SURVEY AND SUMMARY: Saccharomyces cerevisiae basic helix-loop-helix proteins regulate diverse biological processes

Basic helix–loop–helix (bHLH) proteins are among the most well studied and functionally important regulatory proteins in all eukaryotes. The HLH domain dictates dimerization to create homo- and heterodimers. Dimerization juxtaposes the basic regions of the two monomers to create a DNA interaction surface that recognizes the consensus sequence called the E-box, 5′-CANNTG-3′. Several bHLH proteins have been identified in the yeast Saccharomyces cerevisiae using traditional genetic methodologies. These proteins regulate diverse biological pathways. The completed sequence of the yeast genome, combined with novel methodologies allowing whole-genome expression studies, now offers a unique opportunity to study the function of these bHLH proteins. It is the purpose of this review to summarize the current knowledge of bHLH protein function in yeast.     

A network of yeast basic helix-loop-helix interactions

The Ino4 protein belongs to the basic helix–loop–helix (bHLH) family of proteins. It is known to form a dimer with Ino2p, which regulates phospholipid biosynthetic genes. Mammalian bHLH proteins have been shown to form multiple dimer combinations. However, this flexibility in dimerization had not been documented for yeast bHLH proteins. Using the yeast two-hybrid assay and a biochemical assay we show that Ino4p dimerizes with the Pho4p, Rtg1p, Rtg3p and Sgc1p bHLH proteins. Screening a yeast cDNA library identified three additional proteins that interact with Ino4p: Bck2p, YLR422W and YNR064C. The interaction with Bck2p prompted us to examine if any of the Bck2p-associated functions affect expression of phospholipid biosynthetic genes. We found that hyperosmotic growth conditions altered the growth phase regulation of a phospholipid biosynthetic gene, CHO1. There are two recent reports of initial whole genome yeast two-hybrid interactions. Interestingly, one of these reports identified five proteins that interact with Ino4p: Ino2p, Hcs1p, Apl2p, YMR317W and YNL279W. Ino2p is the only protein in common with the data presented here. Our finding that Ino4p interacts with five bHLH proteins suggests that Ino4p is likely to be a central player in the coordination of multiple biological processes.     

Identifying DNA-binding proteins using structural motifs and the electrostatic potential

Robust methods to detect DNA-binding proteins from structures of unknown function are important for structural biology. This paper describes a method for identifying such proteins that (i) have a solvent accessible structural motif necessary for DNA-binding and (ii) a positive electrostatic potential in the region of the binding region. We focus on three structural motifs: helix–turn-helix (HTH), helix–hairpin–helix (HhH) and helix–loop–helix (HLH). We find that the combination of these variables detect 78% of proteins with an HTH motif, which is a substantial improvement over previous work based purely on structural templates and is comparable to more complex methods of identifying DNA-binding proteins. Similar true positive fractions are achieved for the HhH and HLH motifs. We see evidence of wide evolutionary diversity for DNA-binding proteins with an HTH motif, and much smaller diversity for those with an HhH or HLH motif.     

Genetic Mapping of Four Mouse bHLH Genes Related toDrosophilaProneural Geneatonal

It has been shown that mammalian neurogenesis is partly controlled by multiple basic helix–loop–helix (bHLH) genes, as inDrosophila.Recently, mouse homologs ofDrosophila atonal,a proneural gene encoding a bHLH protein required for chordotonal organ and photoreceptor development, have been characterized to obtain further insights into the molecular nature of mammalian neurogenesis. Here, to assess their potential involvement in genetic neural disorders, we have determined genetic map positions for four mouseatonal-related genes,Atoh1, Atoh2, Atoh3,andNdrf,which encode MATH-1, MATH-2, MATH-3, and NDRF, respectively. Interspecific backcross analysis indicated thatAtoh1andAtoh2were located in separate positions of Chr 6 and thatAtoh3andNdrfwere mapped to Chr 10 and Chr 11, respectively. Thus, these structurally related genes are located separately on multiple chromosomes.     

ATBS1 INTERACTING FACTORs negatively regulate Arabidopsis cell elongation in the triantagonistic bHLH system

We recently demonstrated that cell elongation in plants is regulated by a triantagonistic bHLH system, in which three bHLH proteins, Activator of Cell Elongation 1 (ACE1), Arabidopsis ILI1 binding BHLH 1 (AtIBH1) and Paclobutrazol Resistance 1 (PRE1), competitively regulate the expression of genes for cell elongation. Here we show that ATBS1 Interacting Factor 2 (AIF2), AIF3 and AIF4 interact with PRE1 and ACE1, similar to AtIBH1, and also negatively regulate cell elongation in the triantagonistic bHLH system. The expression of each AIF is constitutive or induced by light, but AtIBH1 expression is dependent on BR signaling and developmental phase. These results indicate that AIFs and AtIBH1 may play different roles in cell elongation in different signaling pathways.     

Identification of the C-terminal activation domain of the NeuroD-related factor (NDRF)

NeuroD-related factor (NDRF) is a basic helix–loop–helix (bHLH) protein whose expression is restricted to the central nervous system, and is considered to be responsible for maintenance of differentiated neurons as well as neurogenesis. NDRF structurally resembles NeuroD in the bHLH region and can induce neurogenesis ectopically in ectodermal cells of the Xenopus embryo. In this study, we delineated the functional domains of NDRF. Using GAL4/NDRF fusion proteins, we identified the C-terminal activation domain (C-AD) in NDRF between amino acid positions 294 and 383. This region was highly homologous to one part of the activation domain of NeuroD. We further investigated the transactivational function of C-AD in the mouse type 1 inositol 1,4,5-trisphosphate receptor promoter, which has an NDRF site. Truncation of C-AD resulted in reduction of the activation function, whereas the DNA-binding specificity was not affected. These results suggest that C-AD has a stimulatory function in the mammalian nervous system.     

LONGIFOLIA1 and LONGIFOLIA2, two homologous genes, regulate longitudinal cell elongation in Arabidopsis.

Plants have diversified their leaf morphologies to adapt to diverse ecological niches. The molecular components responsible for regulating leaf morphology, however, have not been fully elucidated. By screening Arabidopsis activation-tagging lines, we identified a dominant mutant, which we designated longifolia1-1D (lng1-1D). lng1-1D plants were characterized by long petioles, narrow but extremely long leaf blades with serrated margins, elongated floral organs, and elongated siliques. The elongated leaves of the mutant were due to increased polar cell elongation rather than increased cell proliferation. Molecular characterization revealed that this phenotype was caused by overexpression of the novel gene LNG1, which was found to have a homolog, LNG2,in Arabidopsis. To further examine the role of the LNG genes, we characterized lng1 and lng2 loss-of-function mutant lines. In contrast to the elongated leaves of lng1-1D plants, the lng1 and lng2 mutants showed slightly decreased leaf length. Furthermore, the lng1-3 lng2-1 double mutant showed further decreased leaf length associated with less longitudinal polar cell elongation. The leaf widths in lng1-3 lng2-1 mutant plants were similar to those in wild type, implying that the role of LNG1 and LNG2 on polar cell elongation is similar to that of ROTUNDIFOLIA3 (ROT3). However, analysis of a lng1-3 lng2-1 rot3-1 triple mutant and of a lng1-1D rot3-1 double mutant indicated that LNG1 and LNG2 promote longitudinal cell elongation independently of ROT3. Taken together, these findings indicate that LNG1 and LNG2 are new components that regulate leaf morphology by positively promoting longitudinal polar cell elongation independently of ROT3 in Arabidopsis.