Muscle differentiation (MyoD) Flashcards
What is the function of MyoD? Outline MyoD.
MyoD is a transcription factor that can induce skeletal muscle differentiation in cells from many different lineages.
MyoD belongs to a much larger class of DNA-binding proteins containing a basic helix-loop-helix (bHLH) domain. Soon after the discovery of MyoD, three closely related genes were identified: Myf5, myogenin, and MRF4.
In vitro, each myogenic regulatory factor (MRF) efficiently binds to consensus CANNTG sites (E boxes), which are present in the promoters and enhancers of muscle-specific genes.
What happens when MyoD is expressed in non-skeletal muscle cells?
When expressed in primary fibroblasts or in a wide variety of other cell types, suchas pigment, nerve, fat and liver, MyoD can convert these cells to skeletal muscle.
What was the first evidence that a single gene can initiate
a complex program of differentiation, acting as a master
switch?
In 1979, Taylor and Jones demonstrated that treating the mouse fibroblast cell line 10T1/2 with the demethylating agent 5-azacytidine generated clones with a skeletal muscle phenotype.
- This finding indicated that DNA demethylation was sufficient to induce skeletal muscle gene expression in these cells. When genomic DNA was isolated from these muscle clones and stably transfected into untreated 10T1/2 cells, myogenic colonies were generated at a frequency that was consistent with the presence of a single locus that could convert the fibroblasts into skeletal muscle cells.
The same cell system was then used to clone the cDNA for the myogenic determination gene Myod. When expressed in primary fibroblasts or in a wide variety of other cell types, such as pigment, nerve, fat and liver, Myod can convert these cells to skeletal muscle.
In at least some primary cell types, Myod
transcriptionis activelysuppressed. How was this discovered? How is Myod suppressed?
While the demethylation of the Myod locus is sufficient to activate its expression in 10T1/2 cells, the Myod gene is not methylated in primary fibroblasts and is not expressed in these cells.
- Heterokaryon formation between primary fibroblasts, which have an unmethylated Myod gene, and 10T1/2 cells, which contain the trans-acting factors necessary for the expression of a transfected unmethylated Myod gene, did not result in the expression of the unmethylated fibroblast Myod gene, indicating that expression of the Myod gene is specifically suppressed in primary fibroblasts.
It was subsequently shown that the homeobox factor Msx1 recruits the linker histone H1B to the Myod enhancer element to repress its transcription. Therefore, in at least some primary cell types, Myod transcription is actively suppressed by a combination of Msx1 and linker histones.
The ability of Myod to convert fibroblasts and other cell types into skeletal muscle strongly indicated that it might have a central role in what process?
Myogenesis
Describe the functional domains of Myod.
Myod (red) forms a heterodimer with an E-protein (green) through the helix-loop-helix domains (helix1 and helix2). The adjacent basic regions (also in an alpha helical conformation) contact the DNA. In Myod, the basic region also contains the ‘myogenic code’. This consists of three residues that are conserved in all of the myogenic bHLH proteins (Myod, Myf5, Myog and Mrf4), which do not directly affect DNA binding but are necessary to activate the transcription of specific muscle genes by either interacting with co-factors or inducing confomational change, or both.
Myod has a single transcriptional activation domain (AD), and a histidine- and cysteine-rich (H/C) region that contains a tryptophan residue that is needed for Myod to interact with the Pbx/Meis complex. The helix 3 region is also required for Myod to cooperatively bind to the Pbx/Meis complex at the Myogenin (Myog) promoter. The E-protein has two independent activation domains (AD1 and AD2) and a domain that can repress the function of either activation domain.
In addition to Myod, what other highly related bHLH proteins are expressed in skeletal muscle?
In addition to Myod, the highly related bHLH proteins Myf5, Mrf4 and Myogenin (Myog) are also expressed in skeletal muscle, and each has a crucial role in muscle cell specification and differentiation.
What is the role of the Eprotein sub-family?
The Eprotein sub-family of bHLH proteins (Tcf3, Tcf4 and Tcf12) has a crucial role in lymphocyte differentiation, and its family members also function as heterodimer partners for many of the tissue-restricted bHLH proteins, such as Myod and Neurod proteins.
What is the result of skeletal muscle bHLH protein knockouts in mice?
- Myod -/-
- Myf5 -/-
- Myod -/- + Myf5 -/-
- Myog -/-
- Myf4 -/-
- Myod -/-: normal muscle development.
- Myf5 -/-: normal muscle development.
- Myod -/- + Myf5 -/-: disrupting both genes results in the absence of myoblasts, indicating that these genes are necessary to establish a viable muscle lineage.
- Myog -/-: myoblasts are present, but disruption prevents muscle differentiation in vivo.
- Myf4 -/-: presence of myotubes, but no myofiber formation.
What is the early expression of these different genes associated with development of?
- Myf5
- Myod
- Early expression of Myf5 is prominent in the epaxial myotome, where it drives the differentiation of the back, intercostal and abdominal wall muscles.
- The early expression of Myod is most prominent in the hypaxial myotome, where it drives the differentiation of the limb, tongue and diaphragm muscles, and the muscles of branchial archderived tissue.
It has been well documented that signaling from the surrounding tissues regulates the expression of the myogenic bHLH genes in the somite: what molecules are involved?
Sonic hedgehog (Shh) from the notochord and floor-plate, Wnt signaling from the dorsal neural tube (see image), and Bmp4 signaling from the adjacent lateral plate mesoderm combine to initiate and restrict myogenesis to the muscle-forming region of the dermamyotome.
Once expressed, how does Myod regulate skeletal muscle cell differentiation? What protein regions are involved?
Myod forms heterodimers with the nearly ubiquitous E-protein sub-family of bHLH proteins through the interaction of the HLH domains. The basic regions act as sequence-specific DNAbinding domains that recognize a binding site with the simple core consensus sequence of CANNTG, termed an E-box, and show additional preferences for internal and flanking sequences. Myod has a single amino-terminal acidic-activation domain (as determined by its fusion to the heterologous DNA-binding domain of the Gal4 protein), whereas E-proteins have a more complex mix of activation and repression domains.
Therefore, the simple model of the transcriptional activity of the myogenic bHLH proteins is that they activate gene transcription** by binding to the **Eboxes**
in the **regulatory regions** of genes that are expressed in **skeletal muscle.
However, Eboxes occur frequently in the genome, not just in the regulatory regions of genes expressed in skeletal muscle so something must limit the potential of these proteins to promiscuously activate genes. Also, skeletal muscle genes are not all expressed simultaneously. Therefore, temporal specificity and promoter specificity must be superimposed on this simple model of a transcription factor and its binding sites.
How does Myod express promoter specificity?
The presence of certain binding sites paired with an E-box confers promoter-specific activity to Myod, or, by extension, to Neurod or the Eproteins, depending on the availability of the cooperating transcription factors.
- MyoD homodimers bind to transcription enhancer elements on DNA (CANNTG). For MyoD there are usually two enhancer specific elements upstream of muscle specific genes that undergo cooperative binding (binding of the first element enhances binding of second). The positive heterodimers are 10x more likely to bind these enhancers than the homodimers.
In addition to cooperative binding, co-factor interaction or sequence-specific DNA/protein interactions might alter the conformation of the Myod complex to effectively expose activation regions.
Cells in the dermomyotome are multipotent and committed to particular cell fate in response to paracrine signaling. These cells specified as ______ __________ _____ (MPCs) then become determined as undifferentiated _________. They delaminate to form the myotome and differentiate into multinucleated ________. Studies of the molecular mechanisms regulating muscle development were triggered by the pioneering discoveries of the MyoD family of _____-_____-____-_____ (bHLH) transcription factors in the late 1980s. Four members of the MyoD family of myogenic regulatory factors (MRFs), ____, ____, ____, and ________, are master regulators of myogenic determination and differentiation. Upstream of MyoD family proteins, transcription factor networks involving ____ induce myogenic specification of MPCs.
Cells in the dermomyotome are multipotent and committed to particular cell fate in response to paracrine signaling. These cells specified as muscle progenitor cells (MPCs) then become determined as undifferentiated myoblasts. They delaminate to form the myotome and differentiate into multinucleated myotubes. Studies of the molecular mechanisms regulating muscle development were triggered by the pioneering discoveries of the MyoD family of basic-helix-loop-helix (bHLH) transcription factors in the late 1980s. Four members of the MyoD family of myogenic regulatory factors (MRFs), MyoD, Myf5, MRF4, and myogenin, are master regulators of myogenic determination and differentiation. Upstream of MyoD family proteins, transcription factor networks involving Pax3 induce myogenic specification of MPCs.
What are the extracellular signalling molecules and transcription factors involved in muscle development?
Extracellular signaling molecules, including Wnts, Sonic hedgehog (Shh), and BMPs, which are secreted from the surrounding environment, regulate the determination and differentiation of the sclerotome and each region of the dermomyotome. These signaling proteins induce transcription factor networks in stem or progenitor cells to determine osteogenic or myogenic fate.
In contrast, Wnt1 and Wnt3a, secreted from the dorsal neural tube, together with Shh, which is secreted from the floor plate of the neural tube and the notochord, determine myogenic fate in the epaxial dermomyotome. On the other hand, Wnt7a, secreted from the dorsal ectoderm, specifies the myogenic fate in the hypaxial dermomyotome.
Among the MyoD family of MRFs, Myf5, MRF4, and MyoD serve as myogenic determination factors.
Upstream of the myogenic regulatory factor (MRF) cascade, other transcription factor networks induce specification as muscle progenitor cells (MPCs) or the expression of MRFs.
- The paired domain/homeodomain transcription factors, Pax3 and Pax7, mark MPCs and play crucial roles in myogenic specification. Pax3 directly and indirectly induces Myf5 expression.
- The homeodomain transcription factors Six1/Six4 are coexpressed with Pax3 during mouse somitogenesis. Six1/Six4 directly induce not only Pax3 expression but also Myf5, Myod, and Myog expression
Do myotubes form based on the division or fusion model? How was this discovered?
Fusion model.
This was investigated using a chimeric mouse: the fertilised mouse zygote forms a four-celled blastomeres, which are split in two, and two divisions from different mice can be joined to form a chimera. Isocystrate enzymes are made from two polypeptides and can made from AA or BB genotypes. In non-chimeric mice the enzymes are always AA or BB, in chimeric fusion mice individual muscle cells contain AA, BB, and AB phenotypes due to different nuclei residing in the same cell. This showed that there is the presence of a hybrid enzyme. This means the cells are generated through cell fusion, and not nuclei division without cell division.
How was MyoD discovered?
Subtractive hybridisation.
In 1979, Taylor and Jones demonstrated that treating the mouse fibroblast cell line 10T1/2 with the demethylating agent 5-azacytidine generated clones with a skeletal muscle phenotype.
This finding indicated that DNA demethylation was sufficient to induce skeletal muscle gene expression in these cells. When genomic DNA was isolated from these muscle clones and stably transfected into untreated 10T1/2 cells, myogenic colonies were generated at a frequency that was consistent with the presence of a single locus that could convert the fibroblasts into skeletal muscle cells.
mRNA and cDNA were comared to find the novel element. This led to the identification of a single cDNA, named MyoD, which was capable of converting a variety of cell types (e.g., fibroblasts, chondrocytes, neurons, amniocytes) to myoblasts.
