Nucleosomes and the chromatin fiber.

During the past year and a half, significant progress has been made in understanding the structure and dynamics of nucleosomes and the chromatin fiber, the mechanism of action of the core histone amino termini, the structure and function of histone variants, and the function of linker histones in the chromatin fiber.

The H3-H4 N-terminal tail domains are the primary mediators of transcription factor IIIA access to 5S DNA within a nucleosome.

Reconstitution of a DNA fragment containing a Xenopus borealis somatic type 5S rRNA gene into a nucleosome greatly restricts the binding of transcription factor IIIA (TFIIIA) to its cognate DNA sequence within the internal promoter of the gene. Removal of all core histone tail domains by limited trypsin proteolysis or acetylation of the core histone tails significantly relieves this inhibition and allows TFIIIA to exhibit high-affinity binding to nucleosomal DNA. Since only a single tail or a subset of tails may be primarily responsible for this effect, we determined whether removal of the individual tail domains of the H2A-H2B dimer or the H3-H4 tetramer affects TFIIIA binding to its cognate DNA site within the 5S nucleosome in vitro. The results show that the tail domains of H3 and H4, but not those of H2A and/or H2B, directly modulate the ability of TFIIIA to bind nucleosomal DNA. In vitro transcription assays carried out with nucleosomal templates lacking individual tail domains show that transcription efficiency parallels the binding of TFIIIA. In addition, we show that the stoichiometry of core histones within the 5S DNA-core histone-TFIIIA triple complex is not changed upon TFIIIA association. Thus, TFIIIA binding occurs by displacement of H2A-H2B-DNA contacts but without complete loss of the dimer from the nucleoprotein complex. These data, coupled with previous reports (M. Vettese-Dadey, P. A. Grant, T. R. Hebbes, C. Crane-Robinson, C. D. Allis, and J. L. Workman, EMBO J. 15:2508-2518, 1996; L. Howe, T. A. Ranalli, C. D. Allis, and J. Ausio, J. Biol. Chem. 273:20693-20696, 1998), suggest that the H3/H4 tails are the primary arbiters of transcription factor access to intranucleosomal DNA.

cis-Diamminedichloroplatinum (II) modified chromatin and nucleosomal core particle probed with DNase I.

Chromatin and nucleosomal core particles were modified with cis-diamminedichloroplatinum (II) and the nucleoprotein complexes then digested with DNase I. Limited digestion of the modified chromatin containing cis-Pt(NH3)2Cl2 mediated cross-links involving the non-histone chromosomal proteins (Scovell et al. (1987) Biochem. Biophys. Res. Commun. 142, 826-835) does not release the low mobility proteins and excises only about 20% of the high mobility proteins 1, 2, and E. This supports previous findings that the low mobility proteins are involved primarily in protein-protein cross-links. In addition, the covalent cross-links between DNA and the high mobility proteins 1, 2, and E are relatively inaccessible to DNase I, in marked contrast to their accessibility to micrococcal nuclease. Furthermore, gels of the denatured DNA fragments obtained from digestion of both modified chromatin and nucleosomal core particle reveal virtually no difference in the 10n base repeat pattern, indicating no detectable change in the DNA-protein interactions upon DNA modification. This suggests that the predominant modification produced on core particle DNA, whether contained within higher order chromatin structure or in the core particle itself, is one which does not significantly alter the helical twist of the DNA within these nucleoprotein assemblies.

Paroxysmal supraventricular tachycardia in the general population.

OBJECTIVES: We sought to determine the epidemiology and clinical significance of paroxysmal supraventricular tachycardia (PSVT) in the general population. BACKGROUND: Current knowledge of PSVT has been derived primarily from otherwise healthy patients referred to specialized centers. METHODS: We used the resources of the Marshfield Epidemiologic Study Area, a region covering practically all medical care received by its 50,000 residents. A review of 1,763 records identified prevalent cases as of July 1, 1991 and all new cases of PSVT diagnosed from that day until June 30, 1993. A mean follow-up period of 2 years was completed in all incident patients. Patients without other cardiovascular disease were labeled as having "lone PSVT." RESULTS: The prevalence was 2.25/1,000 persons and the incidence was 35/100,000 person-years (95% confidence interval, 23 to 47/100,000). Other cardiovascular disease was present in 90% of males and 48% of females (p = 0.0495). Compared with patients with other cardiovascular disease, those with lone PSVT were younger (mean 37 vs. 69 years, p = 0.0002), had a faster PSVT heart rate (mean 186 vs. 155 beats/min, p = 0.0006) and were more likely to have their condition first documented in the emergency room (69% vs. 30%, p = 0.0377). The onset of symptoms occurred during the childbearing years in 58% of females with lone PSVT versus 9% of females with other cardiovascular disease (p = 0.0272). CONCLUSIONS: There are approximately 89,000 new cases/year and 570,000 persons with PSVT in the United States. In the general population, there are two distinct subsets of patients with PSVT: those with other cardiovascular disease and those with lone PSVT. Our data suggest etiologic heterogeneity in the pathogenesis of PSVT and the need for more population-based research on this common condition.

Structure of the TFIIIA-5 S DNA complex.

The missing-nucleoside experiment, a recently developed approach for determining the positions along a DNA molecule that make energetically important contacts with protein, has been used to investigate the structure of the complex of transcription factor IIIA with a somatic 5 S RNA gene from Xenopus borealis. We detect three distinct regions of the 5 S promoter that are contacted by TFIIIA, corresponding to the A-box, intermediate element and C-box regions previously identified by mutagenesis experiments. The advantage of the missing-nucleoside experiment over mutagenesis is that additional information, directly related to the structure of the complex, is obtained. Of most importance is that contacts to each strand of DNA are determined independently, and can be assigned unambiguously as interactions with TFIIIA. Throughout the binding site the strongest contacts are made with the non-coding strand of the 5 S gene. The two groups of contacts at either end of the binding site (boxes A and C) are comprised of sets of approximately ten contiguous nucleosides for which the contacts are reflected, without stagger, from one strand to the other. In contrast, contacts in the center of the promoter (the intermediate element) are staggered about five base-pairs in the 5' direction with respect to each strand. These results, when analyzed in conjunction with the hydroxyl-radical footprint of the complex, support a model in which TFIIIA wraps around the DNA in the major groove of the helix for one turn at the two ends of the complex in boxes A and C, and lies on one side of the DNA helix in the center of the complex at the intermediate element.

The footprint of chromosomal proteins HMG-14 and HMG-17 on chromatin subunits.

The position of chromosomal proteins HMG-14 and HMG-17 in nucleosome cores and in chromatosomes lacking linker histones has been mapped by hydroxyl radical footprinting. Both the nucleosome core and the H1/H5 depleted chromatosome can specifically bind two molecules of HMG-14/-17. The path of HMG-14 on the surface of chromatin subunits is indistinguishable from that of HMG-17. The bound HMGs protect the DNA from hydroxyl radical cleavage 25 base-pairs from the end of the DNA in nucleosome cores and in each of the two major grooves of the DNA flanking the nucleosomal dyad axis. Thus, in both cores and H1/H5-depleted chromatosomes the proteins bridge two adjacent DNA strands on the surface of the particles. The sites occupied by HMG near the end of the chromatosome-length particles are distinct from those occupied by the H1/H5 linker histones. In the region of the dyad axis the binding sites of HMGs overlap those of the linker histones. The placement of HMG-14/-17 near the nucleosomal dyad axis raises the possibility that interactions between histone H1 and HMGs may affect the transcriptional potential of chromatin.

Nucleosome structural changes due to acetylation.

We have measured the helical repeat of acetylated nucleosomes reconstituted with HeLa cell histone octamers and the 5 S RNA gene positioning sequence from Xenopus borealis. The hydroxyl radical footprinting experiments show that the helical repeat of nucleosome-bound DNA is unchanged upon acetylation, and that the extent of DNA-protein contacts remains unaltered. These results, combined with previous determinations of the linking number change in minichromosomes as a function of the extent of acetylation, are analyzed with surface linking theory. We demonstrate that the shape of the nucleosome changes upon acetylation, in such a way as to decrease the number of times the DNA winds around the nucleosome central axis.

Chemical probes of DNA structure in chromatin.

Understanding the way genes work requires detailed knowledge of the organization of DNA in the chromatin complex. The difficulties associated with the study of this large macromolecular assembly present an interesting challenge to both biologists and chemists.

Chromatin remodeling by cell cycle stage-specific extracts from Physarum polycephalum.

Remodeling of chromatin is an essential process allowing the establishment of specific genetic programs. The slime mold Physarum polycephalum presents the attractive advantage of natural synchrony of the cell cycle in several million nuclei. Whole-cell extracts prepared at precise stages during the cell cycle were tested for the ability to induce remodeling in erythrocyte nuclei as monitored by microscopy, protamine competition assays, micrococcal nuclease digestions, and release of histone H5. Extracts derived from two specific cell cycle stages caused opposite types of changes in erythrocyte nuclei. An increase in chromatin compaction was imparted by extracts prepared during S-phase while extracts harvested at the end of G2-phase caused increases in nuclear volume, DNA accessibility, and release of linker histone. We also found that late G2 extracts had the ability to alter the DNase I digestion profile of mononucleosomes reconstituted in vitro in a classical nucleosomes remodeling assay. The relevance of these finding to the Physarum cell cycle is discussed.

Site-directed cleavage of DNA by a linker histone--Fe(II) EDTA conjugate: localization of a globular domain binding site within a nucleosome.

The globular domain of linker histones specifically recognizes and binds to the nucleosome core. However, the exact location of the binding site of the globular domain has not been definitively elucidated. To address this issue, a linker histone has been specifically modified at a site adjacent to the globular domain with a radical-based DNA cleavage reagent. The linker histone-Fe(II) EDTA conjugate was bound to reconstituted nucleosomes containing a Xenopus 5S RNA gene, and the resulting cleavage of DNA was used to precisely map the location of the linker histone binding site. The results indicate that the binding site is located on the inside of the superhelical gyre of DNA, just inside the periphery of the nucleosome core region. The implications of these results for the binding of linker histones within native chromatin complexes are discussed.

A novel labeling technique reveals a function for histone H2A/H2B dimer tail domains in chromatin assembly in vivo.

During S phase in eukaryotes, assembly of chromatin on daughter strands is thought to be coupled to DNA replication. However, conflicting evidence exists concerning the role of the highly conserved core histone tail domains in this process. Here we present a novel in vivo labeling technique that was used to examine the role of the amino-terminal tails of the H2A/H2B dimer in replication-coupled assembly in live cells. Our results show that these domains are dispensable for nuclear import but at least one tail is required for replication-dependent, active assembly of H2A/H2B dimers into chromatin in vivo.

The missing nucleoside experiment: a new technique to study recognition of DNA by protein.

We report a new technique for quickly determining which nucleosides in a DNA molecule are contacted by a sequence-specific DNA-binding protein. Our method is related to the recently reported "missing contact" experiment [Brunelle, A., & Schleif, R. F. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 6673-6679]. We treat the DNA molecule with the hydroxyl radical to randomly remove nucleosides. The ability of protein to bind to gapped DNA is assayed by gel mobility shift. Nucleosides important to protein binding are identified by sequencing gel electrophoresis. The missing nucleoside experiment can be used to scan a DNA molecule at single-nucleotide resolution in one experiment. The bacteriophage lambda repressor-OR1 and cro-OR1 complexes were analyzed to evaluate the method. For both proteins, the most important contacts are located in the protein monomer that binds to the consensus half of the operator. These contacts correspond well to those found by mutational studies, and in the cocrystal structure of the lambda repressor-operator. The missing nucleoside data show that the amino-terminal arms of lambda repressor make energetically important contacts with positions 7 and 8 and the central dyad base pair of the operator. The amino-terminal arm that makes the most extensive contacts to DNA appears to be the one that emanates from the repressor monomer that binds to the consensus half of the operator, in agreement with the cocrystal structure. The lambda cro protein does not have an amino-terminal arm, and the missing nucleoside experiment clearly shows a lack of contacts to DNA in the central region of the operator in this complex.

Assembly into chromatin and subtype-specific transcriptional effects of exogenous linker histones directly introduced into a living Physarum cell.

The apparent diversity of linker histone subtypes may be related to their specific roles in defining functional states of chromatin in vivo. We have developed a novel method to study constitutive peptides throughout the cell cycle and have demonstrated that an exogenous linker histone could be introduced into a living cell of the slime mold Physarum polycephalum. Here, we have used this method to assess the functional differences between three somatic linker histone subtypes in vivo, and to demonstrate the general applicability of this method. Exogenous linker histone proteins H1 degrees, H5 and H1 were directly absorbed into living cell segments of the naturally synchronous Physarum macroplasmodia at precise cell cycle stages. Fluorescence microscopy, native nucleoprotein gels and immunoblotting of nuclei and chromatin with subtype-specific antibodies revealed that exogenous linker histones were efficiently transported into nuclei and were integrated into chromatin. The immunoreactivity of a preparation of anti-H1 degrees antibodies that are blocked from binding to specific H1 degrees epitopes in native chromatin indicates that the exogenous linker histones were similarly associated into Physarum chromatin. Interestingly, linker histones were found to be less stably associated with Physarum chromatin during S-phase than during G(2)-phase. Furthermore, we show that exogenous linker histones incorporated in early G(2)-phase inhibited transcription and that the level of inhibition correlates with the apparent role of the linker histone subtype in regulating transcription in cells where it normally occurs.