Visualization of Early Chromosome Condensation: a Hierarchical Folding, Axial Glue Model of Chromosome Structure
Total Page：16
File Type：pdf, Size：1020Kb
JCBArticle Visualization of early chromosome condensation: a hierarchical folding, axial glue model of chromosome structure Natashe Kireeva,1 Margot Lakonishok,1 Igor Kireev,1 Tatsuya Hirano,2 and Andrew S. Belmont1 1Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801 2Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724 urrent models of mitotic chromosome structure are appear until late prophase, after formation of uniformly based largely on the examination of maximally con- condensed middle prophase chromosomes. Instead, SMC2 C densed metaphase chromosomes. Here, we test these associates throughout early and middle prophase chroma- models by correlating the distribution of two scaffold com- tids, frequently forming foci over the chromosome exterior. ponents with the appearance of prophase chromosome Early prophase condensation occurs through folding of folding intermediates. We confirm an axial distribution of large-scale chromatin fibers into condensed masses. These topoisomerase II␣ and the condensin subunit, structural resolve into linear, 200–300-nm-diameter middle prophase maintenance of chromosomes 2 (SMC2), in unextracted chromatids that double in diameter by late prophase. We metaphase chromosomes, with SMC2 localizing to a 150– propose a unified model of chromosome structure in which 200-nm-diameter central core. In contrast to predictions of hierarchical levels of chromatin folding are stabilized late radial loop/scaffold models, this axial distribution does not in mitosis by an axial “glue.” Introduction Understanding the structural and molecular basis of mitotic predictions in terms of the functional mechanisms under- chromosome condensation remains a basic challenge in cell lying chromosome condensation and the types of folding biology. Historically, three general experimental approaches intermediates that would be observed in prophase during have been pursued to circumvent technical difficulties associ- early stages of chromosome condensation. Unfortunately, a The Journal of Cell Biology ated with direct structural analysis of fully compact, native major deficit in our understanding of mitotic chromosome metaphase chromosomes. These three approaches have led to condensation is the lack of good structural data concerning three alternative conceptual classes of models for metaphase these early prophase stages and correlation of these conden- chromosome architecture—radial loop, hierarchical folding, sation stages with the dynamics of chromosomal proteins and network models—which are quite different in terms of implicated in the process of chromosome condensation. the structural motifs postulated as giving rise to chromosome A series of experiments, combining removal of all histone condensation (Swedlow and Hirano, 2003). Current mi- and most nonhistone proteins with more gentle extraction croscopy methods cannot distinguish between these differ- methods involving selective extraction of histone H1 and/or ent predicted structural motifs in metaphase chromosomes chromatin decondensation by lowering ionic conditions, has (Belmont, 1998). However, these models make very different led to radial loop models of chromosome organization (Paulson and Laemmli, 1977; Laemmli et al., 1978). In these models, loops of radially organized 30-nm chromatin fibers are an- The online version of this article includes supplemental material. chored to an axial chromosome structure, or chromosome Address correspondence to Andrew S. Belmont, Dept. of Cell and Struc- “scaffold” (Marsden and Laemmli, 1979), formed by a spe- tural Biology, University of Illinois at Urbana-Champaign, B107 CLSL, cial class of nonhistone proteins, including topoisomerase II␣ 601 S. Goodwin Ave., Urbana, IL 61801. Tel.: (217) 244-2311. Fax: (217) 244-1648. email: [email protected] (Earnshaw and Heck, 1985; Gasser et al., 1986) and struc- N. Kireeva and I. Kereev’s present address is Department of Electron tural maintenance of chromosomes 2 (SMC2) (Saitoh et al., Microscopy, A.N. Belozersky Institute for Physical Chemical Biology, 1994). Anchoring of these loops to the chromosome scaffold Moscow State University, Moscow 119899, Russia. Key words: chromosome structure; condensins; topoisomerase II; mitosis; Abbreviations used in this paper: SMC2, structural maintenance of chromo- SMC somes 2; TEM, transmission electron microscopy. © The Rockefeller University Press, 0021-9525/2004/09/775/11 $8.00 The Journal of Cell Biology, Volume 166, Number 6, September 13, 2004 775–785 http://www.jcb.org/cgi/doi/10.1083/jcb.200406049 775 776 The Journal of Cell Biology | Volume 166, Number 6, 2004 is proposed to involve special scaffold/matrix associated re- In hierarchical models of chromosome folding, 10- and gion (SAR/MAR) DNA sequences (Razin, 1996). A variation 30-nm chromatin fibers are postulated to fold progressively of the basic radial loop model is the radial loop/helical coil into larger fibers that coil to form the final metaphase chro- model in which a prophase chromatid, itself organized ac- mosomes (Sedat and Manuelidis, 1978; Zatsepina et al., cording to the original radial loop model, is then helically 1983; Belmont et al., 1987; Belmont and Bruce, 1994). In folded to form the final metaphase chromosome (Rattner and contrast to radial loop models, chromosome condensation in Lin, 1985; Boy de la Tour and Laemmli, 1988). However, hierarchical models is not dependent on formation of a core more recent experiments suggest that metaphase chromo- protein scaffold, and therefore the temporal pattern of chro- somes displaying this apparent helical folding are seen only in mosome condensation will not necessarily coincide with hypercondensed metaphase chromosomes isolated from mi- scaffold assembly. Successive helical coiling and folded chromo- totically arrested cells (Maeshima and Laemmli, 2003). nema models are examples of this group of models (Sedat From a functional perspective, radial loop models postu- and Manuelidis, 1978; Belmont and Bruce, 1994). Ultra- late that the chromatin folding above the level of the 30-nm structural analysis of chromosome decondensation between chromatin fiber is guided by nonhistone scaffold protein in- telophase and early G1 indicated one or more levels of com- –100ف teractions with specific DNA sequences. The original model paction between the 30-nm chromatin fiber and an postulated an interconnected protein structure, but an alter- 130-nm chromonema fiber, which itself folded into telo- native possibility of distributed islands of scaffold proteins phase chromosomes (Belmont and Bruce, 1994). However, anchoring local clusters of radial loops was introduced based no comparable work was done to analyze intermediates of on electron microscopy localization of topoisomerase II in prophase chromosome condensation. Recently, a novel im- swollen chromosomes (Earnshaw and Heck, 1985). The pre- aging method was used to selectively label two transgene diction from these models is not only that mitotic chromo- chromosome regions approximately half a chromosome some condensation will be dependent on these scaffold pro- band in size (Strukov et al., 2003). Ultrastructural analysis of teins, but also that the temporal pattern of scaffold assembly immunogold staining of this region within intact metaphase will coincide with or precede the appearance of chromosome chromosomes suggested the existence of a subunit of native nm in size. These results 250ف condensation above the level of the 30-nm chromatin fiber. metaphase chromosomes Immunostaining of unextracted chromosomes and in vivo suggest a hierarchical folding model, but do not distinguish observations have confirmed the existence of an axial core between a helical coil/radial loop model versus nonradial distribution in native metaphase chromosomes for scaffold loop hierarchical models. Still missing is a description of proteins topoisomerase II␣ and condensins (Earnshaw and folding levels lying between the 30-nm fiber and the postu- Heck, 1985; Tavormina et al., 2002; Maeshima and Laem- lated 250-nm coiling subunit. mli, 2003; Ono et al., 2003). A requirement for condensins Meanwhile, chromosome micromanipulation experiments for chromosome assembly was first indicated in a cell-free have challenged predictions from both the radial loop and extract prepared from Xenopus eggs (Hirano et al., 1997). hierarchical models. Brief nuclease treatment leads to a loss Genetic analyses have demonstrated an in vivo role for con- of metaphase chromosome elasticity, arguing against a core densin subunits in chromosome organization and segrega- protein scaffold dominating chromosome mechanical prop- tion (for review see Swedlow and Hirano, 2003); however, erties (Poirier and Marko, 2002). Furthermore, mechanical the exact defects in mitotic chromosome structure have not stretching experiments show elastic extension of metaphase been clearly delineated, and significant chromosome com- chromosomes to several times their normal length without paction has been observed after condensin subunit knock- obvious changes in diameter or the sequential uncoiling of downs. Knockdown of SMC2 condensin subunit expression different folding levels as predicted in hierarchical models by either RNA interference or a conditional knockout re- (Poirier et al., 2000). These results have led to the proposal veals defects in metaphase chromosome structure when chro- of a chromatin network model for chromosome organiza- mosome spreads are prepared after hypotonic treatment