Metabolic properties of histones from rat liver and thymus gland

The arginine-rich histone, H3, isolated from avian erythrocytes, can dimerize by forming a disulfide linkage between the single cysteine sulfhydryl residues at position 110 of the H3 polypeptide chain. The H3 dimer can be substituted for undimerized H3 in experiments in which the nucleosome is reconstituted from DNA and mixtures of the four "core" histones, H2A, H2B, H3, and 114. We report here that reconstituted nucleosomes containing H3 dimer are indistinguishable, by a number of criteria, either from native nucleosomes or from reconstitutes containing H3 monomer. The criteria include the pattern of susceptibility of the complex to nucleases, the amount of DNA supercoiling induced by histone binding, and the hydrodynamic properties of reconstituted nucleosome "core" preparations.The results suggest that the residues in the neighborhood of position 110 on each H3 molecule are in close contact in the nucleosome. If, as has been proposed, the nucleosome has a dyad axis, then the disulfide bridge between H3 molecules must lie on this axis.An understanding of the biological activity of chromatin will require detailed knowledge of the internal architecture of its fundamental repeating subunit, the nucleosome.Considerable evidence has accumulated that the DNA in the 140-base-pair nucleosome "core" is wrapped around (1, 2) a histone octamer consisting of two of each of the core histones H2A, H2B, H3, and H4 (3). Studies of histone-DNA interactions have shown that the arginine-rich histones, H3 and H4, play a central role in nucleosome organization (4-10). We are therefore interested in obtaining further information about the way in which these histones interact with each other within the nucleosome. An obvious approach is to use chemical crosslinking reagents to estimate the proximity of functional groups in the proteins; there have been many such studies of histone interactions in chromatin (3). However, most crosslinking reagents can react at more than one site, and so far it has not been possible to identify the specific amino acid residues within the histones that are involved in the crosslinking.There is one crosslinking reaction that may avoid some of these problems: the formation of intermolecular disulfide bonds between cysteine residues. This is a particularly useful reaction for the study of histones because H3 is the only histone that has cysteine and in organisms other than higher mammals there is only one such residue per molecule (11)(12)(13)(14). The cysteine at this one position, residue 110 of H3, has been preserved, with the single exception of yeast H3 (15), throughout evolution.It is well known (11,12,16) that H3 molecules can dimerize in vitro through a disulfide bond between the cysteine residues at position 110. We report here that such a dimer is capable of entering into the formation of nucleosomes. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §...

Section: Resultsmentioning

confidence: 99%

Histone H3 disulfide dimers and nucleosome structure.

Camerini-Otero¹,

Felsenfeld²

1977

“…The presence of adenine in a nuclear basic protein fraction was reported in rat thymus by Ord and Stocken (24). As a consequence of analyzing protease digests, the same group reported recently that ADP-ribose is present in association with histone F1 and proposed that the attachment is via a serinephosphate residue (13 (14).…”

Section: Discussionmentioning

confidence: 96%

Natural occurrence of poly(ADP-ribosyl) histones in rat liver.

Ueda¹,

Omachi²,

Kawaichi³

et al. 1975

126

Poly(ADP-ribose) bound to histones has been isolated from rat liver. When [14Clribose was administered intraperitoneally to rats at a dosage of 300-750Aig (100-250 A&Ci)/100 g, approximately 1% of the radioactivity was recovered in the acid (5% CLCCOOH)-insoluble material of the liver nuclei 2 hr after injection.Of the acid-insoluble radioactivity, 4. Poly(ADP-ribose), a macromolecule synthesized from NAD in chromatin (1-5), has been assumed to be covalently bound to nuclear proteins, principally histones (6-8). Although this poly(ADP-ribosyl)ation of proteins has been well established in vitro using various nuclear preparations (for reviews, see refs. 9 and 10), there is no report that has demonstrated the existence of the protein-bound polymer in vivo. Doly and Mandel (11) (14), on the other hand, briefly described the occurrence of proteinbound ADP-ribose in rat liver nuclei. Neither of these reports, however, presented any evidence for the existence of polymerized t ADP-ribose attached to nuclear proteins.Recently, we carried out a series of experiments to determine the conditions for labeling NAD in vivo and, thereby, poly(ADP-ribose). The results, which have been presented proved to be useful for following adenine-derived compounds. In this communication, new data obtained by employing these precursors are presented which enable us to report the first unequivocal evidence for the natural existence of poly-(ADP-ribosyl) histones in mammalian tissues. spectively. In the main experiments reported herein, 0.5 ml of the ribose solution and 0.25 ml of the adenine solution were mixed and injected intraperitoneally into each rat. The animal was put in a metabolic chamber and maintained for 2 hr under ventilation with CR2-free air. The animal was then etherized and the liver was quickly removed. The liver was homogenized in either 3 volumes of ice-cold 20% Cl3CCOOH (or 5% HCl04) or 5 volumes of 0.25 M sucrose containing 3.3 mM CaCl2 with the aid of a Potter-Elvehjem type tissue grinder. The acid homogenate was centrifuged for 10 min at 10,000 X g. The precipitate was treated with 20% C13CCOOH and centrifuged as before. This washing procedure was repeated three more times so that the final material was contaminated by less than 0.5% of the total acid-soluble radioactivity. The sucrose homogenate was centrifuged for 5 min at 800 X g, and the precipitate obtained (crude nuclei) was subjected to acid washing as was applied to the acid homogenate. The acid-insoluble material prepared by either of these procedures was extracted with 5 volumes (per original liver weight) of 0.25 N HCl by stirring for 30 min at 4°. After the mixture was centrifuged for 15 min at 15,000 X g, the supernatant fraction was collected and the precipitate was reextracted as above. Five extracts' obtained in this way were combined, concentrated approximately 50-fold with the aid of a Diaflo (Amicon) ultrafiltration device equipped with a UM-2 membrane, and centrifuged for 60 min at 105,000 X g. The final, slightly yellow supernatant fracti...

“…It is intriguing to revisit earlier literature (33,34) aimed at determining whether the cysteines in histone H3 variants ''sense'' changes in the redox state of the nucleus. If so, does the proximity of the two cysteines at the interface between homotypic H3 dimers within each nucleosome play a stabilizing role in the architecture of the chromatin polymer that, in turn, impacts on the regulation of gene expression?…”

Section: Cysteines Of H3 Variants and Their Potential Role In Nuclearmentioning

confidence: 99%

Histone H3 variants and their potential role in indexing mammalian genomes: The “H3 barcode hypothesis”

Hake

Allis

2006

389

315

In the history of science, provocative but, at times, controversial ideas have been put forward to explain basic problems that confront and intrigue the scientific community. These hypotheses, although often not correct in every detail, lead to increased discussion that ultimately guides experimental tests of the principal concepts and produce valuable insights into long-standing questions. Here, we present a hypothesis, the ''H3 barcode hypothesis.'' Hopefully, our ideas will evoke critical discussion and new experimental approaches that bear on general topics, such as nuclear architecture, epigenetic memory, and cell-fate choice. Our hypothesis rests on the central concept that mammalian histone H3 variants (H3.1, H3.2, and H3.3), although remarkably similar in amino acid sequence, exhibit distinct posttranslational ''signatures'' that create different chromosomal domains or territories, which, in turn, influence epigenetic states during cellular differentiation and development. Although we restrict our comments to H3 variants in mammals, we expect that the more general concepts presented here will apply to other histone variant families in organisms that employ them.histone H3 variants H3.1, H3.2, H3.3 ͉ ''barcode hypothesis'' ͉ epigenetic memory ͉ cell differentiation