Helix-loop helix (HLH) proteins are an evolutionarily-conserved family of transcription factors which have complex dimerization and DNA-binding characteristics. For both Drosophila and human HLH proteins, high affinity DNA-binding heterodimers form between different HLH proteins. In flies, a system of 10 interacting HLH genes has been found to control neuronal cell determination. These 10 HLH genes consist of 5 activators and 5 repressors, which control different subsets of the neuronal pattern. The proposed research will test the hypothesis that the HLH activator proteins function to form DNA-binding homodimers and heterodimers, and that the HLH repressor proteins function by binding to the activator proteins to form non-DNA-binding heterodimers. The HLH proteins also will be tested for their ability to activate transcription of reporter genes in cultured cells. This biochemical analysis of HLH protein function will be complemented by analysis of HLH protein regulation of a candidate in vivo target gene, T5. Transcriptional regulation of T5 by HLH proteins will be tested by analysis of T5 transcript expression in HLH mutants. In addition, mutants with different parts of the T5 5' domain deleted will be examined for pattern of T5 transcript expression. Function of putative regulatory regions will be confirmed by P element transformation of flies with reporter genes under control of those regions of the T5 cis DNA. Binding of HLH proteins to T5 regulatory DNA also will be assayed. An eventual goal will be site-directed mutagenesis of specific sites which bind HLH proteins in vitro and are likely to regulate T5 in vivo. In humans, at least some HLH genes are proto-oncogenes. Two human HLH genes are consistently translocated in patients with acute lymphoblastic leukemia. These severe mutations are likely to perturb the normal protein- protein and protein-DNA interactions of these transcription factors. The proposed research will determine the characteristics of the HLH system in flies; this will likely provide a better understanding of the human system, and its role in development and disease.
