The decision to proliferate or differentiate is extremely important for any complex multicellular organism. Improper or unregulated proliferation can lead to a dangerous tumorgenic phenotype, while inappropriate differentiation can severely disrupt the body plan. Myc, max, and mad are three genes from vertebrate cancer biology known to regulate cell proliferation and differentiation. These three genes encode basic/helix-loop-helix/leucine zipper (B/HLH/LZ) transcriptional regulators. The MYC protein has oncogenic potential and can induce unregulated cell proliferation, whereas the MAD protein has an anti-proliferative cell differentiation function. Neither MYC nor MAD can function alone, since both proteins need to dimerize with MAX to bind DNA. MAX, however, can form homodimers in the absence of MYC or MAD. Finally, while in vitro MYC/MAX, MAD/MAX, and MAX/MAX dimers seem to all bind to the same DNA site (CACGTG), only MYC/MAX dimers activate transcription. MAD/MAX dimers recruit the transcriptional repressor protein SIN3 and repress transcription. Despite extensive work on these genes, little is known of the genetic pathway within which these genes act to control proliferation and differentiation. The invariant cell number and lineage program of C. elegans makes it ideal to study the control of cell proliferation and differentiation. Therefore, we have initiated a search for myc, max, and mad-like genes in the nematode and have identified three new genes. These three genes,
cml-1, 2 and 3 (C. elegans myc/max/mad like genes) all encode B/HLH/LZ proteins. The genes
cml-1 and 2 were first predicted by the C. elegans Genome Sequencing Project as ORFs similar to the vertebrate mad gene. Using these genomic sequences, cDNA clones were obtained and used to make fusion proteins. When the CML-1 protein was used to screen a C. elegans mixed stage cDNA lambda
gt11 expression library (courtesy of A. Fire), a new B/HLH/LZ protein was identified, CML-3. The
cml-3 gene shows extensive similarity to the vertebrate max gene. To identify new interacting proteins, we are currently screening the expression library with CML-2 and CML-3. Band shift analysis with the three proteins showed that the dimerization properties of CML-2 and 3 have diverged from their vertebrate homologues. The band shift experiments showed that CML-1 protein formed a tight heterodimer with CML-3, while CML-2 protein would only form homodimers. CML-1 will not form homodimers, a result similar to vertebrate MAD. Surprisingly, however, CML-3, which is similar to vertebrate MAX, will not homodimerize in vitro. Temporal RT-PCR analysis on polyA selected RNA from staged nematodes, indicates that
cml-1 and 3 are expressed at the same time postembryonically; weakly during the L1 stage and strongly during the L2, L3 and L4 stages, a result consistent with their ability to form a strong heterodimeric pair. The
cml-2 gene is strongly expressed during the postembryonic L2 and L3 stages and weakly by L4. The expression of these genes during these postembryonic stages is consistent with the role vertebrate mad/max genes have in cell differentiation, since during the L1, L2, L3, and L4 stages several cell lineages differentiate into adult specific structures. Current studies are underway to identify the spatial expression patterns of these genes and elucidate their loss of function and gain of function phenotypes. Finally using these genes it may be possible to identify a nematode myc gene and therefore gain a better understanding of the genetic pathway that regulates proliferation and differentiation.