One family of regulatory transcription factors found in all metazoans is the ets family of proteins. ETS genes number 8 in Drosophila, 10 in C. elegans, and 25 in humans. All ETS proteins conserve an 85 amino acid DNA binding ETS domain that folds into a winged-helix-tum-helix structure. Biochemical and structural studies of a subset of ETS family members demonstrate that the ETS domain binds the core DNA sequence 5’-GGA(A/T)-3’. It is proposed that all ETS family members bind enhancer elements containing a core GGA sequence.
ETS proteins can perform a variety of functions, despite conserving a highly similar DNA binding domain. This has been demonstrated by gene disruption studies in model organisms. In mice, for instance, seven ETS genes have been targeted for disruption, including ETS-1, ETS-2, PU.l, TEL, FU-1, SP1-B, and ER81, and distinct phenotypes were observed for mutation of each gene. For example, targeting of TEL resulted in defects in bone marrow hematopoiesis, whereas targeting of ETS-1 resulted in defects in T cell and NK cell development. Notably, these distinct phenotypes arose despite the fact that ETS-1 and TEL are co-expressed in some of the same hematopoietic cells. Genetic studies in Drosophila also point to unique functions for co-expressed ETS proteins. For example, both yan and pnt are expressed in the Drosophila eye and are required for normal R7 photoreceptor development. However, yan is a negative regulator of eye development whereas pnt is a positive regulator.
Members of the ETS family display specificity with respect to the genes they regulate and the molecular mechanisms by which they function. This family can therefore be used to investigate the molecular features that define specificity among a gene family.
Many studies of ETS family members have focused on the similarities and differences in the regulation of DNA binding by the ETS domain. These studies have revealed subtle differences in the preferences for DNA binding sequences outside the 5’-GGA-3’ core. Mechanisms have also been identified that modulate the affinity of ETS domain binding. For example, ETS-1 DNA binding activity is regulated by phosphorylation, autoinhibition, and cooperative interactions with other DNA binding proteins. These features show restrictive conservation and could contribute to the unique functions of the ETS-1 protein. The work described in this thesis addresses the question of specificity by examining the function of two features conserved in a limited number of ETS proteins. The first is the Pointed (PNT) domain, a region of 80 amino acids conserved among approximately 40% of ETS proteins. The function of the PNT domain is not well-established, but it is proposed to mediate protein-protein interactions. The second emphasis of study is the regulation of mitogen-activated protein kinase (MAPK) phosphorylation of the ETS proteins ETS-1, ETS-2, and Pnt-P2. Phosphorylation is important for the transactivation function of these ETS proteins, but how phosphorylation is regulated is not well-understood.