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.     

Antigenic properties of heterogeneous nuclear ribonucleoprotein particles weakly binding nonhistone proteins and proteins binding single-stranded DNA (SSB proteins) from Ehrlich ascites tumor]

Antigenic properties of the proteins of heterogeneous nuclear ribonucleoprotein particles, (hnRNP), weakly bound nonhistone chromatin proteins (WB(N)P) and single-strand DNA-binding proteins (SSB proteins) from chromatin and extrachromatin fraction of the Ehrlich ascites tumor cells have been comparatively studied. The chromatin and extrachromatin SSB proteins displayed similar mobility in the tube and slab SDS/PAGE, had the same ssDNA-binding capacity and similarly stimulated the replicative synthesis in permeable cells. However, the chromatin SSB proteins contained 1.4 times higher phosphate amount than the extrachromatin ones (3.1 and 2. 2. moles phosphorus per 1 mole protein, respectively). The study of four protein groups with the use of a rabbit antiserum to/against extrachromatin SSB proteins (titer 1:13000 by enzyme immunoassay) showed that the chromatin and the extrachromatin SSB proteins have similar antigenic properties. One fraction of the hnRNP proteins was also reactive with the antiserum, whereas the WB(N)P displayed no cross-reactivity. The specificity of the ferm "SSB proteins" as applied to eukaryotic cells, their affinity with hnRNP proteins and differences from the HMG proteins are discussed.     

Effect of HMG protein 17 on the thermal stability of control and acetylated HeLa oligonucleosomes.

Many studies have implicated histone acetylation and HMG proteins 14 and 17 in the structure of active chromatin. Studies of the binding of HMG 14 and 17 to chromatin core particles have shown that there are two binding sites for HMG 14 or 17 located within 20-25 bp of the DNA ends of the core particles [13-15]. Such binding sites may result from the free DNA ends in the core particle being available for the binding of HMG 14 and 17. We have studied the effects of the binding of HMG 17 on the thermal denaturation of DNA in mono, di and trinucleosomes. In each case the binding of 1 HMG 17 molecule per nucleosome reduces the DNA premelt region by 50%, while the binding of 2 HMG 17 molecules per nucleosome abolishes the premelt region. From this it is concluded that there are two HMG 17 binding sites per nucleosome which are located between the entry and exit points to the nucleosome and the strongly complexed central DNA region. Highly acetylated mono, di and trinucleosomes have been isolated from butyrate treated HeLa S3 cells. For this series of acetylated oligonucleosomes, it has been found that there are also two HMG 17 binding sites per acetylated nucleosome.     

Studies on the association of the high mobility group non-histone chromatin proteins with isolated nucleosomes.

Nucleosomes have been isolated from rabbit thymus by sucrose gradient centrifugation, and their high mobility group (HMG) protein content analysed by electrophoresis on polyacrylamide gels. The results suggest that proteins HMG 14 and HMG 17 are associated with the core particle of the nucleosome, and that there are two or more sub-populations of both HMG 1 and HMG 2 molecules. One sub-population appears to be fairly tightly bound to the nucleosome, while another is rapidly released from the chromatin by digestion with micrococcal nuclease. The latter fraction may participate in a higher order folding of the nucleosomes.     

Preferential in vitro binding of high mobility group proteins 14 and 17 to nucleosomes containing active and DNase I sensitive single-copy genes

High mobility group (HMG) proteins 14 and 17 bind to mononucleosomes in vitro, but the exact nature of this binding has not been clearly established. A new method was developed to allow direct membrane transfer of DNA from HMG 14/17 bound and unbound nucleosomes, which have been separated by acrylamide gel electrophoresis. Hybridization analysis of membranes obtained by this method revealed that the HMG 14/17 bound nucleosomes of avian erythrocytes and rat hepatic tumor (HTC) cells were enriched, about 2-fold, in actively transcribed genes and also inactive but DNase I sensitive genes. Nucleosomes containing inactive, DNase I resistant genes were bound by HMG 14/17, but not preferentially. Several factors that have been reported to greatly influence the binding of HMG 14/17 to nucleosomes in vitro were tested and shown to not account for the preferential binding to DNase I sensitive chromatin. These factors include nucleosomal linker DNA length, single-stranded DNA nicks, and DNA bulk hypomethylation. An additional factor, histone acetylation, was preferentially associated with the HMG 14/17 bound chromatin fraction of avian erythrocytes, but it was not associated with the HMG 14/17 bound chromatin fraction of metabolically active HTC cells. The latter finding was true for all kinetic forms of histone acetylation.     

The phosphorylation of high mobility group proteins 14 and 17 and their distribution in chromatin

The phosphorylation of the high mobility group (HMG) proteins has been investigated in mouse Ehrlich ascites, L1210 and P388 leukemia cells, human colon carcinoma cells (HT-29), and Chinese hamster ovary cells. HMG 14 and 17, but not HMB 1 and 2, were phosphorylated in the nuclei of all cell lines with a serine being the site of modification for both proteins in Ehrlich ascites cells. Phosphorylation of HMG 14 and 17 was greatly reduced in cultured cells at plateau phase in comparison to log phase cells, suggesting that modification of HMG 14 and 17 is growth-associated. However, phosphorylation was not linked to DNA synthesis, since incorporation of 32P did not vary through G1 and S phase in synchronized Chinese hamster ovary cells. Treatment of HT-29 or Ehrlich ascites cells with sodium butyrate reduced HMG phosphorylation by 30 and 70%, respectively. The distribution of the phosphorylated HMG proteins in chromatin was examined using micrococcal nuclease and DNase I. 32P-HMG 14 and 17 were preferentially associated with micrococcal nuclease-sensitive regions as demonstrated by the release of a substantial fraction of the phosphorylated forms of these proteins under conditions which solubilized less than 3% of the DNA. Short digestions with DNase I did not show a marked release of 32P-HMG 14 or 17.     

Comparison of the NH2-Terminal sequences of chick Type I preprocollagen chains synthesized in an nRNA-dependent reticulocyte lysate

The histone variants and high-mobility-group (HMG) proteins of a transcribing fraction of chromatin, described in the preceding paper of this journal, have been analysed qualitatively and quantitatively by a combination of one-dimensional and two-dimensional gel electrophoresis. The stoichiometry of the four core histones (all variants included) in this fraction is equimolar and is not detectably different from that in the nontranscribing fraction or in total chromatin. The molar ratio of histone H1 to the core histones is markedly lower, by approximately 72%, than that in the nontranscribing fraction. A minor histone variant identified as M1 (an H2A variant) is detected only in the transcribing fraction, while variant H3.1 is found only in the non-transcribing fraction. Proteins A24, HMG1 and HMG2 are essentially absent from the transcribing fraction; HMG14 is found uniquely in this fraction, while HMG17 occurs at a relatively lower level.     

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.     

Histone H1 and HMG 14/17 are deposited nonrandomly in the nucleus.

We have studied the assembly of histone H1 and the high mobility group nonhistones 14/17 by isopycnic analysis after crosslinking density labeled MSB cell nuclei or chromatin. Carbodiimide crosslinking produces dense poly-H1 and hybrid density H1-H2A histone dimers, indicating that new H1 is deposited nonrandomly, albeit nonconservatively relative to new core histones. Core histone-HMG crosslinking with succinimidyl propionate yields dense HMG 14 in uniformly dense particles and new HMG 17 crosslinked to both dense and light protein, implying that HMG 14 and 17 each deposit nonrandomly; but differently with respect to new core octamers. Propionimidate crosslinking yields dense H1-HMG 17 dimers, suggesting that the interactions of new 14/17 with H1 (new HMG 14-old H1, new HMG 17-new H1) are reciprocal to their interactions with the core histones.     

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 binding sites for large and small high-mobility-group (HMG) proteins. Studies on HMG-nucleosome interactions in vitro

Studies in vitro of binding high-mobility-group (HMG) proteins to nucleosomal particles that differ in their DNA contents reflect several aspects pertinent to their function in vivo. Two molecules of HMG 14 or 17 are accommodated by particles with 140 or 180 base pairs of DNA whereas HMG 1 or 2 are only bound by the larger specimens irrespective of the presence of HMG 14/17. It is concluded that one molecule of HMG 1 or 2 binds to the 40 base pairs of linker DNA whereas the HMG 14 or 17 molecules associate with the nucleosomal core. At physiological ionic strength, HMG 14 binding is cooperative, probably by triggering a conformational change in the nucleosomal particle. The phenomenon has been studied by two independent techniques. Besides the common gel-electrophoretic system, a centrifugation assay is described, which permits the derivation of a Hill coefficient nH = 1.3 and dissociation constants in the range of 30-90 nM at 0.15 M NaCl, pH 6.8.     

Release of nucleosomes from nuclei by bleomycin-induced DNA strand scission

Mononucleosomes were released from both isolated mammalian (hog thyroid) and protozoan (Tetrahymena) nuclei by the bleomycin-induced DNA-strand breaking reaction. Trout sperm nuclei, on the other hand, were protected from the bleomycin-mediated DNA degradation. The mononucleosomes released from the bleomycin-treated nuclei contained the core histones H2A, H2B, H3, and H4; while HMG1 and HMG2 proteins, in addition to the core histones, were detected in the mononucleosomes obtained by micrococcal nuclease digestion of nuclei. HMGs, but not H1 histone, were dissociated into the supernatant by cleavage of chromatin DNA with bleomycin, whereas both HMGs and H1 were found in that fraction by digestion of nuclei with micrococcal nuclease. HMG1 and HMG2 were exclusively dissociated from chromatin with 1 mM bleomycin under the solvent condition where the DNA strand-breaking activity of the drug is repressed. These observations suggest the possibility that bleomycin preferentially binds to linker DNA regions not occupied by H1 histone in chromatin and exclusively dissociates HMG proteins and breaks the DNA strand. The results of the effects on bleomycin-induced DNA cleavage of nuclei of various drugs including polyamines, chelating agents, intercalating antibiotics such as mitomycin C or adriamycin, and radical scavengers are also presented.     

The characterisation of 1SF monomer nucleosomes from hen oviduct and the partial characterisation of a third HMG14/17-like in such nucleosomes.

Nucleosomes released from oviduct nuclei during brief micrococcal nuclease digestions are enriched in transcribed sequences (bloom K.S. and Anderson, J.N. (1978) Cell, 15, 141-150). Such nucleosomes released into this 1Sf supernatant fraction are enriched in proteins HMG14, 17 and a third lower molecular weight protein which we show in this paper to be related to HMG14 and 17. This protein, which we call HMGY, runs as a doublet on polyacrylamide gels. A similar doublet is present in smaller quantities in chicken erythrocyte nuclei. Monomer nucleosomes in the 1SF supernatant have been separated by polyacrylamide gel electrophoresis into two main bands. The slower moving band contains the three HMG proteins HMG14, 17 and Y but lacks histone H1.     

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.