The -117 mutation in Greek HPFH affects the binding of three nuclear factors to the CCAAT region of the gamma-globin gene.

The Greek form of hereditary persistence of fetal hemoglobin (HPFH) is associated with a point mutation immediately upstream of the distal of the two CCAAT elements of the A gamma-globin gene. Three proteins present in nuclear extracts of erythroleukemia cells bind to this CCAAT region and contact the nucleotide mutated in Greek HPFH. The ubiquitous CCAAT-binding factor CP1 interacts preferentially with the proximal CCAAT sequence. An erythroid cell-specific factor, referred to as NF-E, binds with a higher affinity to the distal CCAAT region and interacts only with sequences flanking the CCAAT motif. The third protein is the vertebrate homologue of the sea urchin CCAAT displacement protein and recognizes sequences in both CCAAT elements and their flanking sequences. While the point mutation in Greek HPFH slightly strengthens the binding of CP1 and the CCAAT displacement protein, the same base change strongly reduces the binding of NF-E to the distal CCAAT region, suggesting a possible role of NF-E in the repression of gamma-globin genes in adult erythroid cells.     

Detection of two tissue-specific DNA-binding proteins with affinity for sites in the mouse beta-globin intervening sequence 2.

To identify proteins from uninduced murine erythroleukemia nuclear extracts which specifically bind to sequences from the DNase I-hypersensitive region within the mouse beta-globin intervening sequence 2 (IVS2), a gel electrophoretic mobility shift assay was used. Two distinct sequence-specific binding proteins were detected. The specific binding sites for these factors were delineated by both DNase I protection footprinting and methylation interference. Factor B1 bound specifically to two homologous sites, B1-A and B1-B, approximately 100 base pairs apart within the IVS2 and on opposite strands. These two regions could interact with factor B1 independently. Factor B1 was limited to cells of hematopoietic lineages. Factor B2 bound to a site approximately 5 base pairs away from the B1-A site and was limited to cells of the erythroid lineage. The limited tissue distribution of these factors and the locations of their binding sites suggest that one or both of these factors may be involved in the formation of the tissue-specific DNase I-hypersensitive site in the IVS2 of the mouse beta-globin gene.     

Three-dimensional structure of complexes of single-stranded DNA-binding proteins with DNA. IKe and fd gene 5 proteins form left-handed helices with single-stranded DNA

Specimen-tilting in an electron microscope was used to determine the three-dimensional architecture of the helical complexes formed with DNA by the closely related single-stranded DNA binding proteins of fd and IKe filamentous viruses. The fd gene 5 protein is the only member of the DNA-helix-destabilizing class of proteins whose structure has been determined crystallographically, and yet a parameter essential to molecular modeling of the co-operative interaction of this protein with DNA, the helix handedness, has not been available prior to this work. We find that complexes formed by titrating fd viral DNA with either the fd or IKe gene 5 protein have a left-handed helical sense. Complexes isolated from Escherichia coli infected by fd virus are also found to be left-handed helical; hence, the left-handed fd helices are not an artefact of reconstitution in vitro. Because the proteins and nucleic acid of the complexes are composed of asymmetric units which cannot be fitted equivalently to right-handed and left-handed helices, these results rule out a previous computer graphics atomic model for the helical fd complexes: a right-handed helix had been assumed for the model. Our work provides a defined three-dimensional structural framework within which to model the protein-DNA and protein-protein interactions of two structurally related proteins that bind contiguously and co-operatively on single-stranded DNAs.     

The deletion of the distal CCAAT box region of the A gamma-globin gene in black HPFH abolishes the binding of the erythroid specific protein NFE3 and of the CCAAT displacement protein.

Non-deletion Hereditary Persistence of Fetal Hemoglobin (HPFH) is characterized by great elevation of the synthesis, in adult age, of fetal hemoglobin (HbF), of either the A gamma or G gamma type. Strong genetic evidence indicates point mutations in the G gamma- or A gamma-globin promoter as responsible for overexpression of the mutated gene. Here we report that a 13 nucleotides deletion in the CCAAT box region of the A gamma-globin promoter, associated with greater than 100 fold overexpression of the gene, abolishes the in vitro binding of the ubiquitous factors CP1 and CDP (CCAAT displacement protein) and of the erythroid specific protein NFE3. Loss of NFE3 binding is consistent with a similar effect of the -117 G greater than A HPFH mutation, suggesting a possible role of NFE3 as a negatively acting factor. In addition, loss of CDP binding indicates that this alteration might also contribute to the HPFH phenotype in this particular case, suggesting possible heterogeneity of the mechanisms causing HPFH.     

The trihelical bundle subdomain of the GGA proteins interacts with multiple partners through overlapping but distinct sites

The Golgi-localized, gamma-adaptin ear-containing, ARF-binding (GGA) proteins are monomeric clathrin adaptors that mediate the sorting of cargo at the trans-Golgi network and endosomes. The GGAs contain four different domains named Vps27, Hrs, Stam (VHS); GGAs and TOM1 (GAT); hinge; and gamma-adaptin ear (GAE). The VHS domain recognizes transmembrane cargo, whereas the hinge and GAE regions bind clathrin and accessory proteins, respectively. The GAT domain is a polyfunctional module that interacts with various partners including the small GTPase ARF, the endosomal fusion regulator Rabaptin-5, ubiquitin, and the product of the tumor susceptibility gene 101 (TSG101). Previous x-ray crystallographic analyses showed that the GAT region is composed of two subdomains, an N-terminal helix-loop-helix containing the ARF binding site, and a C-terminal triple alpha-helical (trihelical) bundle. In this study, we define the Rabaptin-5 binding site on the GGA1-GAT domain and its relationship to the binding sites for ubiquitin and TSG101. Our observations show that Rabaptin-5, ubiquitin, and TSG101 bind to overlapping but distinct binding sites on the trihelical bundle. The different GAT binding partners engage in both competitive and cooperative interactions that may be important for the function of the GGAs in protein sorting.     

A novel C-T transition within the distal CCAAT motif of the G gamma-globin gene in the Japanese HPFH: implication of factor binding in elevated fetal globin expression.

Hereditary persistence of fetal hemoglobin (HPFH) is a condition characterized by the continued expression of the fetal globin gene in adulthood. Both deletional and nondeletional forms have been described. We studied one Japanese family with two different nondeletional forms of HPFH. Analysis of polymorphic restriction sites in the beta-globin gene cluster suggested that one affecting both G gamma and A gamma globin expression in two members of the family could be associated with unknown conditions not linked to the beta-globin gene loci. Characterization by the polymerase chain reaction (PCR) of another form producing a G gamma-HPFH phenotype in two other members demonstrated a novel C-T transition at the nucleotide -114 within the distal CCAAT motif of the G gamma-globin gene. Using gel retardation assays on various nuclear extracts, we also demonstrated that this novel mutation abolishes the binding of the ubiquitous CCAAT binding factor, CP1 to the distal CCAAT motif of the gamma-globin gene but does not affect the binding of any erythroid specific factor, thereby suggesting a possible role for CP1 in the developmental regulation of fetal globin expression.     

Nuclear proteins that bind the human gamma-globin gene promoter: alterations in binding produced by point mutations associated with hereditary persistence of fetal hemoglobin.

The molecular mechanisms responsible for the human fetal-to-adult hemoglobin switch have not yet been elucidated. Point mutations identified in the promoter regions of gamma-globin genes from individuals with nondeletion hereditary persistence of fetal hemoglobin (HPFH) may mark cis-acting sequences important for this switch, and the trans-acting factors which interact with these sequences may be integral parts in the puzzle of gamma-globin gene regulation. We have used gel retardation and footprinting strategies to define nuclear proteins which bind to the normal gamma-globin promoter and to determine the effect of HPFH mutations on the binding of a subset of these proteins. We have identified five proteins in human erythroleukemia cells (K562 and HEL) which bind to the proximal promoter region of the normal gamma-globin gene. One factor, gamma CAAT, binds the duplicated CCAAT box sequences; the -117 HPFH mutation increases the affinity of interaction between gamma CAAT and its cognate site. Two proteins, gamma CAC1 and gamma CAC2, bind the CACCC sequence. These proteins require divalent cations for binding. The -175 HPFH mutation interferes with the binding of a fourth protein, gamma OBP, which binds an octamer sequence (ATGCAAAT) in the normal gamma-globin promoter. The HPFH phenotype of the -175 mutation indicates that the octamer-binding protein may play a negative regulatory role in this setting. A fifth protein, EF gamma a, binds to sequences which overlap the octamer-binding site. The erythroid-specific distribution of EF gamma a and its close approximation to an apparent repressor-binding site suggest that it may be important in gamma-globin regulation.     

A new method for the purification of DNA-binding proteins with sequence specificity

We describe a technique for a rapid and efficient isolation and purification of proteins binding to defined DNA sequences. Cloned double-stranded DNA was covalently coupled to m-aminobenzyloximethylcellulose in order to purify proteins which recognize and bind to specific sequences on the DNA. The purification of two DNA-binding proteins from Drosophila melanogaster is demonstrated using the respective cloned DNA sequences.     

The G1021A substitution in the RYR1 gene does not cosegregate with malignant hyperthermia susceptibility in a British pedigree.

A single base change in the RYR1 gene encoding the skeletal muscle ryanodine receptor (calcium-sensitive calcium-release channel of the sarcoplasmic reticulum), resulting in the substitution of G1021 by A, has been proposed to underlie malignant-hyperthermia (MH) susceptibility in as many as 10% of cases in the European population. As part of our mutation-screening program in MH-susceptible (MHS) individuals, we have investigated this substitution in individuals from 151 unrelated British MHS families and have detected G1021A heterozygotes in 7 families. This mutation was not found in 156 unrelated MH-negative (MHN) individuals. We also examined eight families with central core disease (CCD): the mutation did not occur in any family members of any disease status (affected or unaffected for CCD, MHS, or MHN). In one large family, the G1021A mutation was found but did not show complete cosegregation with MH susceptibility: it occurred in only 7/12 MHS individuals in the kinship, and susceptibility was inherited from parents who were G1021 homozygotes, as well as from parents who were heterozygotes. On the basis of these findings, it is clearly unreliable at present to offer presymptomatic DNA testing for MH status, even in families in which a mutation has been detected.     

A functional glucocorticoid-responsive unit composed of two overlapping inactive receptor-binding sites: evidence for formation of a receptor tetramer.

An unusual glucocorticoid-responsive element (called GRE A) was found to mediate the induction of the cytosolic aspartate aminotransferase gene by glucocorticoids and was bound by the glucocorticoid receptor in a DNase I footprinting assay. GRE A consists of two overlapping GREs, each comprising a conserved half-site and an imperfect half-site. The complete unit was able to confer glucocorticoid inducibility to a heterologous promoter (delta MTV-CAT). Mutation of any of the half-sites, including the imperfect ones, abolished inducibility by the hormone, demonstrating that each of the isolated GREs was inactive. In electrophoretic mobility shift assays, purified rat liver glucocorticoid receptor (GR) formed a low-mobility complex with GRE A, presumably containing a GR tetramer. When purified bacterially expressed DBD was used, low-mobility complexes as well as dimer and monomer complexes were formed. In inactive mutated oligonucleotides, no GR tetramer formation was detected. Modification of the imperfect half-sites in order to increase their affinity for GR gave a DNA sequence that bound a GR tetramer in a highly cooperative manner. This activated unit consisting of two overlapping consensus GREs mediated glucocorticoid induction with a higher efficiency than consensus GRE.