Cullin4 RING finger ubiquitin ligase (CRL4) is a key regulator of DNA repair and replication. DDB1 (damaged DNA binding) is the adapter component of the CRL4 complex, and binds diverse substrate recognition subunits (SRSs) to facilitate ubiquitin-mediated degradation of cellular targets. Inactivation of the CRL4 components CUL-4 or DDB-1 produces a fully penetrant germline defect. The DNA content of the germ cells suggests an arrest or elongation of G1 phase, and there is no evidence of the DNA re-replication that is observed in somatic cells of the
cul-4 or
ddb-1 mutants. Notably, nucleoli in the mutant germ cells exhibit an abnormal, globular morphology. We have identified DCAF-1 (DDB1 and CUL4-associated factor) as the SRS that is specific for the CRL4 germline function. The DCAF-1 protein does not appear to be critical for somatic development, as
dcaf-1 mutants or
dcaf-1 (RNAi) animals develop normally to become sterile adults. We noticed that
ncl-2(
e1896) mutants have a similar germ cell nucleolar morphology defect, and the
ncl-2 gene is located in the same chromosomal region as the
dcaf-1 gene. Significantly,
dcaf-1 and
ncl-2 mutations fail to complement each other, implying that they are the same gene. Transmission electron microscopy reveals that
ncl-2 mutant germ cells possess significantly fewer ribosomes than wild type, suggesting a profound defect in ribosome biogenesis. Recently we observed that reducing the activity of FOG-1 rescues the
dcaf-1 germ cell nucleolar morphology defect. FOG-1 is a cytoplasmic polyadenylation element binding (CPEB) protein that is required for sperm specification. Inactivation of FOG-3, which similarly functions in spermatogenesis, also rescues the nucleolar morphology defect. We are currently investigating the previously unrecognized role of FOG-1 and FOG-3 in ribosome biogenesis and nucleolar morphology in order to better understand the molecular mechanisms underlying this novel function of the CRL4DCAF-1 complex. 1181B Identification of novel target genes of CeTwist homodimers. Nirupama Singh, Peng Wang, Ann Corsi. Dept. of Biology, The Catholic University of America, Washington D.C. Our laboratory is interested in the protein, Twist, a transcription factor that functions as a heterodimer and homodimer in the developing mesoderm in many organisms. Mutations in the human gene encoding the Twist protein and in genes that encode its downstream target genes lead to craniosynostotic diseases characterized by premature fusion of skull sutures. Twist functions as a homodimer and heterodimer in humans; however a detailed understanding of the effect on target genes for each dimer is not known. We are interested in investigating how CeTwist, coded for by
hlh-8, functions in mesoderm development of C. elegans. Previous work in our laboratory provides some evidence that CeTwist functions as both a homodimer and heterodimer. We have already identified a number of target genes of CeTwist heterodimers but not of homodimers. In C. elegans, having a group of target genes that respond to each dimer will help to understand how CeTwist regulates individual target genes. Our aim is to find novel target genes of CeTwist homodimers. Finding novel target genes of CeTwist homodimers may also help us to identify human homologues which are mutated in related craniosynostotic syndromes but have not been identified yet. To find homodimer targets, the approach we are taking is to overexpress CeTwist by using an inducible heat shock promoter and finding the potential target genes that are overexpressed using Affymetrix oligonucleotide microarrays. Using this approach, we found a total of 106 genes which were upregulated. From this list, 22 genes were given high priority for further study on the basis of their expression level, and homology in other organisms. Using transcriptional GFP reporters, we are testing the expression pattern of these genes. We are interested in a pattern that may partially overlap with the CeTwist expression pattern. So far, three potential target genes based on expression pattern have been identified and are being examined further. Our next step is to examine carefully the expression pattern of the remaining constructs to determine which genes are the best candidates to be CeTwist homodimer target genes and to validate the three probable target genes through further experiments.