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G Felsenfeld

Publications and source records attributed to G Felsenfeld.

At least 73 records · Page 4Linked to original sources
PubMed1990

Properties of BGP1, a poly(dG)-binding protein from chicken erythrocytes.

The chicken beta A-globin gene contains in the neighborhood of its 5' promoter a (dG)-homopolymer sequence 16 base pairs long. The 66 kD protein BGP1 (beta globin protein 1), isolated from chicken erythrocytes, has been shown to bind specifically to this sequence. We describe further purification of BGP1, measure its affinity for the beta A-globin promoter binding site, and analyze its binding properties. The minimal binding sequence is seven dG residues; methylation interference studies show that each of these residues contacts BGP1. Binding competition experiments employing (dG).(dC) oligomers of varying lengths also consistent with (dG)7 as a minimum recognition sequence. All of the data can be explained by a model in which BGP1 binds to any contiguous set of seven (dG) residues, so that the effective constant for binding to (dG)n is proportional to n minus 6. This behavior may be typical of proteins that bind specifically to repeated sequences.

Animals↗
PubMed1990

Structure and evolution of a human erythroid transcription factor.

Vertebrate erythroid cells contain a tissue-specific transcription factor referred to as Eryf 1 (ref. 1), GF-1 (ref. 2) or NF-E1 (ref. 3), for which binding sites are widely distributed in the promoters and enhancers of the globin gene family, and of other erythroid-specific genes. Aberrant binding of the human factor to a mutant site has been implicated in one form of hereditary persistence of fetal haemoglobin (HPFH; ref. 2). The complementary DNAs for both the chicken cEryf 1 (ref. 11) and mouse mEryf 1 (ref. 12) encoding genes have recently been cloned. We report here the cloning of the cDNA for the human Eryf 1 encoding gene. The central third of the hEryf 1 cDNA, containing two 'finger' motifs, is almost identical to that of chicken or mouse. The amino-and carboxy-terminal thirds of the human protein are similar to those of mouse, but are strikingly different from the corresponding domains in chicken. The evidence indicates that these erythroid regulatory factors evolved from a common precursor composed of two distinct kinds of repeated domains, which subsequently evolved at greatly different rates.

Amino Acid Sequence↗
PubMed1990

Developmental regulation of topoisomerase II sites and DNase I-hypersensitive sites in the chicken beta-globin locus.

We have mapped DNase I-hypersensitive sites and topoisomerase II (topo II) sites in the chicken beta-globin locus, which contains four globin genes (5'-rho-beta H-beta A-epsilon-3'). In the 65 kilobases (kb) mapped, 12 strong hypersensitive sites were found clustered within the 25-kb region from 10 kb upstream of rho to just downstream of epsilon. The strong sites were grouped into several classes based on their tissue distribution, developmental pattern, and location. (i) One site was present in all cells examined, both erythroid and nonerythroid. (ii) Three sites, located upstream of the rho-globin gene, were present at every stage of erythroid development, but were absent from nonerythroid cells. (iii) Four sites at the 5' ends of each of the four globin genes were hypersensitive only in the subset of erythroid cells that were transcribing or had recently transcribed the associated gene. (iv) Another three sites, whose pattern of hypersensitivity also correlated with expression of the associated gene, were found 3' of rho, beta H, and epsilon. (v) A site 3' of beta A and 5' of epsilon was erythroid cell specific and present at all developmental stages, presumably reflecting the activity of this enhancer throughout erythroid development. We also mapped the topo II sites in this locus, as determined by teniposide-induced DNA cleavage. All strong teniposide-induced cleavages occurred at DNase I-hypersensitive sites, while lesser amounts of cleavage were observed in transcribed regions of DNA. Most but not all of the DNase I-hypersensitive sites were topo II sites. These data are consistent with the hypothesis that, in vivo, topo II preferentially acts on nucleosome-free regions of DNA but suggest that additional topo II regulatory mechanisms must exist.

Animals↗
PubMed1989

The erythroid-specific transcription factor Eryf1: a new finger protein.

The erythroid-specific transcription factor Eryf1 binds to DNA sites within regulatory regions of every member of both the alpha- and beta-globin families in chicken. The distribution of these sites suggests that Eryf1 may serve as a general "switch" factor for erythroid development. We have cloned the cDNA for Eryf1 and show that the corresponding mRNA is present in all erythroid lineages, but is absent from non-erythroid cells. We demonstrate that the cDNA encodes the specific Eryf1 binding activity found in erythrocytes. Eryf1 is a basic 38 kd protein containing a pair of highly similar "fingers" with the motif Cys-x-x-Cys-x17-Cys-x-x-Cys. The amino acid sequences of these regions bear no resemblance to those found in other regulatory proteins with a similar arrangement of cysteine residues. Our evidence suggests, furthermore, that transition metal ions are unusually tightly bound, or may not be necessary for the sequence-specific DNA binding of Eryf1.

Amino Acid Sequence↗
PubMed1989

Developmental modulation of protein binding to beta-globin gene regulatory sites within chicken erythrocyte nuclei.

We describe the interaction of two adjacent binding sites in the chicken beta-globin gene promoter with regulatory factors present in erythroid cells. One of these sites is a palindromic sequence (Pal) that binds a member of the nuclear factor 1 family; the other is the CACCC sequence found in most adult beta-globin promoters. Transfection of primary erythrocytes with plasmids carrying the gene coupled to truncated promoters reveals that the Pal site inhibits and the CACCC site stimulates expression. Nuclease protection experiments on intact nuclei show that at early stages of embryonic development, the CACCC site is occupied and the Pal site is vacant, but as development progresses, the Pal site is filled gradually and the CACCC site loses its bound protein. Beyond day 15 of development, Pal is completely occupied and CACCC is empty in vivo. Parallel DNase I footprinting and gel retardation studies in vitro show that nuclear extracts contain sharply increasing Pal-binding activity as development proceeds, but CACCC-binding activity falls off only slightly. We show that the two factors bind to their sites in vitro in an anticooperative manner and conclude that this could account for the observed changes in site occupancy in vivo. Our results suggest that the Pal factor may play a role in the shutdown of adult beta-globin expression late in erythroid development.

Animals↗
PubMed1989

Identification and characterization of a chicken alpha-globin enhancer.

We identify and describe the properties of an enhancer within the chicken alpha-globin gene cluster. This cluster consists of one gene (pi) expressed only in primitive erythrocytes and two (alpha A and alpha D) expressed in both primitive and definitive cell lineages. The genes are linked together in the order 5'-pi-alpha D-alpha A-3' and occupy a region about 10 kilobase pairs long. The enhancer is located at the 3' end of the cluster, about 750 base pairs 3' to the alpha A translation stop site. When assayed by transfection into either primitive or definitive primary chicken erythrocytes, this element stimulated expression from plasmids containing the alpha D- or alpha A-globulin gene promoters. Except for sites in the alpha-globin promoters, no other stimulatory activity was observed in DNA taken from other regions of the alpha-globin locus. Moderate resolution DNase I hypersensitivity studies as well as DNase I footprinting revealed three regions of protein binding, each containing a similar core DNA sequence within the enhancer element. Gel mobility shift studies demonstrated that all three regions bind the recently identified erythrocyte-specific factor, EryfI, which has binding sites in the regulatory regions of all chicken globin genes. Our data suggest that the enhancer we have identified may act in vivo only on the alpha A gene; expression of the alpha D gene is affected by another EryfI site located in the alpha D promoter. Such a mechanism would be consistent with the observed relative abundances of alpha A- and alpha D-globin in vivo. The simplicity of these regulatory elements may reflect the limited repertoire of expression of these genes during development.

Animals↗
PubMed1988

An erythrocyte-specific DNA-binding factor recognizes a regulatory sequence common to all chicken globin genes.

We have identified a protein present only in erythroid cells that binds to two adjacent sites within an enhancer region of the chicken beta-globin locus. Mutation of the sites, so that binding by the factor can no longer be detected in vitro, leads to a loss of enhancing ability, assayed by transient expression in primary erythrocytes. Binding sites for the erythroid-specific factor (Eryf1) are found within regulatory regions for all chicken globin genes. A strong Eryf1 binding site is also present within the enhancer of at least one human globin gene, and proteins from human erythroid cells (but not HeLa cells) bind to both the chicken and the human sites.

Animals↗
PubMed1988

Mutational analysis of the chicken beta-globin enhancer reveals two positive-acting domains.

We report a mutational analysis of the chicken beta-globin enhancer, using transient expression in primary embryonic erythrocytes. A series of 27 scanning mutants differing only by consecutive 4-base substitutions was tested. Previous experiments, using protection from DNase I digestion, demonstrated four regions in the enhancer that bind factors. Mutations in two of the four regions (denoted I and III) have no effect on enhancer activity. The other two regions (II and IV) mediate all of the positive-acting activity. Region II appears to consist of two subregions, mutation in either of which leads to the same loss in activity as mutation of both. Like region II, mutation of either half of region IV results in reduced enhancer activity. However, in contrast to region II, mutation of both halves of region IV results in more loss of enhancer activity than either single mutation. These data suggest that the half-sites in region II must interact in order to cause enhancement, whereas the half-sites in region IV act independently. Triplications of regions II or IV are able to enhance to a similar degree as the parental enhancer, but single copies of regions II or IV enhance only weakly. Thus, as measured by transient expression in embryonic erythrocytes, the beta-globin enhancer may involve the action of as few as three DNA-binding proteins acting at two sites.

Acetyltransferases↗
PubMed1988

Bidirectional control of the chicken beta- and epsilon-globin genes by a shared enhancer.

An enhancer specific to erythroid cells was identified previously in the 3' flanking sequence of the chicken adult beta-globin gene and shown to act on the beta-globin promoter. This enhancer lies between the adult beta-globin gene and the embryonic epsilon-globin gene, about equidistant from the two promoters. To determine whether this enhancer acts also on the epsilon-globin promoter, we constructed plasmids containing the enhancer and either the beta- or the epsilon-globin promoter fused to the bacterial chloramphenicol acetyltransferase gene. Primary chicken erythrocytes of both primitive and definitive lineages were transfected with these plasmids. We show that the enhancer is able to stimulate expression from the epsilon-globin promoter as well as the beta-globin promoter. Levels of expression change with the developmental stage of the cell in a way that is partially consistent with the observed developmental regulation of the beta- and epsilon-globin genes in vivo. There appear to be no other enhancer elements either 5' of the epsilon-globin gene or within 6 kilobase pairs of its 3' end. Thus, the enhancer between the beta- and epsilon-globin genes apparently serves to regulate both genes.

Animals↗
PubMed1988

An erythrocyte-specific protein that binds to the poly(dG) region of the chicken beta-globin gene promoter.

The promoter region of the chicken adult beta-globin gene contains a sequence of 16 deoxyguanosine residues located at a nucleosome boundary in tissues where the gene is inactive. In definitive erythrocytes that express the beta-globin gene, the nucleosome is displaced, the G-string and adjacent sequences are occupied by sequence-specific DNA-binding proteins, and a nuclease hypersensitive domain is generated in this region. To gain insight into the role of the G-string in this series of events, we have examined the proteins that bind to it. Using the gel mobility shift assay and a monoclonal antibody that blocks specific binding to the G-string, we have identified a specific protein, BGP1, that is found only in chicken erythroid cells and appears at the same time, or shortly before, the changes in chromatin structure. The antibody interacts strongly with BGP1 and cross-reacts weakly with Sp1. Although both BGP1 and Sp1 require Zn2+ for their DNA-binding activity, these proteins differ in their binding-site specificities, chromatographic properties, and molecular weights. In contrast to Sp1, which is found in a wide variety of cell types, BGP1 is restricted to erythrocytes and is most abundant in definitive erythrocytes. Thus, its presence corresponds to the tissue- and stage-specific occupancy of the G-string in vivo.

Animals↗
PubMed1987

Effect of Z-DNA on nucleosome placement.

Histone octamers were reconstituted on plasmids carrying the alternating nucleotide sequence (G-C)15. The plasmids, radioactively labeled at one of two neighboring sites near the (G-C) insert, were digested with micrococcal nuclease. Nucleosome core particles were isolated and the monomer DNA subjected to restriction analysis. Quite different results are obtained if the reconstitution is carried out with relaxed plasmids, in which the (G-C) insert is in the B form, or with supercoiled plasmids, where it is in the Z form. With supercoiled plasmids, there is a marked reduction (compared with relaxed plasmids) in the abundance of labeled monomers, the result of a large decrease in core particles carrying any (G-C) sequence. Some core particles formed on supercoiled (Z) plasmids are positioned either just outside the (G-C) sequence, or with the sequence occupying the terminal position within the core particle. In contrast, monomers obtained from relaxed plasmids incorporate the (G-C) sequence in the B form more or less randomly in the interior of the core particle; species showing discrete positioning make only a minor contribution. We conclude that DNA in the Z form cannot be incorporated within core particles, except at their termini, and that a transition from the B to the Z form in vivo might result in a significantly altered local placement of nucleosomes.

Base Composition↗
PubMed1987

Analysis of the tissue-specific enhancer at the 3' end of the chicken adult beta-globin gene.

In an earlier paper we identified a tissue-specific enhancer in the 3' flanking region of the chicken adult beta-globin gene. In this paper we analyze the properties of this enhancer. Deletion analysis and transient expression assays show that the domain responsible for activation of transcription is at most 136 base pairs long. Specific factors that bind to discrete sequences within the enhancer DNA are found in extracts of embryonic and adult erythrocytes and in brain. These factors are specific for the tissue or the erythrocyte developmental stage and protect at least five discrete regions in or near the enhancer against DNase I digestion in "footprinting" experiments. Four of these regions reside wholly within the 136-base-pair functional enhancer domain, which also comprises a site in chromatin that is hypersensitive to nucleases. The nature of the binding sites and the program of appearance of the factors during development suggest that a subset of these interactions may be responsible for the developmental specificity of the enhancer.

Animals↗
PubMed1987

Effects of DNA supercoiling on the topological properties of nucleosomes.

In the nucleosome core particle, at least 145 base pairs of DNA are bound to the histone octamer in a superhelical conformation. We have asked what effect the presence of these particles has on the ability of DNA gyrase to supercoil DNA. Synthetic minichromosomes, constructed by reconstituting complexes of core histones with the closed circular plasmid pBR322, were treated with various amounts of DNA gyrase. We have found that the maximum level of supercoiling that is attainable is nearly identical for protein-free plasmids and for plasmids half-saturated with core histones, even though supercoiling does not result in a loss of histones from the complex. It appears that, at sufficiently high levels of supercoiling, the core particle is disrupted in such a way that the DNA bound to histones is no longer constrained.

Chromosomes↗
PubMed1987

The mechanism of osmotic transfection of avian embryonic erythrocytes: analysis of a system for studying developmental gene expression.

We have undertaken a study of the mechanism of DNA transfer into primary chicken erythrocytes by a method named osmotic transfection. The cells are subjected to controlled osmotic swelling in NH4Cl and then ruptured in a lower osmotic strength solution containing DNA and DEAE-dextran. The osmotic rupture results in transient formation of a single hole in the cell membrane, which is followed within hours by recovery of near normal levels of RNA and protein synthesis. The association of DNA with the cells is much greater for ruptured than for unruptured cells or for cells that have been lysed and resealed before DNA is added. Transient formation of pores in the cell membrane is apparently essential for high rates of macromolecular transfer into the cell. DEAE-dextran increases the amount of DNA associated with the cells, especially after cell rupture. Our understanding of the mechanism has allowed us to extend the application of osmotic transfection to essentially all developmental stages of avian erythroid differentiation. Osmotic transfections were done with plasmids containing the chloramphenicol acetyl transferase (cat) gene placed between the chicken beta-globin promoter and the 3' beta-globin enhancer. The pattern of CAT expression at sequential developmental stages parallels that of the endogenous gene, showing that osmotically transfected cells appear to retain developmental fidelity. The approach provides a convenient, sensitive, and flexible system for the study of transient gene expression as a function of development.

Acetyltransferases↗