ABSTRACT
The polyomavirus enhancer binding protein 2alpha (PEBP2alpha) is a DNA binding transcriptional regulatory protein that binds conserved sites in the polyomavirus enhancer, mammalian type C retroviral enhancers and T-cell receptor gene enhancers. Binding of PEBP2alpha and homologous proteins to the consensus DNA sequence TGPyGGTPy is mediated through a protein domain known as the runt domain. Although recent NMR studies of DNA-bound forms of the runt domain have shown an immunoglobulin-like (Ig) fold, the identification of residues of the protein that are involved in DNA binding has been obscured by the low solubility of the runt domain. Constructs of the mouse PEBP2alphaA1 gene were generated with N- and C-terminal extensions beyond the runt homology region. The construct containing residues Asp90 to Lys225 of the sequence (PEBP2alpha90-225) yielded soluble protein. The residues that participate in DNA binding were determined by comparing the NMR spectra of free and DNA-bound PEBP2alpha90-225. Analysis of the changes in the NMR spectra of the two forms of the protein by chemical shift deviation mapping allowed the unambiguous determination of the regions that are responsible for specific DNA recognition by PEBP2alpha. Five regions in PEBP2alpha90-225 that are localized at one end of the beta-barrel were found to interact with DNA, similar to the DNA binding interactions of other Ig fold proteins.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , DNA/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolism , Animals , Base Sequence , Binding Sites , DNA/chemistry , DNA/genetics , DNA-Binding Proteins/genetics , Immunoglobulins/chemistry , Mice , Models, Molecular , Nitrogen/metabolism , Nuclear Magnetic Resonance, Biomolecular , Osmolar Concentration , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Protons , Sequence Homology, Amino Acid , Solubility , Temperature , Transcription Factor AP-2 , Transcription Factors/geneticsABSTRACT
wingless and decapentaplegic signal during endoderm induction in Drosophila to regulate expression of the homeotic gene Ultrabithorax. Here, we define a minimal wingless response sequence in the midgut enhancer of Ultrabithorax. We show that this sequence is recognized by the murine transcription factor LEF-1 (lymphocyte enhancer binding factor 1) in a ternary complex with armadillo protein, the cytoplasmic target of the wingless signaling pathway. In stable transformants, transcriptional stimulation of the Ultrabithorax enhancer by LEF-1 depends on armadillo. Furthermore, overexpression of LEF-1 bypasses the need for wingless signaling and causes phenotypes in the midgut, notum, and wing that mimic wingless hyperstimulation. Finally, efficient transcriptional stimulation by LEF-1 in the midgut depends also on the decapentaplegic response sequence and is limited spatially by decapentaplegic signaling. Thus, LEF-1 coordinates inputs from multiple positional signals, consistent with its architectural role in regulating the assembly of multiprotein enhancer complexes.
Subject(s)
DNA-Binding Proteins/genetics , Drosophila Proteins , Insect Proteins/physiology , Proto-Oncogene Proteins/physiology , Signal Transduction/physiology , Trans-Activators , Transcription Factors/genetics , Animals , Armadillo Domain Proteins , DNA-Binding Proteins/metabolism , Drosophila , Enhancer Elements, Genetic/physiology , Gene Expression Regulation, Developmental/physiology , Insect Proteins/metabolism , Intestines/chemistry , Intestines/physiology , Larva/chemistry , Larva/physiology , Lymphoid Enhancer-Binding Factor 1 , Molecular Sequence Data , Phenotype , Sequence Homology, Nucleic Acid , Transcription Factors/metabolism , Transforming Growth Factor beta/physiology , Wnt1 ProteinABSTRACT
LEF-1 is a transcription factor that participates in the regulation of the T-cell receptor alpha (TCR alpha) enhancer by facilitating the assembly of multiple proteins into a higher order nucleoprotein complex. The function of LEF-1 is dependent, in part, on the HMG domain that induces a sharp bend in the DNA helix, and on an activation domain that stimulates transcription only in a specific context of other enhancer-binding proteins. With the aim of gaining insight into the function of context-dependent activation domains, we cloned ALY, a novel LEF-1-interacting protein. ALY is a ubiquitously expressed, nuclear protein that specifically associates with the activation domains of LEF-1 and AML-1 (CBF alpha2, PEBP2 alpha(B), which is another protein component of the TCR alpha enhancer complex. In addition, ALY can increase DNA binding by both LEF-1 and AML proteins. Overexpression of ALY stimulates the activity of the TCR alpha enhancer complex reconstituted in transfected nonlymphoid HeLa cells, whereas down-regulation of ALY by anti-sense oligonucleotides virtually eliminates TCR alpha enhancer activity in T cells. Similar to LEF-1, ALY can stimulate transcription in the context of the TCR alpha enhancer but apparently not when tethered to DNA through an heterologous DNA-binding domain. We propose that ALY mediates context-dependent transcriptional activation by facilitating the functional collaboration of multiple proteins in the TCR alpha enhancer complex.
