Bimolecular fluorescence complementation (BIFC) analysis of AP-1 dimers in Caenorhabditis elegans

Yeong-Shiuh John Shyu, Purdue University

Abstract

Activator protein 1 (AP-1) is one of the most studied basic region-leucine zipper (bZIP) family of transcription factors. AP-1 refers to a group of functionally and structurally similar members of Fos, Jun, ATF, Maf, and JDP subfamily of transcription factors. These AP-1 proteins are capable of forming heterodimers or homodimers to bind DNA and regulate transcription. Although AP-1 proteins have been studied over the past two decades, the cellular and developmental roles of individual AP-1 dimers remain to be difficult to characterize. We have developed an improved bimolecular fluorescence complementation (BiFC) assay for direct visualization of AP-1 dimerization in living cells and in living Caenorhabditis elegans. Furthermore, we have taken advantage of the dominant negative nature of the bZIP domains and the irreversibility of BiFC complexes to develop a novel BiFC-based dominant dimeric repressor (BiFC-DDR) system for functional analysis of AP-1 dimers in C. elegans. Using the BiFC-DDR, we have identified the C. elegans ATF-1 homodimer as a critical regulator of L2/L3 larval development. The microarray analysis in conjunction with the BiFC-DDR has identified 188 genes that might be regulated by ATF-1 homodimers. Preliminary characterization of these target genes has suggested that the genes encoding the vacuolar ATPase, the mitochondrial ATPase, and the ATP synthase might be the target genes of ATF-1 homodimers responsible for L2/L3 larval development. This success attests the general applicability of the BiFC-DDR for functional identification of AP-1 dimers and target genes in vivo.

Degree

Ph.D.

Advisors

Hu, Purdue University.

Subject Area

Genetics

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