The MyoD Gene Family: A Nodal Point During 
Specification of Muscle Cell Lineage 
Harold M. Weintraub, M.D., Ph.D. — Investigator 
Dr. Weintraub is also a Full Member in the Division of Basic Sciences at the Fred Hutchinson Cancer 
Research Center and Affiliate Professor of Pathology and Zoology at the University of Washington, Seattle. 
He received his M.D.-Ph.D. degree from the University of Pennsylvania School of Medicine and completed 
his postdoctoral studies at the Medical Research Council Laboratory of Molecular Biology in Cambridge, 
England. Before joining the staff at the Hutchinson Center, Dr. Weintraub was in the Department of 
Biochemical Sciences at Princeton. He is a member of the National Academy of Sciences and the American 
Academy of Arts and Sciences. Among his many honors are the Eli Lilly Award and the Richard Lounsbery 
Award from the National Academy of Sciences. 
THE MyoD gene converts many differentiated 
cell types into muscle. MyoD is a member of 
the protein family characterized by the basic 
helix-loop-helix, a 68-amino acid domain in 
MyoD that is necessary and sufficient for myo- 
genesis. MyoD binds cooperatively to muscle- 
specific enhancers and activates transcription. 
The helix-loop-helix motif is responsible for di- 
merization, and, depending on its dimerization 
partner, MyoD activity can be controlled. 
MyoD senses and integrates many facets of the 
cell state. The gene is expressed only in skeletal 
muscle cells and their precursors; in nonmuscle 
cells it is repressed by specific genes. MyoD acti- 
vates its own transcription, perhaps stabilizing 
commitment to myogenesis. Despite this seem- 
ingly overwhelming evidence that MyoD is cru- 
cial for myogenesis in vertebrates, recent experi- 
ments with Michael Krause and with Andrew Fire 
show that zygotic deletions of MyoD in worms 
result in embryos that retain the capacity to acti- 
vate muscle cell differentiation. 
Muscle-Specific Transcriptional 
Activation by MyoD 
Our laboratory has focused on the mechanism 
by which MyoD activates transcription. Previous 
experiments showed that when the 13-amino 
acid basic region of the ubiquitously expressed 
basic helix-loop-helix gene E12 replaces the 
corresponding basic region of MyoD, the result- 
ing MyoD-E12Basic chimeric protein can bind 
specifically to muscle-specific enhancers in vitro 
and form dimers with El 2, but cannot activate a 
cotransfected reporter gene or convert lOTVi 
cells to muscle. Back mutation of this chimeric 
protein (with the corresponding residues in 
MyoD) reestablishes activation. A specific ala- 
nine is involved in increasing DNA binding, and a 
specific threonine is required for activation. 
A reporter gene containing MyoD-binding sites 
located downstream from the transcription start 
site was used to show that MyoD-E12Basic can 
bind in vivo and thereby inhibit expression of the 
reporter. In vivo binding is also supported by the 
fact that the addition of the "constitutive" VPl6 
activation domain to MyoD-El 2Basic restores full 
trans-activation potential. The normal MyoD acti- 
vation domain maps within the amino-terminal 
53 residues and can be replaced functionally by 
the activation domain of VP16. 
The activity of the MyoD activation domain is 
dramatically elevated when deletions are made 
almost anywhere in the rest of the MyoD mole- 
cule, suggesting that the activation domain in 
MyoD is usually masked. Surprisingly, MyoD- 
E12Basic can activate transcription in CVl and 
B78 cells (but not in IOT1/2 or 3T3 cells), sug- 
gesting that the activation function of the basic 
domain requires a specific factor present in CVl 
and B78 cells. We propose that the masked MyoD 
activation domain requires, in order to function, 
the participation of another factor that recog- 
nizes the basic region. 
By replacing the MyoD basic region and the 
adjacent four-residue junction region with helix 
1 into the corresponding region of El 2, we have 
recently shown that this small section of MyoD is 
sufficient for myogenesis. Our work suggests that 
only three residues, A114, Tn,, and K124, are 
uniquely critical for "recognition factor" func- 
tion and subsequent activation of myogenic gene 
transcription. 
Control of MyoD Activity 
A variety of transforming agents, including a 
variety of growth factors, the oncogenes src, ras, 
fos,jun,fps, erbA, myc, and El A, and such chem- 
ical agents as butyrate and phorbol esters, inhibit 
myogenic differentiation. Most of these reagents 
can inactivate the expressed MyoD protein; in ad- 
dition, several (such as ras and fos) also inhibit 
MyoD transcription. Whether this is a secondary 
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