The nuclease sensitivity of active genes.

Brief micrococcal nuclease digestion of chick embryonic red blood cells results in preferential excision and solubilization of monomer nucleosomes associated with beta-globin sequences and also 5'-sequences flanking the beta-globin gene. Both regions are DNAse-I sensitive in nuclei. Such salt-soluble nucleosomes are enriched in all four major HMG proteins but HMG1 and 2 are only weakly associated. These nucleosomes appear to have lost much of the DNAse-I sensitivity of active genes. The HMG14 and 17-containing salt-soluble nucleosomes separated by electrophoresis are not DNAse-I sensitive and contain inactive gene sequences as well as active sequences. Reconstitution of HMG proteins onto bulk nucleosomes or chromatin failed to reveal an HMG-dependent sensitivity of active genes as assayed by dot-blot hybridization and it was found that the DNAse-I sensitivity of ASV proviral sequences as assayed by dot-blot hybridization was not HMG-dependent. These results indicate that higher order chromatin structures might be responsible for nuclease sensitivity of active genes.     

Chromatin structure of genes during the cell cycle

K562 ceils were separated by centrifugal elutriation into 5 fractions each of which had a unique distribution of ceils at different phases of the cell cycle. A study of DNAseI sensitivity of the e- and -globin genes, which are expressed in these cells, and the light chain immunoglobulin gene, which is not expressed, in nuclei from each of the 5 fractions was undertaken. There was no detectable difference in the arrangement of DNAseI-hypersensitive sites (DHS) in chromatin of each of the three genes in the different fractions.     

High mobility group nonhistone chromosomal proteins of the developing sea urchin embryo

The high mobility group or HMG proteins are nonhistone chromosomal proteins that have been found in relatively high amounts in nuclei of many tissues. A number of studies have shown that some of these proteins are preferentially associated with actively transcribed regions of the genome and may play a role in maintaining these regions in an active state. In this study, we undertook an investigation of the high mobility group proteins from the sea urchin, Stronglyocentrotus purpuratus. Initially the putative sea urchin HMGs were extracted from isolated nuclei of hatching blastula-stage embryos with 5% perchloric acid (PCA). The major proteins in this extract were characterized according to their electrophoretic mobility, amino acid composition, and association with isolated deoxyribonucleoprotein particles. The results indicate there is only one "major" sea urchin HMG protein, termed P2 in this paper. An estimate of the amount of P2 in relation to the inner histones, however, was low compared to what has been found for other HMG proteins. Of the other major 5% PCA-extractable proteins, one was identified as the cleavage stage H1. Another protein apparently resulted from H3 contamination in the 5% PCA extract, and the fourth major protein did not have all the characteristics of an HMG. In particular, it was not found associated with nucleosomal particles. The HMG proteins from other developmental stages were then examined. Five percent PCA extracts of nuclei from unfertilized eggs, 2-cell, 16-cell, hatching blastula, gastrula, and pluteus stages were analyzed on SDS- and acetic acid-urea gels. This analysis indicated that P2 exists in two different forms differing slightly in charge. The less basic form was found in the egg, 2-cell and 16-cell extracts. At the hatching blastula stage, both forms were present and by pluteus stage, the more basic form predominated. It appears that P2 is undergoing a developmental change from a less to more basic form. The presence of P2 in the 5% PCA extract of egg nuclei is proof that P2 does not initially appear sometime during embryogenesis but is already in the egg nucleus prior to fertilization.     

The distribution of nuclear proteins and transcriptionally-active sequences in rat liver chromatin fractions

Chromatin fractions from rat liver nuclei digested by nucleases were separated by differential solubility into several fractions. Material solubilized during digestion (predominantly monomer nucleosomes and polynucleosomes) had the highest HMG14 + 17/DNA ratios but were not enriched in active gene sequences (albumin and c-Ha-ras1 genes). Material soluble in a low ionic strength buffer containing 0.2 mM MgCl2 (monomer nucleosomes and polynucleosomes) contained in addition to the histones, HMG14 and 17 plus a 41K non-histone protein. This fraction was depleted in active gene sequences and enriched in inactive sequences. The insoluble material was highly enriched in active sequences and had the lowest HMG14 + 17/DNA ratio. This fraction could be further fractionated into a histone-containing 2 M NaCl-soluble fraction and a 2 M NaCl-insoluble matrix-bound fraction, both of which were enriched in active sequences. The results show that the HMG proteins do not partition with active sequences during fractionation of chromatin. The 41K protein may be associated with inactive chromatin fraction.     

Fractionation by high-performance liquid chromatography of the low-molecular-mass high-mobility-group (HMG) chromosomal proteins present in proliferating rat cells and an investigation of the HMG proteins present in virus transformed cells

The low-molecular-mass high-mobility-group (HMG) proteins from young rat thymus nuclei were fractionated by high-performance liquid chromatography. Two proteins analogous to calf HMG14 and HMG17 were found together with a third major component HMGI similar to that found in HeLa cells [Lund et al. (1983) FEBS Lett. 152, 163-167]. HMGI has as amino acid composition similar to but distinct from HMG14 and HMG17. The three proteins form a family of proteins with HMG14 having an amino acid composition intermediate between HMG17 and HMGI. HMGI is present in proliferating fibroblasts and embryos but is present in very low levels in rat liver, a non-dividing tissue, supporting the notion that HMGI is required for proliferating cells. Fibroblasts transformed with avian sarcoma virus have high levels of HMGI and an additional band HMGI' but the presence of HMGI and HMGI' is not dependent on a functional src gene product.     

Interaction of wheat high-mobility-group proteins with four-way-junction DNA and characterization of the structure and expression of HMGA gene

Plant high-mobility-group (HMG) chromosomal proteins are the most abundant and ubiquitous nonhistone proteins found in the nuclei of higher eukaryotes. There are only two families of HMG proteins, namely, HMGA and HMGB in plants. The cDNA encoding wheat HMGa protein was isolated and characterized. Wheat HMGA cDNA encodes a protein of 189 amino acid residues. At its N terminus, there is a histone H1-like structure, which is a common feature of plant HMGA proteins, followed by four AT-hook motifs. Polymerase chain reaction results show that the gene contains a single intron of 134 bp. All four AT-hook motifs are encoded by the second exon. Northern blot results show that the expression of HMGA gene is much higher in organs undergoing active cell proliferation. Gel retardation analysis show that wheat HMGa, b, c and histone H1 bind to four-way-junction DNA with high binding affinity, but affinity is dramatically reduced with increasing Mg(2+) and Na(+) ion concentration. Competition binding studies show that proteins share overlapping binding sites on four-way-junction DNA. HMGd does not bind to four-way-junction DNA.     

Analysis of age-associated alteration in the synthesis of HMG nonhistone proteins of the rat liver

HMG proteins were extracted with 5% PCA or 0.35 M NaCl from whole tissue, nuclei or chromatin of the liver of young (19 weeks) and old (118 weeks) male rats. They were resolved on acetic acid-urea polyacrylamide gel. The electrophoretic patterns of the major HMG proteins 1, 2, 14 and 17 of both ages are similar. The in vitro synthesis of HMG 1 and 2 decreases, but that of HMG 14 and 17 increases considerably in the liver of old rats. The synthesis of different HMG proteins is modulated differentially by spermine, butyrate, dexamethasone and 3-aminobenzamide in the liver of young and old rats. These findings suggest that HMG proteins contribute to alterations in the organization of chromatin and expression of genes during aging.     

The high mobility group proteins HMG 14 and 17, do not prevent the formation of chromatin higher order structure.

The high mobility group proteins, HMG 14 and 17, have been associated with the chromatin of active genes (refs 1-8), although how they function is not known. We use sedimentation and electric dichroism to investigate the effect of HMG 14 and 17 on the condensation of chicken erythrocyte chromatin into higher order structure. We find no evidence that excess HMG 14 and 17 induce an extended configuration, either in bulk chromatin or in the chromatin of the chicken beta-globulin gene.     

The DNA sequence specificity of HMG boxes lies in the minor wing of the structure.

To establish the basis of sequence-specific DNA recognition by HMG boxes we separately transferred the minor and major wings from the sequence-specific HMG box of TCF1 alpha into their equivalent position in the non-sequence-specific box 2 of HMG1. Thus chimera THT1 contains the minor wing (of 11 N-terminal and 25 C-terminal residues) from the HMG box of TCF1 alpha and the major wing (the 45 residue central section) from HMG1 box 2, whilst the situation is reversed in chimera HTH1. The structural integrity of the two chimeric proteins was established by CD, NMR and their binding to four-way junction DNA. Gel retardation and circular permutation assays showed that only chimera THT1, containing the TCF1 alpha minor wing, formed a sequence-specific complex and bent the DNA. The bend angle was estimated to be 59 degrees for chimera THT1 and 52 degrees for the HMG box of TCF1 alpha. Our results, in combination with mutagenesis and other data, suggests a model for the DNA binding of HMG boxes in which the N-terminal residues and part of helix 1 contact the minor groove on the outside of a bent DNA duplex.     

The effect of a high mobility group protein (HMG 17) on the structure of acetylated and control core HeLa cell chromatin

The effect of binding a high mobility group protein (HMG 17) on the stability and conformation of acetylated and control HeLa high molecular weight core chromatin (stripped of H1 and non-histone chromosomal proteins) was studied by circular dichroism and thermal-denaturation measurements. Previously it had been shown that conformational differences exist between native whole chromatin derived from butyrate-treated (acetylated) and control HeLa cells and that these conformational differences disappear by removing H1 and non-histone chromosomal proteins (Reczek, P.R., Weissman, D., Huvos, P.E. and Fasman, G.D. (1982) Biochemistry 21, 993–1002). The circular dichroism spectra and the thermal denaturation profiles of control and acetylated core chromatin were found to be similar. The circular dichroism properties of HMG 17 reconstituted highly acetylated and control core chromatin indicated the same alteration of chromatin structure at low ionic strength (1 mM sodium phosphate/0.25 mM EDTA, pH 7.0). The magnitudes of the decrease in ellipticity were proportional to the amount of HMG 17 bound and were found to be the same for both the acetylated and control core chromatin. Thermal denaturation profiles confirmed this change in structure induced by HMG 17 on control and highly acetylated core chromatin. The thermal denaturation profiles, which were resolved into three component transitions, exhibited a shifting of hyperchromicity from the lower melting transitions to the higher melting transitions, with a concomitant rise in T_m, on HMG 17 binding to both control and acetylated chromatin. The natures of the interactions of HMG 17 at higher ionic strength (50 mM NaCl/0.25 mM EDTA/1 mM sodium phosphate, pH 7.0) with acetylated and control core chromatin were slightly different, as measured by circular dichroism; however, a decrease in ellipticity was observed for both samples upon binding of HMG 17. These observations suggest that acetylation coupled with HMG 17 binding to core chromatin does not loosen chromatin structure. HMG 17 binding to control and acetylated core chromatin produces an overall stabilization and compaction of chromatin structure.     

Interaction of HMG 14 and 17 with actively transcribed genes

The interaction of HMG 14 and 17 with actively transcribed genes was studied by monitoring the sensitivity of specific genes to DNAase I after reconstitution of HMG-depleted chromatin with HMG 14 and 17. Our experiments lead to the following conclusions: most actively transcribed genes become sensitized to DNAase I by HMG 14 and 17; either HMG 14 or HMG 17 can sensitize most genes to DNAase I; genes transcribed at different rates have about the same affinity for HMG 14 and 17; HMG 14 and 17 bind stoichiometrically to actively transcribed nucleosomes; and HMG 14 and 17 can restore DNAase I sensitivity to purified nucleosome core particles depleted of HMGs. This last observation suggests that during reconstitution, low levels of HMG 14 and 17 can associate with the active nucleosomes in the presence of a 10–20 fold excess of inactive nucleosomes. Consequently, we conclude that besides their association with HMGs, active nucleosomes also have at least one other unique feature that distinguishes them from bulk nucleosomes and insures proper HMG binding during reconstitution.     

Constitutive and light-induced DNAseI hypersensitive sites in the rbcS genes of pea (Pisum sativum)

The chromatin structure of pea (Pisum sativum) rbcS genes in inactive (root), potentially active (dark-grown leaf), and active states (light-grown leaf) was analysed using (a) pancreatic DNAseI to detect general DNAseI sensitivity and DNAseI-hypersensitive sites, and (b) methyl-sensitive restriction endonucleases to probe for cytosine methylation within the promoter region. We showed that within the same organ individual members of the pea rbcS multigene family are differentially sensitive to DNAseI suggesting differential protection in nuclei. During light activation general DNAseI sensitivity increases in some genes, especially their 5 upstream regulatory sequences. DNAseI-hypersensitive sites are constitutively present in 5 upstream regulatory sequences around positions –335, –465, –650, and –945 (5 constitutive domain) and in the coding region around position +340, +450, +530, +640, and +810 (3 constitutive domain). One additional hypersensitive site appears after light induction (inducible site). This region is centred around position –190 and flanked by light-responsive elements (LREs). In spite of changes in the chromatin structure of rbcS genes during their transition from an inactive to an active state, their cytosine methylation at Alu I, Fnu 4HI, Hae III, Sau 3AI and Sau 96I sites in the promoter region remains uniform.