Exchange of proteins during immunofractionation of chromatin

The migration and rearrangement of chromosomal proteins during immunofractionation of chromatin has been investigated. Oligonucleosomes from two different chromatins, chicken erythrocyte or rat liver, were mixed with oligonucleosomes from the other species which had been depleted of histones H1/H5 and high mobility group proteins (HMGs). The mixture was treated with buffers of various ionic strengths and immunofractionated on an anti-H1 degrees/H5 or anti-HMG-17 IgG-Sepharose column. The type of DNA, which was retained as the bound fraction on the column, was determined by slot blot analysis using nick-translated repetitive DNA probes from either chicken or rat. The results indicate that in low ionic strength buffers (i.e., below 40 mM NaCl), there is very little exchange of either histone H5 or HMG-17 among nucleosomes and therefore we suggest that it is possible to fractionate nucleosomes according to their antigenic content.     

Unravelled nucleosomes,nucleosome beads and higher order structures of chromatin: Influence of non-histone components and histone H1

Effects of non-histone components and histone H1 on the morphology of nucleosomes and chromatin were studied by electron microscopy. Soluble rat liver ehromatin was depleted of non-histone components [NH]or non-histone components and H1 [NH and H1] by dissociation and subsequent fractionation in sucrose gradients in the presence of 300 to 350 mm or 500 mm-NaCl, respectively. In reconstitution experiments the depleted samples were mixed either with [NH] or with [NH and H1] or with purified H1. The morphology of the ionic strength-dependent condensation of the samples was monitored by electron microscopy using 0 mm to 100 mm-NaCl. Based on the appearance of the different types of fibres in very low salt (0 mm up to 10 mm-NaCl), namely the zigzag-shaped, the beads-on-a-string or the DNA-like filaments, it is possible to distinguish between nucleosomes, partially unravelled nucleosomes and unravelled nucleosomes, respectively. Only those fibres which were zigzag-shaped at low ionic strength condense at increasing ionic strength into higher order structures of compact fibres. We demonstrate the dependence of the appearance of nucleosomes and chromatin upon its composition and upon the ionic strength of the solvent.[NH] have no detectable influence upon the formation of higher order chromatin structures, but they can prevent the unravelling of nucleosomes at very low ionic strength, presumably by charge shielding.For the appearance of zigzag-shaped fibres and for the condensation into compact fibres with increasing ionic strength, H1 must be present in about native amounts. Partial removal of H1 (about 10%) promotes a change from fibres into tangles. This supports the model that an H1 polymer is stabilizing the higher order chromatin structures.Reconstitution experiments with purified H1 regenerated fibres containing all the features of [NH]-depleted chromatin. Reconstitution experiments with [NH and H1] promoted fibres compatible with control chromatin. Overloading of chromatin with H1 led to additional condensation. The detailed morphology of the reconstituted fibres showed local distortions. One possibility explaining these local distortions would be competition between “main” and “additional” binding sites for histone H1.     

Immunofractionation of DNA sequences associated with HMG-17 in chromatin

Antibodies specific for chromosomal protein HMG-17 were immobilized on CNBr-Sepharose and the resulting immunoaffinity column was used to purify chromatin segments enriched in HMG-17. The DNA was purified from both the nucleosomal fraction which was bound to the column and from the fraction which was not bound, and examined with DNA probes representing repetitive DNA, non-transcribed genes, transcribed genes and inducible genes. The results suggest that HMG-17 is preferentially associated with DNA sequences coding for genes, regardless of whether they are transcribed, and therefore support the notion that HMG-17 confers specific structural characteristics on selected regions in the genome.     

Reconstitution of chromatin higher-order structure from histone H5 and depleted chromatin

Reconstitution of the 30 nm filament of chromatin from pure histone H5 and chromatin depleted of H1 and H5 has been studied using small-angle neutron-scattering. We find that depleted, or stripped, chromatin is saturated by H5 at the same stoichiometry as that of linker histone in native chromatin. The structure and condensation behavior of fully reconstituted chromatin is indistinguishable from that of native chromatin. Both native and reconstituted chromatin condense continuously as a function of salt concentration, to reach a limiting structure that has a mass per unit length of 6.4 nucleosomes per 11 nm. Stripped chromatin at all ionic strengths appears to be a 10 nm filament, or a random coil of nucleosomes. In contrast, both native and reconstituted chromatin have a quite different structure, showing that H5 imposes a spatial correlation between neighboring nucleosomes even at low ionic strength. Our data also suggest that five to seven contiguous nucleosomes must have H5 bound in order to be able to form a higher-order structure.     

Enzymic acetylation of nucleosome histone.

Rat liver chromatin prepared from purified nuclei catalyzed the acetylation of histones in nucleosomes at the same level as that of nuclei. The activity of histone acetyltransferase in chromatin was destroyed by heat treatment at 65 degrees C for 5 min. Histones in exogenously added nucleosomes also served as substrate for the enzyme. The sites of acetylation in the nucleosomes appeared to be in the trypsin-digestable N-terminal regions of histones H4, H3, and H2A, as has been reported in an in vivo system.     

Fractionation of nucleosomes by salt elution from micrococcal nuclease- digested nuclei

The solubilization of nucleosomes and histone H1 with increasing concentrations of NaCl has been investigated in rat liver nuclei that had been digested with micrococcal nuclease under conditions that did not substantially alter morphological properties with respect to differences in the extent of chromatin condensation. The pattern of nucleosome and H1 solubilization was gradual and noncoordinate and at least three different types of nucleosome packing interactions could be distinguished from the pattern. A class of nucleosomes containing 13-- 17% of the DNA and comprising the chromatin structures most available for micrococcal nuclease attack was eluted by 0.2 M NaCl. This fraction was solubilized with an acid-soluble protein of apparent molecular weight of 20,000 daltons and no histone H1. It differed from the nucleosomes released at higher NaCl concentrations in content of nonhistone chromosomal proteins. 40--60% of the nucleosomes were released by 0.3 M NaCl with 30% of the total nuclear histone H1 bound. The remaining nucleosomes and H1 were solublized by 0.4 M or 0.6 M NaCl. H1 was not nucleosome bound at these ionic strengths, and these fractions contained, respectively, 1.5 and 1.8 times more H1 per nucleosome than the population released by 0.3 M NaCl. These fractions contained the DNA least available for micrococcal nuclease attach. The strikingly different macromolecular composition, availability for nuclease digestion, and strength of the packing interactions of the nucleosomes released by 0.2 M NaCl suggest that this population is involved in a special function.     

Involvement of the globular domain of histone H1 in the higher order structures of chromatin

We have attacked H1-containing soluble chromatin by α-chymotrypsin under conditions where chromatin adopts different structures.Soluble rat liver chromatin fragments depleted of non-histone components were digested with α-chymotrypsin in NaCl concentrations between 0 mm and 500 mm. at pH 7, or at pH 10, or at pH 7 in the presence of 4 m-urea. α-Chymotrypsin cleaves purified rat liver histone H1 at a specific initial site (CT) located in the globular domain and produces an N-terminal half (CT-N) which contains most of the globular domain and the N-terminal tail, and a C-terminal half (CT-C) which contains the C-terminal tail and a small part of the globular domain. Since in sodium dodecyl sulfate/polyacrylamide-gel electrophoresis CT-C migrates between the core histones and H1, cleavage of chromatin-bound H1 by α-chymotrypsin can be easily monitored.The CT-C fragment was detected under conditions where chromatin fibers were unfolded or distorted: (1) under conditions of H1 dissociation at 400 mm and 500 mm-NaCl (pH 7 and 10); (2) at very low ionic strength where chromatin is unfolded into a filament with well-separated nucleosomes; (3) at pH 10 independent of the ionic strength where chromatin never assumes higher order structures; (4) in the presence of 4 m-urea (pH 7), again independent of the ionic strength. However, hardly any CT-C fragment was detected under conditions where fibers are observed in the electron microscope at pH 7 between 20 mm and 300 mm-NaCl. Under these conditions H1 is degraded by α-chymotrypsin into unstable fragments with a molecular weight higher than that of CT-C. Thus, the data show that there are at least two different modes of interaction of H1 in chromatin which correlate with the physical state of the chromatin.Since the condensation of chromatin into structurally organized fibers upon raising the ionic strength starts by internucleosomal contacts in the fiber axis (zig-zag-shaped fiber), where H1 appears to be localized, it is likely that in chromatin fibers the preferential cleavage site for α-chymotrypsin is protected because of H1-H1 contacts. The data suggest that the globular part of H1 is involved in these contacts close to the fiber axis. They appear to be hydrophobic and to be essential for the structural organization of the chromatin fibers. Based on the present and earlier observations we propose a model for H1 in which the globular domains eventually together with the N-terminal tails form a backbone in the fiber axis, and the nucleosomes are mainly attached to this polymer by the C-terminal tails.     

Involvement of histone H1 in the organization of the nucleosome and of the salt-dependent superstructures of chromatin

We describe the results of a systematic study, using electron microscopy, of the effects of ionic strength on the morphology of chromatin and of H1-depleted chromatin. With increasing ionic strength, chromatin folds up progressively from a filament of nucleosomes at approximately 1 mM monovalent salt through some intermediate higher- order helical structures (Thoma, F., and T. Koller, 1977, Cell 12:101- 107) with a fairly constant pitch but increasing numbers of nucleosomes per turn, until finally at 60 mM (or else in approximately 0.3 mM Mg++) a thick fiber of 250 A diameter is formed, corresponding to a structurally well-organized but not perfectly regular superhelix or solenoid of pitch approximately 110 A as described by Finch and Klug (1976, Proc. Natl. Acad. Sci. U.S.A. 73:1897-1901). The numbers of nucleosomes per turn of the helical structures agree well with those which can be calculated from the light-scattering data of Campbell et al. (1978, Nucleic Acids Res. 5:1571-1580). H1-depleted chromatin also condenses with increasing ionic strength but not so densely as chromatin and not into a definite structure with a well-defined fiber direction. At very low ionic strengths, nucleosomes are present in chromatin but not in H1-depleted chromatin which has the form of an unravelled filament. At somewhat higher ionic strengths (greater than 5 mM triethanolamine chloride), nucleosomes are visible in both types of specimen but the fine details are different. In chromatin containing H1, the DNA enters and leaves the nucleosome on the same side but in chromatin depleted of H1 the entrance and exit points are much more random and more or less on opposite sides of the nucleosome. We conclude that H1 stabilizes the nucleosome and is located in the region of the exit and entry points of the DNA. This result is correlated with biochemical and x-ray crystallographic results on the internal structure of the nucleosome core to give a picture of a nucleosome in which H1 is bound to the unique region on a complete two-turn, 166 base pair particle (Fig. 15). In the formation of higher-order structures, these regions on neighboring nucleosomes come closer together so that an H1 polymer may be formed in the center of the superhelical structures.     

Psoralen-crosslinking of soluble and of H1-depleted soluble rat liver chromatin

We purified soluble rat liver chromatin and H1-depleted chromatin and photocrosslinked its DNA with psoralen at pH 7. Digestion of this chromatin with micrococcal nuclease produced a normal nucleosomal repeat. Chromatin was photoreacted in the presence of 0 to 700 mM-NaCl and was fractionated in sucrose gradients containing the same NaCl concentrations. The dissociation of H1 occurred as in the non-crosslinked controls and no preferential dissociation of core histones was observed. The samples between 100 and 500 mM-NaCl showed precipitation. In the electron microscope, the fibers appeared indistinguishable from the controls at low ionic strength. In the presence of 40 mM-NaCl, the fibers of the photoreacted chromatin were slightly more compact than the controls, and at 500 mM-NaCl, despite the complete dissociation of H1, there were still apparently intact fibers at this ionic strength. The disruption of the psoralen-treated chromatin fibers occurred only in 600 mM-NaCl, as opposed to 500 mM-NaCl in controls. The DNA of all the photoreacted samples was spread for electron microscopy under denaturing conditions. They revealed, for all the samples, single-stranded bubbles corresponding to 200 to 400 base-pairs in size. H1-depleted chromatin containing stoichiometric amounts of core histones was photoreacted at pH 10 and very low ionic strength. Under these conditions many of the nucleosomes appeared to be unraveled, although to a variable extent. In the electron microscope, the purified DNA from these samples showed extensive crosslinking when spread under denaturing conditions. These observations show that histone-DNA interactions different from those in intact nucleosomes may be created, which allow extensive access of psoralen to the DNA.     

Histone-H1-dependent chromatin superstructures and the suppression of gene activity

I have identified a chromatin particle containing DNA as large as 20-40 kb that migrates as a discrete entity on agarose gels. With increasing nuclease digestion, the particle becomes cleaved in the linker regions between nucleosomes, but remains intact, probably held together by the outer histones, H1 and H5. By hybridization analysis, inactive genes are found in these particles. Active genes (and their flanking sequences) are also found in particles containing H1 and H5, but in contrast to inactive supranucleosome particles, active polynucleosome particles are not held together after cleavage of linker DNA. This suggests that H1 cross-links adjacent nucleosomes in inactive regions and that H1 is bound differently in expressed regions. The results raise the possibility that the marked degree of suppression of repressed, tissue-specific genes may be determined, in part, by their assembly into these inactive supranucleosome structures.     

Genome-wide distribution of macroH2A1 histone variants in mouse liver chromatin

Studies of macroH2A histone variants indicate that they have a role in regulating gene expression. To identify direct targets of the macroH2A1 variants, we produced a genome-wide map of the distribution of macroH2A1 nucleosomes in mouse liver chromatin using high-throughput DNA sequencing. Although macroH2A1 nucleosomes are widely distributed across the genome, their local concentration varies over a range of 100-fold or more. The transcribed regions of most active genes are depleted of macroH2A1, often in sharply localized domains that show depletion of 4-fold or more relative to bulk mouse liver chromatin. We used macroH2A1 enrichment to help identify genes that appear to be directly regulated by macroH2A1 in mouse liver. These genes functionally cluster in the area of lipid metabolism. All but one of these genes has increased expression in macroH2A1 knockout mice, indicating that macroH2A1 functions primarily as a repressor in adult liver. This repressor activity is further supported by the substantial and relatively uniform macroH2A1 enrichment along the inactive X chromosome, which averages 4-fold. Genes that escape X inactivation stand out as domains of macroH2A1 depletion. The rarity of such genes indicates that few genes escape X inactivation in mouse liver, in contrast to what has been observed in human cells.     

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.