Identification of C/EBP basic region residues involved in DNA sequence recognition and half-site spacing preference.

Abstract

C/EBP and GCN4 are basic region-leucine zipper (bZIP) DNA-binding proteins that recognize the dyadsymmetric sequences ATTGCGCAAT and ATGAGTCAT, respectively. The sequence specificities of these and other bZIP proteins are determined by their a-helical basic regions, which are related at the primary sequence level. To identify amino acids that are responsible for the different DNA sequence specificities of C/EBP and GCN4, two kinds of hybrid proteins were constructed: GCN4-C/EBP chimeras fused at various positions in the basic region and substitution mutants in which GCN4 basic region amino acids were replaced by the corresponding residues from C/EBP. On the basis of the DNA-binding characteristics of these hybrid proteins, three residues that contribute significantly to the differences in C/EBP and GCN4 binding specificity were defined. These residues are clustered along one face of the basic region a helix. Two of these specificity residues were not identified as DNA-contacting amino acids in a recently reported crystal structure of a GCN4-DNA complex, suggesting that the residues used by C/EBP and GCN4 to make base contacts are not identical. A random binding site selection procedure also was used to define the optimal recognition sequences for three of the GCN4-C/EBP fusion proteins. These experiments identify an element spanning the hinge region between the basic region and leucine zipper domains that dictates optimal half-site spacing (either directly abutted for C/EBP or overlapping by one base pair for GCN4) in high-affinity binding sites for these two proteins.Basic region-leucine zipper (bZIP) DNA-binding proteins constitute a major class of eukaryotic transcriptional regulatory proteins (28). More than 50 genes encoding these proteins have been isolated from a variety of organisms. Most bZIP proteins can be classified into one of five subfamilies based on their DNA sequence specificities (Fig. 1). The binding sites recognized by each subfamily consist of related but distinct palindromic sequences containing two 5-bp half-sites. These consensus binding sites differ in the sequence of their half-sites and/or in the spacing between half-sites, which can be either directly abutted or overlapping by one base pair. This spacing difference alone distinguishes the binding of the Fos/Jun and ATF/CREB subfamilies.The bZIP DNA-binding domain is characterized by two major features: a helix-permissive segment containing repeated leucine residues spaced at seven-amino-acid intervals (the leucine zipper) and an associated segment of strong net positive charge (the basic region) immediately adjacent to the leucine repeats (33). The leucine repeats are part of an amphipathic a helix that mediates protein dimerization (31,33,34,63), and the paired helices in a bZIP dimer lie in a parallel orientation, as in classical coiled-coil structures (15,50). Recently, the crystal structures of a synthetic leucine zipper peptide (49) and a complete bZIP DNA-binding domain (13) have been solved, proving that the leucine zipper dime...

Directed mutation of the basic domain of v-Jun alters DNA binding specificity and abolishes its oncogenic activity in chicken embryo fibroblasts

“…Some members of this class of proteins exhibit similar binding speci®cities whereas others are completely di erent. For instance the yeast transcription factor GCN4 recognizes the same target sequence as c-Jun (TGACTCA) whereas c/ EBP binds a target much removed from this sequence (ATTGCGCAAT) (Johnson, 1993;Suckow et al, 1993). Mutational studies have demonstrated that the basic region adjacent to the leucine dimerization domain is directly responsible for speci®city of target recognition in this class of proteins.…”

“…Mutational studies have demonstrated that the basic region adjacent to the leucine dimerization domain is directly responsible for speci®city of target recognition in this class of proteins. Substitution of a few critical amino acids in the basic region of GCN4 with those found in c/EBP results in a mutant GCN4 protein in which DNA binding speci®city has been altered from TGACTCA (GCN4 target) to ATTGCG-CAAT (c/EBP target) (Johnson, 1993;Suckow et al, 1993). Mutants such as this have the advantage that they exhibit altered functional speci®city while retaining their structural integrity.…”

Structure, alternative splicing and chromosomal localization of the cystatin-related epididymal spermatogenic gene

“…In addition, seven consensus sites that bind the sex-determining region Y transcription factor [36] and its related DNA-binding protein Sox-5 [37] were present throughout the 1.6 kb of Cres promoter sequences. Other DNAbinding sites in the Cres promoter included those for CCAAT\ enhancer-binding protein [38], cAMP-response-element-binding protein [39] and GATA-1-binding protein [40]. Considering the highly restricted expression of the Cres gene to the testicular haploid germ cells, epididymis [20] and the anterior pituitary gonadotropes (G. Sutton and G. A. Cornwall, unpublished work), the presence of some of these DNA-binding elements is not surprising and might suggest a means by which the Cres gene is regulated.…”

CpG methylation of half-CRE sequences creates C/EBPα binding sites that activate some tissue-specific genes

“…S3 F-I). C/EBPα ChIP-chip showed that C/EBPα preferentially binds promoters containing the known C/EBP consensus site (28) and that 85% of bound promoters are unmethylated (Fig. 2E).…”