THE MyoD GENE FAMILY: A NODAL POINT DURING SPECIFICATION 
OF MUSCLE CELL LINEAGE 
Harold M. Weintraub, M.D., Ph.D. Investigator 
The MyoD gene converts many differentiated cell 
types into muscle. The protein MyoD, a member of 
the basic helix-loop-helix (bHLH) domain, contains 
a 68-amino acid segment that is necessary and suffi- 
cient for myogenesis. It binds cooperatively to mus- 
cle-specific enhancers and activates transcription. 
The helix-loop-helix motif is responsible for dimer- 
ization, and control of MyoD activity depends on its 
dimerization partner. 
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. The protein activates its 
own transcription, perhaps stabilizing commitment 
to myogenesis. Despite this seemingly overwhelm- 
ing evidence that MyoD is crucial for myogenesis in 
vertebrates, recent experiments with Drs. Michael 
Krause and Andrew Fire show that zygotic deletions 
of MyoD in worms result in embryos that retain the 
capacity to activate muscle cell differentiation, pre- 
sumably because there are redundant or alternative 
pathways for myogenic activation. 
Muscle-Specific Transcriptional 
Activation by MyoD 
Dr. Weintraub and his colleagues have been fo- 
cusing on the mechanism by which MyoD activates 
transcription. A major paradox is that other bHLH 
proteins similar to MyoD fail to activate myogenesis. 
For example. El 2 binds to the same sequence as 
MyoD, but it is expressed in all cell types. Previous 
experiments showed that when the 13-amino acid 
basic region of the ubiquitously expressed bHLH 
gene jE'i2 replaces the corresponding basic region of 
MyoD, the resulting MyoD-E12Basic chimeric pro- 
tein could bind specifically to muscle-specific en- 
hancers and form dimers with El 2, but cannot acti- 
vate a cotransfected reporter gene or convert 1 OT^/z 
cells to muscle. Back mutation of this chimeric pro- 
tein (with the corresponding residues in MyoD) 
reestablishes activation. A specific alanine is in- 
volved in increasing DNA binding, and a specific 
threonine is required for activation. These results 
define certain residues in the basic region of MyoD 
as necessary for myogenic activation. When these 
residues are "recognized" as being bound to the 
proper DNA-binding sites, subsequent myogenic ac- 
tivation can occur. 
By replacing the MyoD basic region and the adja- 
cent four-residue junction region with helix 1 into 
the corresponding region of El 2, Dr. Weintraub and 
his colleagues have shown that this small section of 
MyoD (2 1 amino acids) is sufficient for myogenesis. 
Further work suggests that only three residues, Aj , 
Tu5 , and K124, are uniquely critical for "recognition 
factor" function and subsequent activation of myo- 
genic gene transcription. 
Control of MyoD Activity 
A variety of transforming agents inhibit myogenic 
differentiation, including a variety of growth fac- 
tors, oncogenes (src, ras, fos, jun, fps, erbK, myc, 
and El A), and chemical agents such as butyrate and 
phorbol esters. Most of these reagents can inactivate 
the expressed MyoD protein; in addition, several 
(such as ras and fos) also inhibit MyoD transcrip- 
tion. Whether this is a secondary effect due to an 
inhibition of the autoactivation function of MyoD 
protein or a more direct inhibition of MyoD tran- 
scription remains to be determined. Rhabdomyosar- 
coma cells (derived from tumors of patients who 
harbor a genetic predisposition to myogenic tu- 
mors) differentiate poorly but express MyoD, sug- 
gesting that loss of anti-oncogene activity at the 
rhabdomyosarcoma locus can also impinge on 
MyoD action. The specific pathway by which each 
of these oncogenes, anti-oncogenes, and growth fac- 
tors inhibits myogenesis provides a potential clue to 
how MyoD might integrate information coming 
from many aspects of cellular function. 
Recently, in collaboration with the laboratory of 
Dr. Inder Verma (Salk Institute), Dr. Weintraub and 
his colleagues have found that the leucine zipper 
region of the protein expressed by the jun oncogene 
actually binds to the helix-loop-helix region of 
MyoD, both in vivo and in vitro. Similarly, assays for 
recognition factor for MyoD activation show that 
such a factor, which is missing in rhabdomyo- 
sarcoma cell lines, can be provided in trans by 
fusion with lOT'/a cells. Possibly, failure to acti- 
vate myogenesis leads to increased prolifer- 
ation, and then secondary effects that give rise to 
rhabdomyosarcomas . 
Activation of MyoD During Development 
Developmental activation of MyoD is being stud- 
ied in three organisms: mice, worms, and frogs. In 
both mice and worms, deletional analysis has identi- 
fied regulatory sequences upstream of the MyoD 
gene that are important for correct developmental 
GENETICS 283 
