Isolation of a protein scaffold from mitotic HeLa cell chromosomes

Abstract

We have recently shown that, after the histones and most of the nonhistone proteins are gently removed from HeLa metaphase chromosomes, the chromosomal DNA is still highly organized and relatively compact. The structure of these histone-depleted chromosomes is due to the presence of a number of nonhistone proteins that form a central scaffold that retains the approximate size and shape of intact chromosomes and to which the DNA is attached, predominantly forming loops. We now demonstrate that the protein scaffold may be iso ated independently of the DNA by treating HeLa chromosomes with micrococcal nuclease before removing the histones.The chromosomal scaffolds may be isolated by sucrose density gradient centrifugation as a well-defined peak that is stable in 2 M sodium chloride, but is dissociated by treatment with proteases, 4 M urea, or 0.1% sodium dodecyl sulfate. Polyacrylamide gel electrophoresis reveals that the protein content of scaffold preparations is identical to that of histone-depleted chromosomes. Fluorescence microscopy of purified scaffolds in isolation buffer shows that the particles still possess the familiar chromosome morphology. When the scaffolds are examined in the electron microscope, a fibrous structure with the approximate size and shape of intact, paired chromatids is seen. Less than 0.1I% of the chromosomal DNA and virtually no histones are associated with the purified scaffold structures. Mitotic chromosomes are composed of the five histones and a large number of nonhistone proteins that keep the DNA in a highly compact arrangement. Electron microscopy has revealed that the nucleoprotein fiber in mitotic chromosomes is a "knobby" fiber with a diameter of about 200-300 A and appears in a complex, net-like arrangement (e.g., ref. 1). The complexity of this network and the tendency of the chromatin fibers to adhere to each other make it difficult to study the higher-order folding of the nucleoprotein fiber in chromosomes.A great deal has been learned in the last few years about the structural role played by the histones in chromatin, but little is known about a possible involvement of nonhistone proteins in chromosome structure. Starting with the hypothesis that nonhistone proteins-may play a role in determining the superstructure of interphase and metaphase chromosomes, we have recently developed an approach that permits dissection of the structural contribution made by the histones and certain nonhistone proteins. Our general approach toward uncovering a structural role for the nonhistone proteins has been to gently remove the histones from chromosomes by competition with dextran sulfate and heparin, without destroying the continuity of the DNA fiber (2). In this way, all the histones and many nonhistone proteins are removed, leaving behind some tightly bound nonhistone proteins that keep the chromosomal DNA highly organized and compact. This dissecting procedure hasThe costs of publication of this article were defrayed in part by the payment of page charges. This article mu...

Topoisomerase II is a structural component of mitotic chromosome scaffolds.

“…Scaffolds were prepared from 95 mg of chromosomes by standard procedures using 2 M NaCl to extract the bulk of the chromosomal protein (9,14). The scaffolds were resuspended in SDS PAGE sample buffer (10) and electrophoresed in two preparative SDS polyacrylamide gels.…”

“…In 1977 and 1978, Laemmli and co-workers (6)(7)(8) proposed that in mitotic chromosomes these loops are formed as a result of interactions between the nucleohistone fiber and a subgroup of chromosomal nonhistone proteins. A residual structure thought to be enriched in these nonhistone proteins was isolated by extraction of nuclease-digested chromosomes under conditions that solubilized >90% of the chromosomal proteins, including all of the histones (9). Because it retained the size and approximate shape of the mitotic chromosome, this residual structure, was termed the "chromosome scaffold."…”

In Vivo Analysis of Chromosome Condensation inSaccharomyces cerevisiae

“…In a more recent model of chromosome organization, chromatin is folded into fibers around the central axis, which acts as a "glue" (Kireeva et al, 2004). In both of these models, the protein scaffold is proposed to condense linearly during mitosis.The mitotic scaffold was first observed in histone-depleted mammalian chromosomes and components of this protein scaffold have been of considerable interest (Adolphs et al, 1977;Paulson and Laemmli, 1977). In higher eukaryotes, the process of condensing chromosomes before anaphase requires a protein scaffold comprised of at least condensin and topoisomerase II (Earnshaw et al, 1985;Gasser et al, 1986;Adachi et al, 1991;Saitoh et al, 1994;Gimenez-Abian et al, 1995;Hirano et al, 1997;Maeshima and Laemmli, 2003).…”

Tropomyosin is localized in the nuclear matrix and chromosome scaffold of physarum polycephalum

“…The nuclear matrix and chromosome scaffold are the residual fibrillar network structures of the nucleus and chromosome which are depleted of DNA, histone and most of nonhistone proteins [1][2][3][4][5][6]. These two structures are dynamic during the cell cycle and play important roles in various activities essential for the cell life [7][8][9].…”

“…These two structures are dynamic during the cell cycle and play important roles in various activities essential for the cell life [7][8][9]. The main component of nuclear matrix and chromosome scaffold is nonhistone proteins [1][2][3][4], [10]. Although many biochemical and immunocytochemical analyses have been conducted concerning protein composition of the nuclear matrix and chromosome scaffold, only a few proteins (such as topoisomerase II) have been identified [11][12][13][14][15][16].…”