| The heterochronic gene lin-14 affects certain somatic cell-fate choices during post-embryonic development in C. elegans . In lin-14 mutants, cells express stage-specific developmental programs, such as cell divisions or dauer entry, at inappropriate times in development. Loss-of-function mutations in lin-14 result in the precocious execution of L2-specific programs during the first larval stage and the omission of L1-specific programs, while gain-of-function mutations retard the execution of L2-specific programs during the second larval stage and instead cause the re-iteration of L1-specific programs.;I chose to study the molecular mechanisms of lin-14 function using two complementary approaches. First, I characterized lin-14 biochemically by analyzing its association with nuclei in vivo and by performing in vitro DNA binding assays. LIN-14 protein extracted from worms exists in three "forms:" one form is easily-extractable from nuclei by low salt, another form is associated with chromatin, and a third form is associated with the nuclear matrix. Moreover, bacterially-expressed LIN-14 can bind DNA in vitro, and binds preferentially to the consensus sequence GAACA/GC.;Second, I performed microarray analysis to identify genes misregulated under lin-14 loss-of-function or gain-of-function conditions. I identified a number of genes whose transcript levels are regulated either positively or negatively by lin-14, and which could be among lin-14's in vivo targets. Using Northern blot and GFP-reporter fusion analysis I have begun to investigate whether these putative targets are transcriptionally controlled by lin-14. The expression of one of these targets, ins-33, a C. elegans insulin homolog, appears to be controlled by lin-14 at the level of transcription in vivo. LIN-14 protein can bind the ins-33 promoter in vitro at two sites. In addition, the two LIN-14 binding sites within the ins-33 promoter are necessary for lin-14-dependent control of ins-33 transcription in vivo. Thus, lin-14 can function as a transcriptional regulator in the worm, and ins-33 is its first identified direct target. |
