myf5 bind the same sites genome-wide but have distinct functions: Myf5 induces histone acetylation without Pol II recruitment or robust gene activation, whereas MyoD induces histone acetylation, recruits PolII and robustly activates gene transcription. Chip-seq profiling of MyoD, Myf5, Histone H4 acetylation (H4Ac), and Pol II in MyoD-/-; Myf5-/- MEFs (M&M MEFs)
Project description:In this work we compare the molecular functions of Myf5 and MyoD, two highly related bHLH transcription factors that regulate skeletal muscle specification and differentiation. We find MyoD and Myf5 bind the same sites genome-wide but have distinct functions: Myf5 induces histone acetylation without Pol II recruitment or robust gene activation, whereas MyoD induces histone acetylation, recruits PolII and robustly activates gene transcription.
Project description:In this work we compare the molecular functions of Myf5 and MyoD, two highly related bHLH transcription factors that regulate skeletal muscle specification and differentiation. We find MyoD and Myf5 bind the same sites genome-wide but have distinct functions: Myf5 induces histone acetylation without Pol II recruitment or robust gene activation, whereas MyoD induces histone acetylation, recruits PolII and robustly activates gene transcription.
Project description:Gene expression changes induced by MyoD or Myf5 were examined in a double-knockout fibroblast cell line lacking endogenous functional myoD or myf5 genes. Use of this cell line precluded the possibility of auto- or cross-activation of endogenous myoD or myf5. Myogenin or hrGFP were expressed in parallel samples as controls. Following infection with retrovirus - expressing the relevant myogenic regulatory factor (MRF) from the viral LTR promoter and hrGFP through an IRES element in the same mRNA transcript - GFP+ cells were sorted by FACS and harvested for total RNA. Keywords: other
Project description:Gene expression changes induced by MyoD or Myf5 were examined in a double-knockout fibroblast cell line lacking endogenous functional myoD or myf5 genes. Use of this cell line precluded the possibility of auto- or cross-activation of endogenous myoD or myf5. Myogenin or hrGFP were expressed in parallel samples as controls. Following infection with retrovirus - expressing the relevant myogenic regulatory factor (MRF) from the viral LTR promoter and hrGFP through an IRES element in the same mRNA transcript - GFP+ cells were sorted by FACS and harvested for total RNA. Experiment Overall Design: this experiment include 4 samples and 12 replicates
Project description:MyoD and NeuroD2 are master regulators of myogenesis and neurogenesis and bind to a "shared" E-box sequence (CAGCTG) and a "private" sequence (CAGGTG or CAGATG, respectively). To determine whether private-site recognition is sufficient to confer lineage-specification, we generated a MyoD-mutant with the DNA binding specificity of NeuroD2. Our results demonstrate that redirecting MyoD binding from MyoD-private sites to NeuroD2-private sites, despite preserved binding to the MyoD/NeuroD2-shared sites, is sufficient to change MyoD from a master regulator of myogenesis to a master regulator of neurogenesis. RNA-seq profiling of mouse P19 cells transfected with MyoD, NeuroD2 and chimera mutants. The chimeric mutants are MyoD with the bHLH domain replaced with the NeuroD2 bHLH domain.
Project description:We performed Chip-seq analysis of Myogenic Regulatory Transcription Factors (MYF5 and MYOD) in Fusion Negative Rhabdomyosarcoma cell lines. Endogenous MYF5/H3K27ac Chip-seq was performed in Rh18 cells and MYOD-H3K27ac Chip-seq was performed in RD cells, given that these cell lines express these proteins in a mutually-exclusive manner. Analysis revealed a common subset of enhancer and promoter regions bound by these transcription factors that are enriched for cell cycle regulation and embryonic muscle development pathways. Keywords: rhabdomyiosarcoma, Chip-seq, chromatin, Transcription factos, MYF5, MYOD
Project description:Rhabdomyosarcomas (RMS) are characterized by expression of myogenic specification genes, such as MyoD and/or Myf5, as well as their bHLH partners for heterodimerization, the E-proteins. We have shown that expression of a forced heterodimer of MyoD with one of the E2A proteins, E12, leads to differentiation in a RMS cell culture model when exposed to low serum conditions. Experiment Overall Design: RD cells (a type of RMS) were retrovirally infected with either the MyoD~E heterodimer or an empty control vector, differentiated for 24 hours and then RNA collected.
Project description:The pig is an important animal model that is increasingly used for biomedical research. For example, pigs produced using somatic cell nuclear transfer (SCNT) technology are relevant for transplantation strategies either involving xenotransplantation or the development of human organs in pig for exotransplantation. Pigs, however, are less well characterized than other animal models such as rodents. In previous studies, we produced pigs that lacked skeletal muscle as this was the first step in humanizing this lineage, and we characterized these MYF5/MYOD/MYF6 knockdown embryos to E35. In the current studies, we have evaluated the development and myogenesis in mid-stage porcine embryos produced by SCNT (E41-E90). We comprehensively examined mid-stage MYF5/MYOD/MYF6 knockdown pigs produced by deleting MYF5/MYOD/MYF6 from porcine fibroblasts, cloning these fibroblasts, transferring the early embryos to surrogate gilts, and harvesting the resulting fetal pigs at defined gestational ages. Here, we observed that these genetic deletions resulted in a complete absence of skeletal muscle in these embryos and fetal pigs, an absence of ribs, vertebral abnormalities and an absence of a sternum. Importantly, we observed that these embryos failed to progress beyond E62 indicating embryonic lethality. In addition, we examined porcine myogenesis by evaluating wildtype comparators using gross anatomical, morphological, histological, molecular biological analyzes as well as magnetic resonance imaging of both WT and knockdown embryos and fetal pigs between the ages of E41-E90. We have determined that the knockdown phenotype in pigs is more severe than that observed in rodents as mice with this multiplexed deletion were lethal in the immediate postnatal period. These studies provide an important platform for engineering humanized muscle in gene edited pigs.
Project description:Skeletal muscle contains long multinucleated and contractile structures known as muscle fibers, which arise from the fusion of myoblasts into nucleated myotubes during myogenesis. The myogenic regulatory factor (MRF) MYF5 is the earliest to be expressed during myogenesis and functions as a transcription factor in muscle progenitor cells (satellite cells) and myocytes. In mouse C2C12 myocytes, MYF5 is implicated in the initial steps of myoblast differentiation into myotubes. Ribonucleoprotein immunoprecipitation (RIP) analysis showed that MYF5 bound a subset of myoblast mRNAs; prominent among them was Ccnd1 mRNA, which encodes the key cell cycle regulator CCND1 (Cyclin D1). Biotin-RNA pulldown, UV-crosslinking, and gel shift experiments indicated that MYF5 was capable of binding the 3' untranslated region (UTR) and the coding region (CR) of Ccnd1 mRNA. MYF5 silencing in proliferating growing myoblasts revealed that and MYF5 promoted CCND1 translation, and it also modestly increased transcription of Ccnd1 mRNA. Importantly, silencing MYF5 reduced myoblast growth as well as differentiation of myoblasts into myotubes, while overexpressing MYF5 in C2C12 cells upregulated CCND1 expression. We propose that MYF5 enhances early myogenesis in part by coordinately elevating Ccnd1 transcription and Ccnd1 mRNA translation. Four replicates were utilized from either Control (IgG) or MYF5-immunoprecipitated RNA samples from C2C12 cells growing in either growth medium (GM) or differentiation medium (DM) for a total of sixteen samples.