Our model system for studying enhanceosome function is a set of enhancers which activate the expression of the T-cell receptor genes a, b, g and d. The minimal enhancer complex for each of these genes appears to require the coordinated interaction of the same four proteins exemplified by the the enhancer complex for the a gene (TCRa).

Model of the T-cell receptor a enhanceosome. The four proteins shown are arranged on the bent DNA induced by the binding of the protein LEF-1. LEF-1 contains an HMG-box DNA-binding domain which intercalates into the DNA and bends the enhancer sequence. The known interactions between each protein are shown, although more interactions are expected to exist. PEBP2/CBF is a heterodimer of two subunits a and b. a contains the DNA-binding domain while b may bind nonspecifically to DNA to enhance the affinity of the a/DNA complex. ETS-1 is imagined to be a modular protein with a DNA-binding domain and one or more protein-interaction domains. The known interaction between ETS-1 and the homdimeric CREB protein is shown. CREB is a member of the leucine zipper family of DNA-binding proteins.
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LEF-1

The three-dimensional structure of Lef-1 (Love et al. Nature 376:791 (1995)) displayed a similar mechanism of DNA-binding as that observed for SRY. The greater DNA bend angle in the Lef-1/DNA complex is the consequence of an extended C-terminus which traverses the compressed major groove of the DNA, forming contacts with the phosphodiester backbone 5’ and 3’ to the center of the binding site.

PEBP2/CBF

PEBP2/CBF is the prototypical member of a small, but important transcription factor family in mammals. This factor is heterodimer of two subunits. The subunit is 451 amino-acids and contains a highly conserved DNA-binding domain termed the Runt-domain. The Runt-domain is > 90% conserved in mammals and > 70% conserved between lower organisms and mammals. The subunit is considerably smaller, approximately 180 amino-acids, and has an unknown function. The only functional phenotype of b which has been determined is its capacity to enhance DNA-binding by a.

The laboratory recently determined the structure of the Runt-domain bound to DNA, although the interface with DNA was not well-defined. The structure of the Runt-domain was observed to be an S-type immunoglobulin fold. Although unusual for a protein which binds DNA, this type of DNA-binding motif has been seen for a few important factors, to include the Rel-homology family, the STAT proteins and the tumor suppressor p53.

The laboratory also has determined the structure of the b subunit. This protein may be loosely described as belonging to the oligomer-binding (OB) motif family of b-barrel proteins. OB proteins frequently bind oligonucleotides or oligosaccharides in a variety of functional contexts. This particular type of barrel has not previously been seen among transcription factors. However, the anti-codon binding domain of aspartyl tRNA-synthetase does use this fold for RNA binding.

Mapping of the heterodimerization interface by direct analysis of the 44kD ternary protein/DNA complex suggests that b also binds proximal to one of the DNA-binding loops of the Runt-domain. The Runt-domain is both necessary and sufficient for heterodimerization with b.

ETS Proteins

Recently, the laboratory has also determined the structure of the ETS1 DNA-binding domain (DBD)/DNA complex. This 25kD complex is one of the largest structures yet determined by NMR methods. The ETS1-DBD is remarkable in that it has a fold nearly identical to that of prokaryotic CAP protein, however, DNA recognition of the GCGGAAGT motif is accomplished in a very different manner from that of CAP, and reflects more the mode of DNA recognition displayed by the eukaryotic winged helix-turn-helix proteins such as HNF3/forkhead protein.

CREB