Identification of a New Family of Tissue-Specific Basic Helix-Loop-Helix Proteins with a Two-Hybrid System

Abstract

With modified two-hybrid technology, we have isolated a member of a new family of basic helix-loop-helix (bHLH) transcription factors. Thing1 (Th1) was identified in a screen of a mouse embryo cDNA library as a partner for the Drosophila E protein daughterless. RNA in situ hybridization and reverse transcriptase-PCR demonstrate a stage-and tissue-specific distribution for the expression of Th1. Although tissue specific, the expression pattern of Th1 is fairly complex. During development, Th1 mRNA is widely expressed in extraembryonic tissues, portions of the heart, autonomic ganglia, the gut, and pharyngeal arches. At embryonic day 7.5 (E7.5), extraembryonic derivatives show robust Th1 expression. By E8.5, expression in the embryonic heart becomes detectable. During the next 2 days of development, the signal also includes gut and pharyngeal arches. Predominant expression at E13.5 is in neural crest derivatives, especially the autonomic nervous system and adrenal medulla. Expression of Th1 persists in the adult, in which it is localized to the smooth muscle cells of the gut. In vitro, Th1 protein recognizes a set of DNA sites that are more degenerate than has been determined 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 of properties.During development, specific genetic programs are activated by transcription factors which are often stage and tissue specific in their distribution. Understanding the combinatorial code of factors which defines each cell fate during the differentiation process requires identification of new factors with restricted spatial activity. One group of factors with pivotal roles during development are the basic helix-loop-helix (bHLH) proteins. These factors are important in differentiation processes as diverse as skeletal myogenesis, neurogenesis, and hematopoiesis (for reviews, see references 2, 12, 13, 43, 59, and 63).bHLH proteins have been grouped into four classes based on partner choice, DNA-binding ability, and transcriptional activity. (i) E proteins, also called class A, are widely expressed and act as partners for at least two of the other classes (6,42,47). (ii) Tissue-specific bHLH proteins, or class B, pair with E proteins to produce heterodimers. Class A homodimers and A/B heterodimers are thought to function as transcriptional activators (9,46,62). (iii) Id proteins lack a basic region and form a non-DNA-binding complex with class A and some class B proteins (6, 55). (iv) Hairy and Enhancer-of-split [h/E(spl)] bHLH proteins form a fourth class whose interactions with the other three groups are not well characterized. The h/E(spl) family is structurally defined, in part, by a WRPW motif at the C terminus and an absolutely conserved proline in the basic region. These proteins act genetically as repressors of bHLHmediated functions and can bind to DNA v...

UVDDB p127-binding modulates activities and intracellular distribution of Hepatitis B virus X protein

“…Screening of the random WHx mutant library was performed as follows: The L40 yeast recipient strain (Hollenberg et al, 1995) was transformed by pACT-UVDDB and a transformed colony was established in medium lacking leucine (-L). The resulting L40 [pACT-UVDDB] strain was then transformed with library DNA and plated on medium lacking leucine and tryptophane (-LW).…”

Cloning and characterization of APS, an adaptor molecule containing PH and SH2 domains that is tyrosine phosphorylated upon B-cell receptor stimulation

“…The isolated colonies were subsequently assayed for b-galactosidase activity on ®lters. The positive library plasmid DNA was isolated by transformation into HB101 and sequenced as described (Hollenberg et al, 1995). Using oncogenic c-kit kinase domain as bait, we cloned several known and unknown SH2 proteins, which will be described elsewhere.…”

PRC1 associates with the hsp70i promoter and interacts with HSF2 during mitosis

“…The resulting strain was then transformed with a mouse whole embryo cDNA library [16]. To confirm the interaction, the pGBD-HSF2 and pVP16 plasmid containing the partial mPRC1 cDNA, referred to as mPRC1 (118-233), were transformed back into yeast and the ability of HSF2 or HSF1 and the mPRC1 clone to interact was determined by growth on selective media lacking adenine or histidine.…”