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.     

Enrichment of transcribed and newly replicated DNA in soluble chromatin released from nuclei by mild micrococcal nuclease digestion

A chromatin fraction solubilized from mouse myeloma nuclei under near-physiological ionic conditions by very mild micrococcal nuclease digestion at 0°C is enriched at least 7-fold in DNA complementary to total myeloma polyadenylated mRNA, and 15-fold in DNA originating near the replication fork (labeled within 30 s). Newly replicated DNA recovered in solubilized chromatin after brief labeling was incorporated mainly into particles sedimenting with, or faster than, mononucleosomes. A rapid decrease in enrichment of newly replicated DNA in readily released, soluble chromatin with increasing labeling times indicated that newly replicated chromatin matured within 90 s to a form that was partitioned similarly to bulk chromatin by this fractionation method. Previous studies showed that chromatin readily solubilized from myeloma nuclei is enriched in high-mobility-group (HMG) and other non-histone proteins, RNA and single-stranded DNA; and depleted in H1 and 5-methylcytosine, relative to bulk chromatin (Jackson, J.B., Pollock, J.M., Jr., and Rill, R.L. (1979) Biochemistry 18, 3739–3748). Mild digestion of chicken erythrocyte nuclei with micrococcal nuclease yielded a soluble chromatin fraction (1–2% of the total DNA) with similar properties. This fraction was enriched at least 6-fold in DNA complementary to chicken globin mRNA, relative to total erythrocyte DNA.     

Nuclear protein modification and chromatin substructure. 3. Relationship between poly(adenosine diphosphate) ribosylation and different functional forms of chromatin.

The relationship between poly(adenosine diphosphate) ribosylation of nuclear proteins and functionally different forms of chromatin from mid-S-phase HeLa nuclei was investigated. The major observations emerging from this study were that unique nonhistone proteins were modified in mid-S-phase HeLa nuclei. The major acceptor for poly(adenosine diphosphate-ribose) [poly(ADP-Rib)] was an internucleosomal nonhistone protein (protein C; 125 000 molecular weight). Histones H3, H1, H2b, and H2a but not H4 were ADP-ribosylated in S-phase nuclei. Chromatin fragments preferentially released by micrococcal nuclease were enriched in nonhistone proteins, poly(ADP)-ribosylated nuclear proteins, poly(ADP-Rib) polymerase activity and nascent DNA from the DNA replicating fork. In extended forms of chromatin, contiguous to the DNA replicating fork, poly(ADP-Rib) polymerase was maximally active. However, in chromatin distal to the replicating fork (i.e., more condensed structures), nucleosomal histones and histone H1 were not significantly ADP-ribosylated, and poly(ADP-Rib) polymerase activity was depressed two- to threefold. The data suggest that a subset of nucleosomes in extended regions of chromatin is subject to extensive ADP ribosylation.     

Analysis of brain chromatin subunit composition using different endonucleases]

A comparison of the processes of chromatin digestion in brain and liver nuclei by Ca, Mg-dependent and staphylococcal endonucleases demonstrates a similarity of the subunit composition of chromatin from both tissues and reveals the same type of linked DNA regions. However, a formation of low molecular weight DNP fragments during hydrolysis and the DNA spectra of soluble and insoluble DNP fragments suggest that brain chromatin contains these fragments alongside with the regions, which are specific for this particular tissue, predominate in it and are resistant to staphylococcal and, particularly, to Ca, Mg-dependent endonucleases. This is paralleled with a non-histone protein enrichment of different brain chromatin fractions and an expansion of the electrophoretic monomer band towards the fragment with a greater molecular weight. It may be assumed that brain nucleosomes are characterized by a higher size heterogeneity of linked DNA, part of which are mostly covered by non-histone proteins, and/or are characterized by a greater set variety.     

Study of heterogeneity of the chromatin endonuclease fragment by free flow electrophoresis]

The oligomer chromatin fragments relatively uniform in size (8--11 nucleosomes) were prepared by a short-term endonycleolysis. The heterogeneity of these fragments with respect to their electrophoretic mobility was revealed using free flow electrophoresis. The individual fragmentated chromatin subfractions were obtained. These subfractions differed in their protein and RNA content per DNA weight unit, in quantitative ratios of different zones of high molecular weight non-histone proteins and in thermal and alkaline denaturation kinetics. It was also found that the parameters investigated are correlated with electronegativeity of the fragmentated chromatin subfractions.     

Thermal denaturation of DNA in the chromatin fragments released by mild micrococcal nuclease digestion of HTC cell nuclei

In chromatin a minor fraction melts at a temperature lower than deproteinized DNA, which may be assigned to DNA destabilizing proteins. We attempted to localize the destabilized DNA in the various chromatin fragments separated by electrophoresis after a mild micrococcal nuclease digestion. The small released fragments are enriched in coding sequences. About 20% of the DNA extracted from the released nucleosomes are single-stranded, 60% of the DNA in these fragments are digested by nuclease S₁ after incubation at low temperature, which suggests that the DNA destabilizing proteins are present in the released nucleosomes. Hybridization studies have shown that 25% of the DNA in nucleosomes are specific of this class of fragments. DNA destabilizing proteins could be associated with the specific sequences.     

Effect of non-histone proteins on thermal transition of chromatin and of DNA.

The effect of chromatin non-histone protein on DNA and chromatin stability is investigated by differential thermal denaturation method. 1) Chromatin (rat liver) yields a multiphasic melting profile. The major part of the melting curve of this chromatin is situated at temperatures higher than pure DNA, with a distinct contribution due to nucleosomes melting. A minor part melts at temperatures lower than DNA which may be assigned to chromatin non-histone protein-DNA complex which destabilized DNA structure. 2) Heparin which extracts histones lowers the melting profile of chromatin and one observes also a contribution with a Tm lower that of pure DNA. In contrast, extraction on non-histone proteins by urea supresses the low Tm peak. 3) Reconstitution of chromatin non-histone protein-DNA complexes confirms the existence of a fraction of chromatin non-histone protein which lowers the melting temperature when compared to pure DNA. It is concluded that chromatin non-histone proteins contain different fractions of proteins which are causing stabilizing and destabilizing effect on DNA structure.     

Distribution of tightly bound non-histone proteins in chromatin fractions obtained by DNAase II digestion

Digestion of pig liver chromatin with DNAse II afforded three different fractions which were characterized in terms of their DNA, RNA and tightly bound non-histone protein content, their DNA fragment size and their template activity. Two of these fractions are soluble after digestion with DNAase II and have been separated on the basis of their different solubility in MgCl2. A third fraction is not solubilized even after extensive digestion, although the size of its DNA is comparable to that of the enzyme solubilized fractions. The three fractions show qualitatively and quantitatively different distribution of tightly bound non-histone proteins, with specific protein components in each fraction; furthermore the non-solubilized fraction is greatly enriched in proteins tightly bound to DNA. From all the data obtained it can be suggested that the tightly bound proteins of the insoluble fraction may play, directly or indirectly, a role in maintaining an organized chromatin structure.     

Gene-specific differences in the supranucleosomal organization of rat liver chromatin

Rat liver chromatin was separated into a solubilized portion and insoluble nuclear material, and the solubilized portion was fractionated by sucrose gradient sedimentation. The chromatin encompassing three transcribed genes (albumin, tryptophan oxygenase, and alpha-fetoprotein), which are expressed at very different levels, partitions preferentially with insoluble nuclear material and possesses a disrupted nucleosome structure. On the contrary, the chromatin encompassing three inactive genes fractionates into the solubilized chromatin portion and exhibits a canonical nucleosome repeat structure. By sucrose gradient sedimentation, all size classes of inactive chromatin particles are found to contain internal cleavages in the linker region between nucleosomes; they are probably held together by histone H1 and mono- and divalent cations. When the chromatin encompassing two flanking sequences of the tyrosine aminotransferase gene is studied, the 0.35-kilobase upstream-located chromatin exhibits features of active genes, while the 2.55-kilobase upstream-located chromatin partitions preferentially with solubilized chromatin and sediments in internally cleaved particles.     

Partitioning of zinc and copper within subnuclear nucleoprotein particles.

Nuclei from frozen calf thymus suspended in buffer were analyzed for metal content prior to and after repeated washing. After three such extractions about 0.1 micrograms Zn/mg DNA and 0.025 micrograms Cu/mg DNA remained tightly associated with chromatin. This level of metal was essentially unchanged with subsequent washings. Digestion of extracted nuclei with micrococcal nuclease yielded soluble nucleoprotein containing zinc and copper. Metal enriched regions of chromatin appeared to be preferentially solubilized by digestion, and the solubilized metal was only partially dializable either with or without EDTA. Metal profiles generated from gel (A-5m) chromatography analysis of chelated and non-chelated solubilized chromatin were distinctive in that copper was undetectable (by flame AA) while zinc was associated only with low molecular weight products when EDTA was used. In contrast, both metals were detected with higher molecular weight oligonucleosomes in the absence of chelating agents. Additionally, the two metals localized within nucleoprotein peaks and these metal-containing regions were only resolved by gel chromatography when EDTA was omitted throughout the procedure. A discrete Cu-rich species in a region of the profile suggests a subset of Cu-rich nucleoprotein complexes.     

Presence of non-histone proteins in nucleosomes

It has been established that nucleosomes are made of histones and DNA fragments. The purpose of this work to establish whether some non-histone proteins are also present in these chromatin subunits. We have found that nucleosome preparations contain phosphorylated non-histone proteins and protein kinases by sucrose gradient analysis. In order to establish whether these proteins are actually bound to nucleosomes or if they represent unbound or aggregated proteins, the following experiments were performed. (a) Free non-histone proteins and proteins released from chromatin by DNase overdigestion were analyzed by sucrose gradient centrifugation. No phosphoproteins but some phosvitin kinase activity was found in the part of the gradient which contained the nucleosomes. It could be assumed that part of the phosphoproteins are bound to nucleosomes. (b) A digestion of nucleosomes with DNase I suppressed the phosvitin kinase activity in the 11-S region of the gradient. (c) High ionic strength, which extracted non-histone proteins, suppressed the phosvitin kinase activity in the nucleosome region. Part of phosvitin kinase and of nuclear phosphoproteins are therefore bound to nucleosomes and are released by nuclease digestion and by high ionic strength.     

The complete amino-acid sequence of a trout-testis non-histone protein, H6, localized in a subset of nucleosomes and its similarity to calf-thymus non-histone proteins HMG-14 and HMG-17.

The complete amino acid sequence of a basic non-histone protein, H6, isolated from the chromatin of rainbow trout (Salmo gairdnerii) testis cells, has been determined. Protein H6, first described by D. T. Wigle and G. H. Dixon [J. Biol. Chem. 246, 5636--5644 (1971)] was extracted with 5% trichloracetic acid and purified by ion-exchange chromatography on carboxymethyl-cellulose (CM-52). Sequence analysis was performed by automatic Edman degradation of the amino terminus of the intact protein and a series of large fragments derived by cleavage with chymotrypsin, staphylococcal protease and with mild acid to cleave at aspartic acid residues. Protein H6 possesses 69 residues and shows considerable similarities to the 89-residue calf thymus HMG-17 protein previously sequenced [Walker, J. M., Hastings, J. R. B. & Johns, E. W. (1977) Eur. J. Biochem. 76, 461--468]. B. Levy W. and G. H. Dixon [Proc. Natl Acad. Sci. U.S.A. 74, 2810--2814 (1977)] have shown that H6 is selectively solubilized when trout testis nuclei (or chromatin) are digested with DNase I under conditions which preferentially hydrolyze that portion of DNA enriched in transcribed sequences [Levy, W. B. & Dixon, G. H. (1977) Nucleic Acids Res. 4, 883--898]. Recently H6 has been located as a stoichiometric component of a distinct subset of trout testis nucleosomes that are complexed with a core nucleosome comprising 140 base pairs of DNA and the inner histones H2A, H2B, H3 and H4 [Levy, W. B., Connor, W. & Dixon, G. H. (1979) J. Biol. Chem., in the press].     

Non-histone chromatin protein fractions associated with ‘active’ chromatin in embryonic chicken liver

Non-histone chromatin proteins synthesized during chicken embryonic liver development were labeled with [3H]tryptophan and [3H]methionine and characterized by electrophoresis. During embryonic development protein/DNA ratio in chromatin was low (1.30-1.62) but synthesis of non-histone protein was high. Especially one characteristic fraction K (MW 18 000), tightly bound with DNA was preferentially associated with DNAase II sensitive, active transcribed sequences. In 7-day old and adult chicken synthesis of all non-histone proteins was low, fraction K was absent or synthesized only in small amounts in association with non-active sequences, however protein/DNA ratio in chromatin was high (2.30-2.33).     

Multiacetylated forms of H4 are found in a putative transcriptionally competent chromatin fraction from trout testis.

We have examined the distribution of acetylated histones derived from various trout testis chromatin fractions of different composition. Our results indicate that a chromatin fraction, preferentially solubilized by micrococcal nuclease, containing the bulk of the HMG proteins and similar to a fraction released from intact trout nuclei and previously shown to be enriched in transcribed DNA sequences also possesses high levels of multiacetylated species of H4. Histones 2A, 2B and 3 are also acetylated in this particular chromatin fraction. Monoacetylated species of the 4 inner nucleosomal histones appear to be characteristic of the nucleohistone portion of trout testis chromatin.