Mutational analysis of the engrailed homeodomain recognition helix by phage display.

The homeodomain (HD) is a ubiquitous protein fold that confers DNA binding function on a superfamily of eukaryotic gene regulatory proteins. Here, the DNA binding of recognition helix variants of the HD from the engrailed gene of Drosophila melanogaster was investigated by phage display. Nineteen different combinations of pairwise mutations at positions 50 and 54 were screened against a panel of four DNA sequences consisting of the engrailed consensus, a non-specific DNA control based on the lambda repressor operator OR1 and two model sequence targets con-taining imperfect versions of the 5'-TAAT-3' consensus. The resulting mutant proteins could be divided into four groups that varied with respect to their affinity for DNA and specificity for the engrailed consensus. The altered specificity phenotypes of several mutant proteins were confirmed by DNA mobility shift analysis. Lys50/Ala54 was the only mutant protein that exhibited preferential binding to a sequence other than the engrailed consensus. Arginine was also demonstrated to be a functional replacement for Ala54. The functional combinations at 50 and 54 identified by these experiments recapitulate the distribution of naturally occurring HD sequences and illustrate how the engrailed HD can be used as a framework to explore covariation among DNA binding residues.     

An upstream regulatory element of the NCAM promoter contains a binding site for homeodomains.

In the present study, we have analyzed an upstream regulatory element of the neural cell adhesion molecule (NCAM) promoter which is required for full promoter activity. It contains an ATTATTA motif that resembles the core recognition sequence of homeodomain (HD) proteins of the Antennapedia (Antp) and related types. Electrophoretic mobility shift (EMSA) and DNase I footprinting analyses revealed that the Drosophila HDs coded by the Antp and the zerknüllt (zen) genes bind this site in vitro. In contrast, the engrailed (en) protein did not produce a detectable footprint. The functional relevance of the ATTATTA motif was demonstrated by showing that a two-nucleotide exchange curtailed stimulation of an heterologous promoter. An oligonucleotide known to be recognized with high affinity by Antp-like HDs efficiently competed for endogenous factor binding. These results suggest that the NCAM gene may be a target for HD proteins.     

Engineering the loop region of a homeodomain-leucine zipper protein promotes efficient binding to a monomeric DNA binding site

Plant homeodomain-leucine zipper (HD-Zip) proteins, unlike many animal homeodomains (HDs), are unable to bind DNA as monomers. To investigate the molecular basis of their different behavior, we have constructed chimeras between the HD of the sunflower HD-Zip protein Hahb-4 and that of Drosophila engrailed (EN). Analysis of the interaction of these proteins with the pseudopalindromic Hahb-4 binding site and the monomeric EN binding site suggests that the loop located between helix I and helix II (amino acids 21-28) of EN is enough to confer efficient DNA binding activity to the Hahb-4 HD. Accordingly, the combined mutation of residues 24 and 25 of Hahb-4 to those present in EN (S24R/R25Y) originated an HD able to interact with the EN binding site, while single mutations were ineffective. We have also determined that a protein with the leucine zipper and helix III of Hahb-4 fused to the rest of the EN HD binds to the Hahb-4 pseudopalindomic binding site with increased affinity and shows extended contacts with DNA respective to Hahb-4. We conclude that the loop located between helix I and helix II of the HD must be regarded as one of the segments that contribute to the present-day diversity in the properties of different HDs.     

Interchange of DNA-binding modes in the deformed and ultrabithorax homeodomains: a structural role for the N-terminal arm

The deformed (Dfd) and ultrabithorax (Ubx) homeoproteins regulate developmental gene expression in Drosophila melanogaster by binding to specific DNA sequences within its genome. DNA binding is largely accomplished via a highly conserved helix-turn-helix DNA-binding domain that is known as a homeodomain (HD). Despite nearly identical DNA recognition helices and similar target DNA sequence preferences, the in vivo functions of the two proteins are quite different. We have previously revealed differences between the two HDs in their interactions with DNA. In an effort to define the individual roles of the HD N-terminal arm and recognition helix in sequence-specific binding, we have characterized the structural details of two Dfd/Ubx chimeric HDs in complex with both the Dfd and Ubx-optimal-binding site sequences. We utilized hydroxyl radical cleavage of DNA to assess the positioning of the proteins on the binding sites. The effects of missing nucleosides and purine methylation on HD binding were also analyzed. Our results show that both the Dfd and Ubx HDs have similar DNA-binding modes when in complex with the Ubx-optimal site. There are subtle but reproducible differences in these modes that are completely interchanged when the Dfd N-terminal arm is replaced with the corresponding region of the Ubx HD. In contrast, we showed previously that the Dfd-optimal site sequence elicits a very different binding mode for the Ubx HD, while the Dfd HD maintains a mode similar to that elicited by the Ubx-optimal site. Our current methylation interference studies suggest that this alternate binding mode involves interaction of the Ubx N-terminal arm with the minor groove on the opposite face of DNA relative to the major groove that is occupied by the recognition helix. As judged by hydroxyl radical footprinting and the missing nucleoside experiment, it appears that interaction of the Ubx recognition helix with the DNA major groove is reduced. Replacing the Dfd N-terminal arm with that of Ubx does not elicit a complete interchange of the DNA-binding mode. Although the position of the chimera relative to DNA, as judged by hydroxyl radical footprinting, is similar to that of the Dfd HD, the missing nucleoside and methylation interference patterns resemble those of the Ubx HD. Repositioning of amino acid side-chains without wholesale structural alteration in the polypeptide appears to occur as a function of N-terminal arm identity and DNA-binding site sequence. Complete interchange of binding modes was achieved only by replacement of the Dfd N-terminal arm and the recognition helix plus 13 carboxyl-terminal residues with the corresponding residues of Ubx. The position of the N-terminal arm in the DNA minor groove appears to differ in a manner that depends on the two base-pair differences between the Dfd and Ubx-optimal-binding sites. Thus, N-terminal arm position dictates the binding mode and the interaction of the recognition helix with nucleosides in the major groove.     

The Drosophila zeste protein binds cooperatively to sites in many gene regulatory regions: implications for transvection and gene regulation.

The Drosophila zeste protein binds in vitro to several sites in the white, Ultrabithorax, decapentaplegic, Antennapedia, and engrailed genes and to at least one site in the zeste gene itself. The distribution of these sites corresponds often with that of regulatory elements in these genes as defined by mutations or, in the case of white, by molecular analysis. A zeste binding site is frequently found in the immediate vicinity of the promoter. zeste binding sites are composed of two or more zeste recognition sequences T/CGAGT/CG. Isolated consensus sequences do not bind or footprint. Cooperative interactions are involved both in binding to a given site and between proteins bound at independent sites. zeste bound to one DNA molecule can in fact bind simultaneously to another DNA molecule. These results suggest a general role for zeste in bringing together distant regulatory elements controlling the activity of a target gene. In this model, transvection effects are a by-product of normal intragenic zeste action.     

Characterisation of chicken erythroid nuclear proteins which bind to the nuclease hypersensitive regions upstream of the beta A- and beta H-globin genes.

Chicken erythrocyte sequence-specific nuclear DNA-binding proteins, which bind to the 5'-flanking DNAseI hypersensitive sites of the erythrocyte chromosomal beta A- and beta H-globin genes, have been fractionated by HPLC gel filtration. Three beta A-globin gene DNA binding activities (to sites A, B and B' (10-12)) were separated. The erythroid precursor cell line HD3 has beta A-globin gene sites B and B' binding activities, but binding to site A is detected only after the HD3 cells are induced to differentiate. The fractionated protein binds to a redefined site B', which contains at its center the globin CACCC consensus sequence. The chromosomal beta H-globin gene has two 5'-flanking DNAseI hypersensitive sites which bracket two sequences (H and H') bound by erythrocyte and HD3 nuclear protein in vitro. The beta H- and beta A-globin gene binding sites (H and B) contain variants of the sequences bound by Nuclear Factor 1 and the TGGCA-binding protein, and their protein binding activity(ies) co-purify after HPLC gel filtration.     

Sequence-specific DNA-binding proteins which interact with (G + C)-rich sequences flanking the chicken c-myc gene

The interaction of nuclear sequence-specific DNA-binding proteins from definitive chicken erythrocytes, thymus and proliferating transformed erythroid precursor (HD3) cells with the 700-base-pair (700-bp) DNA 5'-flanking region of the chicken c-myc gene was investigated by in vitro footprint analysis. The major HD3 protein-binding activity binds to a site (site V) 200 bp upstream from the 'cap' site but, after further fractionation, a second distinct binding activity is detected to a site (site VIII) which contains both the 'CAAT' and 'SP1-binding' consensus sequences. Protein from thymus and erythrocyte cells which express c-myc at lower levels, bind to seven and eight sites respectively. In common with HD3 cell protein, they both bind to site VIII and, although binding to the sequence at site V is also detected, the footprint protection pattern is sufficiently different (site V') to suggest the involvement of different proteins in terminally differentiated and proliferating cells. The DNA-binding activities were partially fractionated by high-performance liquid chromatography gel filtration and include an erythrocyte-specific protein which binds to a c-myc gene poly(dG) homopolymer sequence similar to that found upstream of the chicken beta A-globin gene.     

pBR322 contains glucocorticoid regulatory element DNA consensus sequences

A computer search of the pBR322 DNA sequence identified five sites matching reported glucocorticoid regulatory element (GRE) DNA consensus sequences and three related sites. A pBR322 DNA fragment containing one GRE site was shown to bind immobilized HeLa S3 cell glucocorticoid receptor and to compete for receptor binding in a competitive binding assay. Conversely, a pBR322 DNA fragment devoid of GRE sites showed barely detectable interaction with glucocorticoid receptor in either of these assays. These results demonstrate the importance of GRE consensus sequences in glucocorticoid receptor interactions with DNA, and further identify a cause for high background binding observed when pBR322 DNA is used as a negative control in studies of glucocorticoid receptor-DNA interactions.     

Structure and function of the enhancer 3'' to the human A gamma globin gene.

An enhancer is located immediately 3' to the A gamma globin gene. We have used DNase I footprinting to map the sites of interaction of nuclear proteins with the DNA sequences of this enhancer. Eight footprints were discovered, distributed over 600 base pairs of DNA. Three of these contain a consensus binding site for the erythroid specific factor GATA-I. Each of these GATA-1 sites had an enhancer activity when inserted into a reporter plasmid and tested in human erythroleukemia cells. Other footprints within the enhancer contained consensus binding sequences for the ubiquitous, positive regulatory proteins AP2 and CBP-1. An Sp1-like recognition sequence was also identified. Synthetic oligonucleotides encompassing two of the footprints generated a slowly migrating complex in gel mobility shift assays. The same complex forms on a fragment of the human gamma globin gene promoter extending from -260 to -200. The DNaseI footprint of this protein complex with the enhancer overlapped a sequence, AGGAGGA, found within the binding site for a protein that interacts with the chicken beta globin promoter and enhancer, termed the stage selector element. We propose that this complex of proteins may be involved in the human gamma globin promoter-enhancer interaction.     

Evolutionary conservation of homeodomain-binding sites and other sequences upstream and within the major transcription unit of the Drosophila segmentation gene engrailed.

The engrailed (en) gene functions throughout Drosophila development and is expressed in a succession of intricate spatial patterns as development proceeds. Normal en function relies on an extremely large cis-acting regulatory region (70 kilobases). We are using evolutionary conservation to help identify en sequences important in regulating patterned expression. Sequence comparison of 2.6 kilobases upstream of the en coding region of D. melanogaster and D. virilis (estimated divergence time, 60 million years) showed that 30% of this DNA occurs in islands of near perfect sequence conservation. One of these conserved islands contains binding sites for homeodomain-containing proteins. It has been shown genetically that homeodomain-containing proteins regulate en expression. Our data suggested that this regulation may be direct. The remaining conserved islands may contain binding sites for other regulatory proteins.