This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.A major adaptive response to being deprived of oxygen and nutrients is induced by hypoxia-inducible factor 1 (HIF-1), a transcriptional regulator, which has been demonstrated to be involved in a host of hypoxia related diseases including myocardial, cerebral, and retinal ischemias, as well as pulmonary hypertension and cancer. HIF-1 is a heterodimer comprised of HIF-1a and HIF-1b (also known as the aryl hydrocarbon receptor translocator, ARNT). Both proteins belong to the basic-helix-loop-helix Per-Arnt-Sim (bHLH PAS) family of transcriptional regulator proteins. While HIF-1a is the hypoxically responsive component of the functional HIF-1 heterodimer, ARNT is expressed constitutively and is functionally promiscuous as a result of dimerizing with a number of other bHLH PAS proteins including the Aryl Hydrocarbon Receptor (AHR). The ARNT/AHR dimer is involved in the transcriptional activation of multidrug resistance response to dioxins and aryl hydrocarbons in eukaryotes. It is believed that, like HIF-1a, AHR exists transiently in the cytoplasm of the cell until it is activated, stabilized and translocated to the nucleus, where it forms a functional dimer with ARNT and activates transcription of specific genes. Specificity of response in these proteins is achieved through variety of coeffector binding domain structures or stability pathways, however, there is also a significant specificity for DNA binding sites achieved through the highly homologous bHLH domains responsible for DNA binding and dimerization. This purpose of the studies outlined in this proposal are to explore the variations in structure in the bHLH domains that lead to specificity for different DNA binding sites for ARNT, HIF-1 and AHR. The structures of the DNA bound forms of these functional dimers will reveal differences in protein-DNA contacts resulting from sequence and structure, and will allow us to begin to delineate the structural requirements for functional specificity in drug resistance versus the hypoxic response.
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