Chemical probes of chromatin structure

108

Rat-liver chromatin has been fractionated into transcriptionally active and inactive regions [Gottesfeld eta. (1974) Proc. Nat. Acad. Sci. USA 71, 2193USA 71, -2197 and the distribution of nuclease-resistant complexes in these fractions has been investigated. About half of the DNA of both fractions is resistant to attack by the endonuclease DNase IL The nuclease-resistant structures of inactive chromatin are DNA-histone complexes (v-bodies) which sediment at 11-13 S. Template-active chromatin yields two peaks of nucleaseresistant nucleoprotein. These complexes sediment at 14 and 19 S, and contain DNA, RNA, histone, and nonhistone chromosomal proteins. Polyacrylamide gel electrophoresis reveals a complex pattern of chromatin proteins, suggesting that the complexes are heterogeneous in composition. A regular repeating unit in chromatin was first suggested from the x-ray diffraction studies of Pardon et al. (1) MATERIALS AND METHODSChromatin Fractionation. Chromatin was prepared from rat liver by the method of Marushige and Bonner (22) and treated with diisopropylfluorophosphate (DFP) to inhibit endogenous protease activity (23). Fractionation was carried out as diagrammed in Fig. 1; details of this method have been published previously (21).Preparation of Chromatin Subunits. Nuclease-resistant subunits of rat-liver chromatin were prepared as follows:DNase II was added to 10 units per A260 unit of chromatin in 25 mM sodium acetate (pH 6.6). Digestion was carried out at 240 and was terminated after 90 min by raising the pH to 7.5 with 0.1 M Tris-HCl (pH 11). Nuclease-resistant subunits from chromatin fraction P1 were prepared by homogenizing the pellet fraction in 25 mM sodium acetate (pH 6.6) and redigesting with DNase as described above for whole chromatin. Undigested chromatin (about 20% of the input DNA) was removed by centrifugation at 27,000 X g for 10-15 min. The supernatant was layered on isokinetic sucrose gradients in SW25. 1 cellulose nitrate tubes. The gradients were formed according to Noll (24); the parameters were CTOP = 15% (weight/volume), CRES = 34.2% (weight/ volume), and VMIX = 31.4 ml. All solutions contained 10 mM Tris-HCl (pH 8). Centrifugation was at 25,000 rpm for [36][37][38][39][40][41][42] hr. Gradients were analyzed with an ISCO UV Analyzer and chart recorder. Fractions from these gradients were rerun on 5-24% isokinetic sucrose gradients. The parameters were CTOP = 5.1% (weight/volume), CRES = 31.4% (weight/volume) and VMIX = 9.4 ml. Centrifugation was in the SW 41 rotor at 39,000 rpm at 40 for 16.5 hr.Subunits were also prepared from chromatin devoid of histone I. Removal of this histone was accomplished by extraction of Virtis-sheared chromatin (45 V, 90 sec) with 0.5 M NaCl at 4°. The resultant nucleohistone was pelleted by centrifugation in the Ti 50 rotor at 50,000 rpm for 18 hr.The pellet was digested with nuclease as described above.Redigestion of Chromatin Fraction S2. Chromatin of fraction S2 was redigested with nuclease in three different ways: the DNase II present...

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confidence: 99%

Structure of transcriptionally active chromatin.

Gottesfeld¹,

Murphy²,

Bonner³

1975

108

“…The existence of such subunits has been inferred from experiments showing that the nuclease digestion of chromatin gives a discrete pattern of polynucleotide products (1)(2)(3)(4) and that specific complexes between various histone molecules can be formed in solutions of isolated histone and in chromatin (5)(6)(7). Un-fortunately, x-ray diffraction patterns have not yielded any information about the existence or the possibilities for the internal structure of the subunit, or the manner in which the subunits associate.…”

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confidence: 99%

Chromatin architecture: investigation of a subunit of chromatin by dark field electron microscopy.

Langmore¹,

Wooley²

1975

Dark field scanning electron microscopy of unstained, unfixed samples of chromatin, histone-l-depleted chromatin, and nucleohistone has been used to identify an apparent subunit of chromatin, namely a disk-shaped structure we term the unit particle, which is probably about 135 A wide and 50 A thick in the hydrated state. The unit particles are found at rather uniform intervals along thin DNA-like fibers. Histone 1 depletion leads to a bimodal distribution of these spacings. Our observations suggest that the unit particle consists of a loop of nucleoprotein, perhaps around a histone core.After many attempts, with limited success, to describe the structure of the eukaryotic genetic material as a continuous structure, investigators are now turning their attention to models of a more discontinuous structure, involving subunits which might be "assembled" into chromatin. (20).The chemical composition and stoichiometry of the samples were characterized using the procedures of Bonner et al. (20). Intact chromatin possessed a total histone/DNA weight ratio of 1.12 I 0.04, an H1/DNA ratio of 0.29 + 0.02, and a non-histone protein/DNA ratio of 0.55 I 0.03. The histone/DNA ratio of HI-depleted chromatin was 0.83 + 0.02; no non-histone proteins were found. The nucleohistone had a ratio of histone/DNA of 1.02 I 0.02 and an Hi/ DNA ratio of 0.14 + 0.02; no non-histone proteins were found.All specimen solutions were "desalted" immediately before preparation, by exclusion on Sephadex G-100 equilibrated with 1 mM NaPO4, pH 7.0. About 2 gl of this Mg/ml solution of chromatin were then placed on a 20-30 A thick hydrophilic carbon film, blotted, and air dried.Electron Microscopy. All specimens were examined at 30 keV in dark field using the high-resolution scanning transmission electron microscope developed in the laboratory of . Microscope operating conditions were as described before, except that we chose not to outgas the specimen by baking (17). The elastically and inelastically scattered electron signals were usually simultaneously stored in digital form directly on magnetic tape, using a 512 X 512 picture element format (16,17). At low instrumental magnification each picture element represented 15.4 A at the specimen; at high magnification each picture element represented 4.96 A. The magnification was determined from

“…Accordingly, we interpret our data as follows: Polycations added to chromatin at concentrations below cation/nucleotide-phosphate ratios of 0.5 will preferentially bind to the interparticle strands and dislocate the weakly bound H-1 histones, which are only partially released from the chromatin and move closer towards the nucleosome, resulting in erroneous methylation. As already indicated, others (18)(19)(20) have reported that polylysines are bound within the chromatin up to the half-saturation point, but, beyond this level, release of histone from chromatin will occur, starting with H-i.…”

Section: Discussionmentioning

confidence: 76%

“…of histones (18)(19)(20) After the incubation, histones were isolated and subjected to Bio-Gel P-10 chromatography. The results shown in Fig.…”

Section: Methodsmentioning

confidence: 99%

Displacement and aberrant methylation in vitro of H-1 histone in rat liver nuclei after half-saturation of chromatin with polycations.

Byvoet¹,

Baxter²,

Sayre³

1978

Radiomethyl incorporation in vitro into Nemethyllysine of histones from rat liver nuclei incubated in the presence of S-adenosyl [methyl-3H]methionine is stimulated if the lycations polylysines, protamines, or histones are added to the incubation mixture. Maximal stimulation occurs at a cation/nucleotide ratio of 0.5. Past this point stimulation dro s, except in the case of very lysine-rich histone H-1, for which the maximal level of incorporation remains constant upon further addition of this histone. Bio-Gel P-10 chromatography, differential precipitation, and gel electrophoresis of radiomethylated histones indicate that although the usual incorporation of radiomethyl into histone H-3 is not affected, active methylation of H-1 occurs in the presence of polycations. Column chromatographic amino acid analysis reveals that the methylation of H-1 will specifically generate Ne-monomethyllysine. Except for this condition, H-1 is never methylated in vivo or in incubated cell nuclei. Because H-1 is the weakest bound histone in chromatin, the above phenomena may be explained by assuming that, within the chromatin, polycations displace the lysine-rich histone towards the nucleosome, which results in its aberrant methylation, assuming that the native nucleosome is the seat of the histone lysine methyltransferase.Methylation of r-NH2 groups on lysine side chains in histones occurs in situ in chromatin at the end of S phase (1-3) on newly synthesized histones (4). The lysine methyltransferase responsible for this process utilizes S-adenosylmethionine as methyl donor (1) and may introduce up to three methyl groups into the c-N position of specific lysine residues in H-S and two in H-4. All methylated lysine residues are located in the polar regions of these nucleosomal (5) histones. Methylated lysine residues appear to be situated 3 or 4 amino acid residues from an acetylated lysine (6) and in H-3 next to a phosphorylated serine (7). In contrast to acetylation and phosphorylation, however, methylation has been found to be irreversible (4). A function for this process has eluded investigators so far (8).Previous studies (8, 9) in this laboratory using rat liver nuclei indicate that uptake of radiomethyl into histones will occur during incubation in vitro of rat liver nuclei or chromatin in the presence of S-adenosyl[methyl-3H]methionine ([3H]AdoMet) and that this uptake is practically confined to that into H-3, the uptake into H-4 being much less.We have reported (9) that a number of carcinogenic agents were inhibitory, to this process, whereas planar compounds able to intercalate between DNA base pairs appeared to be stimulatory (10). We now report that saturation of chromatin with polycations (cation/base-pair ratio = 1) stimulates radiomethylation of histones in vitro. We also present evidence indicating that this stimulation is due to displacement of H-1 histone within the chromatin, resulting in its subsequent aberrant methylation. MATERIALS AND METHODSDi-a-L-lysine and tri-a-L-lysine (neutral hydrochloride salt...