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G Felsenfeld

Publications and source records attributed to G Felsenfeld.

At least 19 recordsLinked to original sources
PubMed1992

A nucleosome core is transferred out of the path of a transcribing polymerase.

We have determined the fate of a nucleosome core on transcription. A nucleosome core was assembled on a short DNA fragment and ligated into a plasmid containing a promoter and terminators for SP6 RNA polymerase. The nucleosome core was stable in the absence of transcription. The distribution of nucleosome cores after transcription was examined. The histone octamer was displaced from its original site and reformed a nucleosome core at a new site within the same plasmid molecule, with some preference for the untranscribed region behind the promoter. These observations eliminate several models that have been proposed for transcription through a nucleosome core. Our results suggest that a nucleosome core in the path of a transcribing polymerase is displaced by transfer to the closest acceptor DNA.

Animals↗
PubMed1992

Chromatin as an essential part of the transcriptional mechanism.

The increasingly detailed biochemical definition of the protein complexes that regulate gene transcription has led to the re-emergence of questions about the role of histones. Much recent evidence suggests that transcriptional activation requires that transcription factors successfully compete with histones for binding to promoters, and that there may be more than one mechanism by which this is achieved.

Animals↗
PubMed1992

The developmental switch in embryonic rho-globin expression is correlated with erythroid lineage-specific differences in transcription factor levels.

During chicken embryogenesis, the rho-globin gene is expressed only in the early developmental stages. We have examined the mechanisms that are responsible for this behavior. The transcription of the rho-globin gene is strongly correlated with the presence during development of primitive erythroid lineage cells, consistent with the idea that the expression of the rho-globin gene is restricted to that lineage. The "switching off" of rho-globin during development thus reflects the change from primitive to definitive cell lineages which occurs during erythropoiesis in chicken. We use transient expression assays in primary erythroid and other cells to show that the information for lineage- and tissue-specific expression of the rho-globin gene is contained in a 456 bp region upstream of the gene's translational start site. DNA-binding studies, coupled with analysis of the effect on expression of deletions and binding site mutations, were used to identify important control elements within this 456 bp region. We find that binding sites for the ubiquitous transcription factor Sp1, and the specific hematopoietic factor GATA-1, are crucial for expression of the gene in primitive erythroid cells. Quantitative analysis shows that nuclei of the primitive erythroid lineage contain 10-fold more of these factors than do the nuclei of definitive cells. We show that in principle these differences in factor concentration are sufficient to explain the lineage-specific behavior that we observe in our assays. We suggest that this may be an important part of the mechanism for lineage-restricted rho-globin expression during chicken erythroid development. Similar mechanisms may be involved in regulation of other (but not all) members of the globin family.

Animals↗
PubMed1992

Developmental regulation of globin gene expression.

We have used the globin family of genes in chicken to study developmental regulation of gene expression, both at the level of individual interaction of trans-acting factors with local promoters and enhancers, and at the level of chromatin structure. Regulation of all members of the alpha- and beta-globin clusters is affected by the erythroid regulatory factor GATA-1. Separate mechanisms exist for regulation of individual members of the family. As an example, we describe the control mechanisms that play a role in the expression of the rho-globin gene, which is expressed only in primitive lineage erythroid cells. In addressing the involvement of chromatin structure in gene activation, we have examined the role of locus control elements, and also considered the way in which RNA polymerase molecules might accommodate to the presence of nucleosomes on transcribed genes.

Animals↗
PubMed1991

Triplex RNA.

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Molecular Structure↗
PubMed1991

Structure and promoter activity of the gene for the erythroid transcription factor GATA-1.

We have cloned the gene for the chicken erythroid transcription factor GATA-1 (formerly Eryf1, NF-E1, or GF-1). The gene is composed of six exons, two of which encode the two finger domains of the protein. Transcription of GATA-1 in chicken embryonic erythroid cells initiates from multiple sites clustered approximately 200 base pairs upstream from the start of protein-coding sequence. A number of sequence motifs for known DNA-binding proteins are found to be protected in DNase I-footprinting experiments by either erythroid or brain nuclear extracts or by both. Notably, a cluster of three GATA-1 sites is protected by the erythroid extract, as well as by purified GATA-1. We find that the upstream region of the gene functions as a powerful promoter when transfected into embryonic erythroid cells. In primary chicken embryo fibroblasts the promoter exhibits lower activity, which is increased when the cells are cotransfected with a second plasmid expressing the GATA-1 cDNA. The results suggest that GATA-1 protein plays an autoregulatory role in its own expression.

Animals↗
PubMed1991

Formation of nucleosomes on positively supercoiled DNA.

A transcribing RNA polymerase is thought to generate positive supercoils in front of the advancing transcription complex and negative supercoils behind. We have examined the possibility that positive supercoils might destabilize nucleosomes, facilitating transcription. We show that histone octamers bind to positively supercoiled DNA, and that after the complex is relaxed, 'classical' nucleosomes are present. We tested the possibility that nucleosomes on positively supercoiled DNA are in an altered (presumably more open) conformation, but revert to the classical structure only on release of this stress. However, circular dichroic spectra, and chemical cross-linking and modification of core histones, all suggest that the complexes initially formed on positively supercoiled DNA are classical nucleosomes. Although such structures are stable, their formation requires the plasmid to become more positively supercoiled, resulting in greater superhelical stress. In contrast, formation of nucleosomes on negatively supercoiled DNA relieves superhelical stress. In an exchange experiment in which equilibrium is achieved, nucleosomes transfer from positively to negatively supercoiled DNA, as predicted from the super-coiling free energies of the reactions. This suggests a mechanism for transcription of a gene assembled into chromatin, in which octamers are sequentially transferred from the region in front of the polymerase to the region behind.

Animals↗
PubMed1991

trans-Activation of a globin promoter in nonerythroid cells.

We show that expression in fibroblasts of a single cDNA, encoding the erythroid DNA-binding protein Eryf1 (GF-1, NF-E1), very efficiently activates transcription of a chicken alpha-globin promoter, trans-Activation in these cells occurred when Eryf1 bound to a single site within a minimal globin promoter. In contrast, efficient activation in erythroid cells required multiple Eryf1 binding sites. Our results indicate that mechanisms exist that are capable of modulating the trans-acting capabilities of Eryf1 in a cell-specific manner, without affecting DNA binding. The response of the minimal globin promoter to Eryf1 in fibroblasts was at least as great as for optimal constructions in erythroid cells. Therefore, the assay provides a very simple and sensitive system with which to study gene activation by a tissue-specific factor.

Animals↗
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↗