The Caenorhabditis elegans Heterochronic Regulator LIN-14 Is a Novel Transcription Factor That Controls the Developmental Timing of Transcription from the Insulin/Insulin-Like Growth Factor Gene ins-33 by Direct DNA Binding

Abstract

A temporal gradient of the novel nuclear protein LIN-14 specifies the timing and sequence of stage-specific developmental events in Caenorhabditis elegans. The profound effects of lin-14 mutations on worm development suggest that LIN-14 directly or indirectly regulates stage-specific gene expression. We show that LIN-14 can associate with chromatin in vivo and has in vitro DNA binding activity. A bacterially expressed C-terminal domain of LIN-14 was used to select DNA sequences that contain a putative consensus binding site from a pool of randomized double-stranded oligonucleotides. To identify candidates for genes directly regulated by lin-14, we employed DNA microarray hybridization to compare the mRNA abundance of C. elegans genes in wild-type animals to that in mutants with reduced or elevated lin-14 activity. Five of the candidate LIN-14 target genes identified by microarrays, including the insulin/insulin-like growth factor family gene ins-33, contain putative LIN-14 consensus sites in their upstream DNA sequences. Genetic analysis indicates that the developmental regulation of ins-33 mRNA involves the stage-specific repression of ins-33 transcription by LIN-14 via sequence-specific DNA binding. These results reinforce the conclusion that lin-14 encodes a novel class of transcription factor.

ACKNOWLEDGMENTS

We are grateful to members of the Ambros laboratory, especially Rosalind Lee, Philip Olsen, and Richard Roy, for technical help and discussion; to Cecilia Henricksson-Spencer for technical assistance at Stockholm University; to Stuart Kim and the Stanford Microarrray Facility for help with the microarray assays; and to N. Hernandez, Y. Gruenbaum, and M. Labouesse for antisera.

This work was supported by PHS grant GM34028 to V.A.