Zinc induces a bend within the transcription factor IIIA-binding region of the 5 S RNA gene.

Binding of Zn2+ to the 5 S RNA gene sequence of Xenopus borealis results in strong bending of the DNA, as inferred from transient electric birefringence data. The effect is specific for Zn2+; several other divalent ions are not able to induce a bend of a similar magnitude. Using five different fragments that span the binding sequence, we are able to estimate a bend magnitude of at least 55 degrees centered at base-pair +65 within the gene. This places the bend within the binding domain of the gene-regulatory protein transcription factor (TF) IIIA. Recent evidence has shown that the protein-DNA complex is also bent. Although our data do not allow us directly to link the two bends, our results suggest that TFIIIA could form a folded structure by stabilizing the same bent conformation that is induced by binding of Zn2+. The chemistry of Zn2+ binding to DNA, and the sequence around the bend center, suggest that the bend is most probably caused by joint co-ordination of Zn2+ to the N-7 groups of stacked purine residues.

Developmental changes in the expression of high mobility group chromosomal proteins.

The high mobility group (HMG) chromosomal proteins may modulate the structure of distinct regions in chromatin, thereby affecting processes such as development and differentiation. Here we report that the levels of the HMG chromosomal proteins and their mRNAs change significantly during erythropoiesis. Erythroid cells from 5-day chicken embryos contain 2.5-10 times more HMG mRNAs than cells from 14-day embryos, whereas circulating cells from adult animals are devoid of HMG and most other mRNAs. Nuclear run-off experiments and Northern analysis of RNA from various developmental stages and from Percoll-fractionated cells indicate that the genes are transcribed in early cells of either the primitive or definitive erythroid lineage. The rate of synthesis of the various HMGs changes during erythropoiesis; in erythroid cells from 7-day embryos the ratio of HMG-14b or HMG-17 to HMG-14a is, respectively, 8 and 10 times lower than in 9-day erythroids. HMG-14a, the major chicken HMG-14 species, is synthesized mainly in primitive cells, while HMG-14b is preferentially synthesized in definitive cells. Thus, the change from primitive to definitive erythroid lineage during embryogenesis is accompanied by a change in the expression of HMG chromosomal proteins. Conceivably, these changes may affect the structure of certain regions in chromatin; however, it is not presently clear whether the switch in HMG protein gene expression is a consequence or a prerequisite for proper differentiation.

Differentiation-dependent alteration in the chromatin structure of chromosomal protein HMG-17 gene during erythropoiesis.

The expression of the gene coding for chromosomal protein HMG-17 is down regulated during chicken erythrocyte maturation. The transcriptional down regulation is associated with major alterations in the chromatin structure of this gene. The 5' region of the gene contains both constitutive and developmental stage-specific deoxyribonuclease I (DNase I) hypersensitive sites. The constitutive sites bracket the "CpG island" present in the gene, which remains hypomethylated throughout the various developmental stages. During erythropoiesis, the gene acquires a distinct structure that, upon digestion with micrococcal nuclease (MNase) yields an unusual repeat. Two nucleosomes, with a 200 base-pair repeat, are positioned immediately downstream from the start of transcription. Immediately downstream and upstream from these nucleosomes, the boundaries between MNase sites change to a 75 base-pair repeat, which indicates an unusual chromatin structure. The differentiation related changes in the DNase I and MNase digestion pattern in the 5' region of the gene suggest that sequences present in the first intron may be involved in gene regulation. The results may be relevant to the regulation of the entire HMG-14/-17 gene family.

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.

Erythroid-specific activation and derepression of the chick beta-globin promoter in vitro.

Transcriptionally active extracts were prepared from chick red cells isolated at different stages of development. The template activity of cloned beta-globin genes is highest in extracts from definitive red cells, where the endogenous gene is normally expressed, and lowest in extracts from primitive red cells or nonerythroid tissues. This system has been used to identify regulatory elements and to assign functions to the proteins that bind within the beta-globin promoter. Regulation of expression is achieved, in part, by factors whose composition changes during red cell development. Two proteins, PAL and CON, bind at adjacent sites but have opposite effects on transcription in vitro. Levels of PAL, a potent repressor, are highest in mature red cells while those of CON, an activator, are highest in actively transcribing red cells. The effect of PAL can be overcome by blocking its binding site with a protein having a similar recognition sequence but a dissimilar function.

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.

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.

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.

Mechanism of oligonucleotide loop formation in solution.

We have studied the tridecadeoxynucleotide CGCGAATTACGCG (I), which contains an additional A at position 9 compared to the dodecanucleotide of which the crystal structure has been determined. Sequence I exhibits no distinct melting curve and also has a concentration-dependent pattern of peaks on reverse-phase chromatography. This behavior is explained by a slow equilibration between loop and duplex forms in solution. We have characterized this equilibrium by proton NMR spectroscopy and shown that it is fully reversible by monitoring the two thymine methyl resonances, each of which occurs in two environments. Lower temperature and higher concentration favor the duplex; the midpoint of the transition is such that the loop predominates at room temperature. We have measured the van't Hoff enthalpy of formation of the duplex and the activation energy by temperature-jump and saturation-transfer experiments. The results are compared with those for the 17-mer sequence CGCGCGAATTACGCGCG (II), which contains two additional base pairs in the stem of the loop. The thermodynamic parameters and the effect of increasing salt concentration on the rate of conversion of the loop and duplex forms lead us to presume that the mechanism of interconversion involves complete strand separation and re-formation rather than cruciform formation and branch migration.

Nucleosome phasing on a DNA fragment from the replication origin of simian virus 40 and rephasing upon cruciform formation of the DNA.

Nucleosomes were reconstituted in vitro from a fragment of DNA spanning the simian virus 40 minimal replication origin. The fragment contains a 27-base-pair palindrome (perfect inverted repeat). DNA molecules with stable cruciform structures were generated by heteroduplexing this DNA fragment with mutants altered within the palindromic sequence (C. Nobile and R. G. Martin, Int. Virol., in press). Analyses of the structural features of the reconstituted nucleosomes by the DNase I footprint technique revealed two alternative DNA-histone arrangements, each one accurately phased with respect to the uniquely labeled DNA ends. As linear double-stranded DNA, a unique core particle was formed in which the histones strongly protected the regions to both sides of the palindrome. The cruciform structure seemed to be unable to associate with core histones and, therefore, an alternative phasing of the histone octamer along the DNA resulted. Thus, nucleosome positioning along a specific DNA sequence appears to be influenced in vitro by the secondary structure (linear or cruciform) of the 27-base-pair palindrome. The formation of cruciform structures in vivo, if they occur, might therefore represent a molecular mechanism by which nucleosomes are phased.

Regulated gene expression in transfected primary chicken erythrocytes.

We describe a method for studying transient gene expression in primary avian erythroid cells that involves controlled osmotic shock, followed by DNA transfection using DEAE-dextran. Cells treated in this way reproducibly express high levels of chloramphenicol acetyltransferase (CAT) when transfected with a plasmid having the cat gene coupled to an appropriate viral promoter. An observed correlation between levels of CAT expression and extent of hemoglobin release during controlled shock makes it possible to choose optimum conditions for expression in erythroid cells at various stages of embryonic development. Using these techniques, we have investigated the effect on CAT expression of fusing to the cat gene various portions of the chicken adult beta-globin (beta A) gene. We show that in 9-day or 12-day embryonic erythrocytes, the promoter activity of the 5' flanking region of the beta A gene (in the absence of any viral promoters) is strongly stimulated by a downstream sequence, located in the region 110-588 base pairs on the 3' side of the poly(A) signal, that acts as an enhancer. Its activity is reduced in 5-day embryonic cells and absent in primary chicken fibroblasts and mouse L cells, suggesting that this transient expression system will be useful in studying developmentally regulated globin gene expression.

Crystallization of a DNA tridecamer d(C-G-C-A-G-A-A-T-T-C-G-C-G).

Crystals of the DNA tridecamer d(C-G-C-A-G-A-A-T-T-C-G-C-G) have been grown by the vapor-diffusion technique with 2-methyl-2,4-pentanediol as precipitant. They are monoclinic space group C2, with a = 79.6 A, b = 43.1 A, c = 24.9 A and beta = 98.7 degrees. Previous nuclear magnetic resonance studies predicted that this tridecamer forms a duplex similar to the B DNA dodecamer, d(C-G-C-G-A-A-T-T-C-G-C-G), except for an extra adenosine residue that is stacked within the helix but remains unpaired: (formula; see text) Preliminary X-ray diffraction studies confirmed that the tridecamer is in the B DNA conformation, consistent with the nuclear magnetic resonance results.

DNA conformation at the 5' end of the chicken adult beta-globin gene.

We have examined the conformation of DNA cloned from the hypersensitive region 5' of the chicken adult beta-globin gene, in order to determine whether the unusual sensitivity to nucleases is related to altered secondary structure of the DNA. The region contains a large number of sites for methylation; we have studied the effect of methylation at HpaII sites on the topological properties of a small plasmid containing the region. We find that methylation does not alter the supercoiling properties of the plasmid under a wide variety of conditions; we find no evidence for conversion of any measurable segment of DNA to the Z conformation, even at high superhelix densities. We have also devised a protocol for mapping precisely the sites sensitive to S1 nuclease. The data are not consistent with the presence of cruciform structures. They reveal, however, a major cutting site located within a tract of 16 consecutive deoxyguanosine residues in the center of the hypersensitive region. Susceptibility to cutting is dependent upon supercoiling, but methylation at HpaII sequences has no effect on the S1 cutting pattern.

DNA stem-loop structures bind poorly to histone octamer cores.

Heteroduplex DNA molecules were generated in which one of the two strands contained a 7-base-pair (double-stranded) stem and 3-base-pair (single-stranded) loop. The heteroduplexes and their corresponding homoduplex parental molecules, each of approximately 260 base pairs, were used for nucleosomes reconstitution. Protection from restriction endonuclease digestion was used to probe the structure of the resulting dinucleosomes. Although 50% or more of the potential cleavage sites in the homoduplex DNAs and in the linear portion of the heteroduplex DNAs were inaccessible to nuclease digestion, no protection of the stem-loop structures was observed. The results imply that a stem-loop structure preferentially occupies the spacer region between nucleosomes, but if it is part of a core particle the stem-loop is always pointed "outward" in such a way as to be accessible to nuclease digestion.

Higher order structure of chromatin: orientation of nucleosomes within the 30 nm chromatin solenoid is independent of species and spacer length.

We have used electric dichroism to study the arrangement of nucleosomes in 30 nm chromatin solenoidal fibers prepared from a variety of sources (CHO cells, HeLa cells, rat liver, chicken erythrocytes, and sea urchin sperm) in which the nucleosome spacer length varies from approximately 10 to approximately 80 bp. Field-free relaxation times are consistent only with structures containing 6 +/- 1 nucleosomes for every 11 nm of solenoidal length. With very few assumptions about the arrangement of the spacer DNA, our dichroism data are consistent with the same orientation of the chromatosomes for every chromatin sample examined. This orientation, which maintains the faces of the radially arranged chromatosomes inclined at an angle between 20 degrees-33 degrees to the solenoid axis, thus appears to be a general structural feature of the higher order chromatin fiber.

Histone hyperacetylation has little effect on the higher order folding of chromatin.

HeLa cells were grown in the presence of 10 mM sodium butyrate and soluble chromatin containing hyperacetylated histones was prepared by mild micrococcal nuclease digestion and sucrose gradient fractionation. Sedimentation and electric dichroism were used to study the cation-induced folding of this acetylated chromatin from the 10 nm filament to the 30 nm solenoid conformation. Although under some conditions acetylated chromatin appears slightly less condensed than control chromatin, the major conclusion is that hyperacetylation of histones does not in itself prevent the formation of the higher order chromatin solenoid.

Effect of the B--Z transition in poly(dG-m5dC) . poly(dG-m5dC) on nucleosome formation.

We have studied the properties of complexes formed between histones and the methylated synthetic polydeoxynucleotide poly(dG-m5dC). poly(dG-m5dC). This polymer undergoes the transition from B DNA to left-handed Z DNA at moderate ionic strength. When the polymer is in the Z form it will bind histones, but nucleosomes are not detected. When the polymer in the B form is combined with equimolar quantities of the four core histones and digested with micrococcal nuclease, particles are formed which behave in all respects as normal nucleosome cores. When these core particles are placed in solvents that would result in conversion of the protein-free polymer to the Z form, no transition is observed. The formation of a nucleosome core particle thus stabilizes the B form, whereas the presence of the Z form prevents nucleosome formation. The results suggest that if Z DNA is present in eukaryotic nuclei, it will serve to disrupt the normal chromatin structure.