IDENTIFICATION OF A NEW FAMILY OF TISSUE-SPECIFIC BASIC HELIX-LOOP-HELIX PROTEINS WITH A 2-HYBRID SYSTEM

With modified two-hybrid technology, we have isolated a member of a ne w family of basic helix-loop-helix (bHLH) transcription factors. Thing 1 (Th1) was identified in a screen of a mouse embryo cDNA library as a partner for the Drosophila E protein daughterless. RNA in situ hybrid ization and reverse transcriptase-PCR demonstrate a stage- and tissue- specific distribution for the expression of Th1. Although tissue speci fic, the expression pattern of Th1 is fairly complex. During developme nt, Th1 mRNA is widely expressed in extraembryonic tissues, portions o f the heart, autonomic ganglia, the gut, and pharyngeal arches. At emb ryonic day 7.5 (E7.5), extraembryonic derivatives show robust Th1 expr ession. By E8.5, expression in the embryonic heart becomes detectable. During the next 2 days of development, the signal also includes gut a nd pharyngeal arches. Predominant expression at E13.5 is in neural cre st derivatives, especially the autonomic nervous system and adrenal me dulla. Expression of Th1 persists in the adult, in which it is localiz ed to the smooth muscle cells of the gut. In vitro, Th1 protein recogn izes a set of DNA sites that are more degenerate than has been determi ned for other bHLH factors, indicating a reduced binding specificity. Transient transfection of NIH 3T3 cells with GAL4-Th1 fusions reveals a repression activity mediated by the Th1 bHLH domain. In combination, these properties define Th1 as a new bHLH protein with a unique set o f properties.