lin-35, a gene originally identified as a class B SynMuv gene, is the sole member of the Retinoblastoma protein (Rb) family in C. elegans. To discover additional functions for Rb family proteins in C. elegans, we have carried out screens to identify mutations that show synthetic interactions with
lin-35 loss of function. Among the isolated alleles were partial LOF mutations in
ubc-18, a ubiquitin conjugating enzyme, and
pha-1, a gene of unknown function. Both mutations lead to early defects in pharyngeal morphogenesis in conjunction with
lin-35 LOF. In addition, two previously isolated suppressors of a
pha-1 temperature sensitive mutation (
e2123),
sup-36 and
sup-37, were found to suppress the synthetic lethality of
lin-35;
pha-1 and
lin-35;
ubc-18 mutants. Based on our genetic data,
sup-36 or
sup-37 may represent mutual targets for negative regulation by the LIN-35, UBC-18, and PHA-1 pathways. To gain insight into the cause of the synthetic lethality and to uncover novel targets for these proteins, we are cloning both suppressor mutations.
sup-36 has been mapped using genetic markers and SNPs to a ~100Kb region on linkage group LGIV. We are currently injecting regional cosmids and using RNAi knockdown to determine the molecular identity of
sup-36. Likewise
sup-37 has been mapped to a small region on LGV and rescue of
sup-37 (as determined by de-suppression of
pha-1;
sup-37 mutants) was obtained through the injection of two adjacent and overlapping cosmids. Interestingly, neither comsid alone is capable of robust rescue, indicating that sequences on both cosmids are required for full function or proper expression of
sup-37. In a separate line of experiments, we have initiated studies to determine the transcriptional targets of
lin-35 as well as the two C. elegans E2F family members,
efl-1 and
efl-2. In one approach, we are using standard microarray analysis to identify genes whose expression is misregulated in the
lin-35 mutant background during development. In addition, to determine the direct regulatory targets of LIN-35 and E2F proteins, we are using the DNA-adenine methylase identification (DAMID) method in conjunction with array analysis. This technique employs the expression of fusion proteins between the chromatin-binding proteins of interest and an active DNA methylase domain from bacteria. Chromosomal regions that become methylated as the result of specific association with fusion proteins can be enriched and used to screen microarrays containing genomic probes, thereby leading to the identification of in vivo targets. Progress on these studies as well as the cloning and characterization of
sup-36 and
sup-37 will be presented.