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Cell Determination and Differentiation: The Muscle Paradigm
How does the genome orchestrate the formation of skeletal muscle?
Much of the current research in developmental biology is focused on identifying
the genes that are involved in determinative events in development and unraveling
the roles of the proteins they encode. Our understanding of the molecular
events leading to functional cells and tissues remains quite sketchy. A
major discovery that has facilitated progress in understanding cell determination
and cell differentiation came with the discovery of a family of myogenic
regulatory factors (MRFs), which are a group of transcription factors
involved in switching on the muscle cell lineage during development. This
mechanism serves as a model for the gene control of cell determination and
The initial member of the MRF family to be discovered was MyoD.
How was MyoD discovered? See Browder et al. (1991), pages 731-737,
especially Figs. 18.4-18.7; Gilbert (1997), pages 349-351; Kalthoff, 1996,
pages 463-467; Wolpert et al., 1998, pages 287-288.
The other members of this family in vertebrates are Myf-5, myogenin
and MRF4. Each of these factors has the potential to turn tissue
culture cells into myoblasts, which can - in turn - fuse with one-another
and differentiate into muscle. Myoblast fusion occurs when growth factors
become limiting and the myoblasts cease dividing (Olson, 1992).
The genes encoding the MRFs are thought to be master regulatory genes, whose
expression initiates a cascade of events that lead to muscle cell differentiation.
They are expressed in a hierarchical fashion during myogenesis. Myf-5 and
MyoD are expressed in cultured myoblasts (and continue to be expressed after
muscle differentiation). Myogenin is expressed after myoblast fusion. It
is an essential intermediate as shown by the prevention of myoblast differentiation
by inhibition of myogenin expression with antisense oligonucleotides (Fig.
1, Florini and Ewton, 1990). MRF4 is expressed only after muscle differentiation.
The MRFs share a region of homology with two functionally significant domains:
the helix-loop-helix (HLH) domain, which facilitates dimerization, and the
basic region, which contains positively charged amino acids that mediate
binding to DNA. These characteristics define a large family of proteins
that function primarily as transcriptional activators. These are the basic
helix-loop-helix (bHLH) proteins.
MRFs form functional entities that bind to DNA by dimerizing with a member
of the ubiquitously-expressed E protein family. This family includes E12,
E47, ITF1 and ITF2. The most prevalent heterodimers
in myotube extracts contain E12, but any of the E proteins can pair with
the MRFs to form a functional heterodimer.
Dimerization is essential for bHLH protein function, but their specificity
of binding to DNA is due to the basic region. Modifications to this region
can either abolish the DNA binding capability of MyoD or eliminate its ability
to activate transcription of muscle-specific genes (see Figs 2, 3 and 6,
Davis et al., 1990). Thus, these mutants act like dominant-negative
inhibitors of wild-type MyoD by competing with it for binding to its partners
and inhibiting its activity.
Nature has produced its own dominant-negative inhibitor of the MRFs. The
interactions of MRFs with DNA can be prevented by members of a family of
HLH factors called "Id", which stands for "inhibitor
of DNA binding". Id proteins lack a basic region. When they bind to
MRF proteins, they impede their ability to bind to DNA and activate transcription
of target genes (Fig. 6, Benezra et al., 1990). The inhibitory role
of Id proteins is supported by the observations that:
- Id proteins are expressed in proliferating myoblasts in culture, but
disappear when the myoblasts differentiate to form myotubes;
- overexpression of Id protein in cultured myoblasts prevents their differentiation
into myotubes (Jen et al., 1992);
- Id transcripts are detected during the gastrula stage of mouse development
before MRF transcripts first appear and are downregulated before MRFs are
expressed (Wang et al., 1992).
Although the roles of Id in embryonic development are uncertain, the
evidence suggests that it is initially an inhibitor of myogenesis and its
downregulation then permits myogenesis to proceed by allowing MRFs to bind
DNA of target genes.
MRF proteins bind to a sequence in the promoter of target genes called the
E box. E boxes contain the sequence CANNTG (where N is any
nucleotide). The genes encoding the MRFs contain an E box, which suggests
that these proteins may regulate their own and one-another's transcription.
Each MRF presumably owes its functional distinctiveness to unique sequences
outside the bHLH domain.
- What is the mode of action of 5-azacytidine?
- What are CpG islands?
- How did the use of 5-azacytidine lead to the discovery of MyoD1?
- How was transfection used to confirm that MyoD1 is sufficient for myogenic
- What are the properties of the protein encoded by MyoD1?
- What is a mobility shift assay, and how was this technology used to
demonstrate the interaction between the MyoD1 protein and the muscle-specific
creatine kinase enhancer?
- Name the members of the myogenic regulatory factor family.
- What properties do the MRFs share?
- What is the relationship between growth and myoblast fusion?
- What is the significance of myoblast fusion?
- Describe the E protein family and discuss their functional relationship
with the MRFs.
- Describe the functional properties of the helix-loop-helix domain and
the basic region of MRFs.
- What is your evidence to back up your interpretation of the role of
the basic region?
- What is Id, and what evidence can you cite regarding its functional
- What is the E box?
Arnold, H.-H. and Winter, B. 1998. Muscle differentiation: more complexity
to the network of myogenic regulators. Curr. Opinion Genet. Develop. 8:
Kelly, R.G., Zammit, P.S., Schneider, A., Alonso, S., Biben, C. and Buckingham,
M.E. 1997. Embryonic and fetal myogenic programs act through separate enhancers
at the MLC1F/3F locus. Develop. Biol. 187: 183-199.
Jen, Y., H. Weintraub and R. Benezra. 1992. Overexpression of Id protein
inhibits the muscle differentiation program: in vivo association of Id with
E2A proteins. Genes & Dev. 6: 1466-1479.
Ludolph, D.C. and S.F. Konieczny. 1995. Transcription factor families: muscling
in on the myogenic program. FASEB J. 9: 1595-1604.
Maroto, M., Reshef, R., Münsterberg, A.E., Koester, S., Goulding, M.
and Lassar, A.B. 1997. Ectopic Pax-3 activates MyoD and Myf-5
expression in embryonic mesoderm and neural tissue. Cell 89: 139-148.
Rawls, A. and Olson, E.N. 1997. MyoD meets its maker. Cell 89: 5-8.
Tajbakhsh, S., Rocancourt, D., Cossu, G. and Buckingham, M. 1997. Redefining
the genetic hierarchies controlling skeletal myogenesis: Pax-3 and
Myf-5 act upstream of MyoD. Cell 89: 127-138.
Links to related material
The NeuroD Family: Neurogenic bHLH Proteins
This material is based substantially upon a recent review article
on this subject (Browder, 1997).
Benezra, R., R.L. Davis, D. Lockshon et al. 1990. The protein
Id: A negative regulator of helix-loop-helix DNA binding proteins. Cell
Browder, L.W. 1997. Gene expression, cell determination and differentiation.
In Principles of Tissue Engineering, edited by R. Lanza, R. Langer,
and W. Chick. R.G. Landes, Austin, Texas. Pages 79-86.
Browder, L.W., C.A. Erickson and W.R. Jeffery. 1991. Developmental Biology.
Third ed. Saunders College Publishing. Philadelphia.
Davis, R.L., P.-F. Cheng, A.B. Lassar and H. Weintraub. 1990. The MyoD DNA
binding domain contains a recognition code for muscle-specific gene activation.
Cell 60: 733-746.
Florini, J.R. and D.A. Ewton. 1990. Highly specific inhibition of IGF-I-stimulated
differentiation by an antisense oligodeoxyribonucleotide to myogenin mRNA.
J. Biol. Chem. 265: 13435-13437.
Olson, E.N. 1992. Interplay between proliferation and differentiation within
the myogenic lineage. Develop. Biol. 154: 261-272.
Wang, Y., R. Benezra and D.A. Sassoon. 1992. Id expression during mouse
development: a role in morphogenesis. Dev Dynamics 194: 222-230.