Preferential and cooperative binding of histone I to chromosomal mammalian DNA.

Abstract

There is a strong preferential binding of histone I to lymphocyte DNA as compared to Escherichia coli DNA when large DNA fragments (2 X 106 daltons) are used. The binding of histone I to lymphocyte DNA and to E. coli DNA is cooperative. The distribution of preferential binding sites has been investigated on fragmented DNA. Most of the 2 X 106 dalton fragments were found to have at least one preferential histone I binding site, whereas most of the 2 X 106 dalton fragments have none.Specific DNA-protein interactions are most probably involved in generating the structure of the chromosome and regulating the activity of the genes. There is a large amount of experimental evidence which points to the acidic chromosomal proteins as likely candidates for specific gene regulators (1). Histones seem to bind less specifically to DNA than acidic chromosomal proteins (2). Histone I, present in almost all eukaryotic chromosomes and responsible for their insolubility under physiological conditions, has been implicated as a participant in the folding of the chromosomes (3, 4). If this is the case, histone I may be expected to interact in a preferential way with certain regions of DNA to generate a specific folding pattern. In this paper experimental evidence for such preferential DNA-histone I interactions will be given. MATERIALS AND METHODSAfter stimulating bovine lymphocytes with Phytohemagglutinin P (DIFCO) and labeling with [3H]thymidine for 64 hr, cell nuclei were treated with RNase and pronase. Escherichia coli H 560 (thy-) was labeled with ['4C]thymine and crude DNA was isolated essentially according to Thomas (5). The following steps were identical for both mammalian and bacterial DNA. DNA was adsorbed onto hydroxyapatite in 2 M NaCl, 5 M urea, 0.15 M potassium phosphate (pH 6.8) and washed three times with the same buffer. DNA was eluted with 0.5 M potassium phosphate (pH 6.8) and freed from residual proteins by phenol extraction. After gel filtration on Bio-Gel A-5m in 0.15 M NaCl, 0.01 M Tris.HCl (pH 7.5), 0.0001 M EDTA, the DNA was fractionated by sucrose gradient centrifugation. The small molecular weight DNA (<8 X 105) was produced by sonication and then separated on a sucrose gradient. The molecular weight of the fractionated DNA was calculated from its sedimentation coefficient so0 , according to Studied (6) using 32P-labeled fd replicative form DNA (a gift from Dr. H. Schaller's laboratory) as reference. When the fractionated DNA samples were analyzed on a second sucrose gradient, the peaks formed showed a Gaussian distribution. In each sample there is a range of fragment sizes which, however, is rather narrow, e.g., 76% of our 2 X 106 daltons sample is composed of DNA pieces of sizes between 0.8 and.4.4 X 106 daltons. The specific activity of the bovine 733 DNA was 7000 3H cpm/,ug; that of E. coli DNA was 1000 14C cpm/,ug. Chromatin was mainly obtained from bovine lymph nodes, but also from bovine liver and HeLa cells. The chromatin was isolated from nuclei (7), dialyzed against 2 M NaCl, 5 M urea, 0....

Interaction of MAR‐sequences with nuclear matrix proteins

“…The preference of histone H1 for a subfraction of the mammalian genomic DNA [Renz, 1975;Diez-Caballero et al, 19891 and even to a discrete region flanking the rat albumin gene has been reported earlier [Sevall, 19881. It has been even suggested that dispersed preferential binding sites for H1 exist in the mammalian genome [Renz, 1975;Diez-Caballero et al, 19891. This speculation is particularly interesting in view of the suggestion that SARs may control the conformation of chromatin domains through their highly specific association with H1 [Izaurralde et al, 19891. In the latter case the specificity of the interaction was demonstrated by the formation of specific precipitable complexes between isolated histone H1 and several SARs.…”

Use of matrix attachment regions (MARs) to minimize transgene silencing

“…It has long been known that H1 histone binds preferentially to AT-rich DNA (Renz, 1975). Later work in Laemmli's group showed that H1 binds cooperatively to MAR elements to nucleate further assembly of H1 molecules and condensation of chromatin Käs et al, 1989).…”

Signals in chicken beta-globin DNA influence chromatin assembly in vitro.

“…It has not been adequately explained, however, how a localized activation event could prevent or alter the presumed default packaging of large stretches of DNA into inactive chromatin or, alternatively, how an active chromatin structure could propagate over a large distance. Thus, the tight binding of activator proteins to their recognition sites on DNA would be expected to generate nucleosome-free DNase-hypersensitive sites in that region, as observed (16), but the concomitant opening of a large adjacent chromatin domain would not be expected, given the known physical chemistry of histone-DNA interactions.It has been suggested that the breaking of cooperative interactions among histone Hi molecules in chromatin at a hypersensitive site might lead to the destabilization (and potential activation) of a large region of chromatin (50).However, although histone Hi binds cooperatively to naked DNA (11,25,35) been demonstrated, and highly cooperative binding appears unlikely (1). Moreover, active chicken ,-globin chromatin from erythroid nuclei exists in a compacted state, consistent with either a fully folded 30-nm fiber containing a local disruption at the nucleosome-free hypersensitive region (5) or an only slightly disrupted fiber (19).…”

“…However, although histone Hi binds cooperatively to naked DNA (11,25,35) been demonstrated, and highly cooperative binding appears unlikely (1). Moreover, active chicken ,-globin chromatin from erythroid nuclei exists in a compacted state, consistent with either a fully folded 30-nm fiber containing a local disruption at the nucleosome-free hypersensitive region (5) or an only slightly disrupted fiber (19).…”