Worm Breeder's Gazette 14(5): 48 (February 1, 1997)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Isolation and characterization of factors that interact with C. elegans MyoD

Morgan Park (mp130c@nih.gov), Robert Littlejohn, Michael Krause

Laboratory of Molecular Biology NIDDK/NIH Building 5, Room B1-04 5 Center Dr MSC 0510 Bethesda, MD 20892-0510

        The MyoD family of transcription factors are involved in the
specification and development of vertebrate skeletal muscle.  These
factors contain a conserved basic helix-loop-helix (bHLH) domain that
directs DNA binding and protein dimerization.  In vertebrates, the MyoD
family members heterodimerize with an ubiquitously expressed E protein
to activate muscle specific expression.  A single homolog of MyoD
(CeMyoD) and E proteins (CeE/DA) has been found in C. elegans.  Although
CeMyoD is expressed in the nuclei of all body wall muscle cells and
their precursor blastomeres, CeE/DA is not expressed in these cells. 
This result raises the question of whether there are other factors that
heterodimerize with CeMyoD to direct proper body wall muscle
development.  To address this question, we have utilized the yeast
two-hybrid system to identify gene products that interact with CeMyoD.

        We fused the bHLH domain of CeMyoD (aa 150-203) to the binding
domain of Gal4 to screen a cDNA library containing fusions to the
activation domain of Gal4 (the library was kindly provided by Bob
Barstead).  We isolated 3 potential interactors after screening
1,000,000 clones from the cDNA library.  We have sequenced the inserts
from these interacting clones.  The following is a summary of each of
the interacting clones:
1) pal-1.
        One of the clones identified in the screen is the homeodomain
protein pal-1.  It is involved in the development of the somatic
daughters of the P lineage;  the C+D lineages give rise to body wall
muscle.  Loss of pal-1 leads to a loss of posterior body wall muscle. 
The link between pal-1 and CeMyoD is not clear since the expression of
pal-1 protein disappears prior to the onset of stable CeMyoD expression
in the embryo.  Additionally, pal-1 expression appears to be restricted
from body wall muscle post-embryonically.  (Thanks to Craig Hunter and
Lois Edgar for sharing information on pal-1).
2) putative GTP binding protein.
        The second clone is similar to a putative GTP binding protein
identified in C. elegans (CGP-1) and in Ascaris (AGP-1).  This clone
lies on Linkage Group V in cosmid TO4H1 and matches two clones from the
Kohara EST library (yk20h4, yk20g2).  We have used the cosmid sequence
to design primers to PCR amplify a 2.5 kB portion of the genomic
sequence upstream of the coding region of the gene to construct GFP and
LacZ reporter constructs.  The reporters are expressed in body wall
muscle, pharyngeal muscle, intestinal muscle, and anal spincter muscle
in adults and larvae.  The earliest expression with these constructs is
seen at the three-fold stage in the pharyngeal and anal spincter
3) cyclin D homolog.
        The sequence from the third clone has matches to three ESTs
(yk118d3, CEESF44, CEESH01), but no matches to sequences in the genomic
database.  The predicted protein sequence is similar to Cyclin D. 
Cyclin D is a G1-cyclin and is involved in decision to differentiate or
to re-enter the cell cycle.  It is thought that Cyclin D acts as a
growth factor sensor to tell cells to exit G1 and differentiate.  In
myogenesis, there is evidence that the myoD family members cooperate
with G1 cyclins (Rb and Cyclin D) to activate skeletal muscle specific
genes and to commit cells to muscle cell development.
        We have generated his-tagged fusion constructs to purify these
proteins for use in generating antibodies, in biochemical studies to
look at the interactions of these proteins with CeMyoD, and in band
shift assays.  We are characterizing the expression of these clones by
in situ hybridization and their possible function by antisense RNA