Topoisomerase II, scaffold component, promotes chromatin compaction in vitro in a linker-histone H1-dependent manner

TopoisomeraseII (Topo II) is a major component of chromosomal scaffolds and essential for mitotic chromosome condensation, but the mechanism of this action remains unknown. Here, we used an in vitro chromatin reconstitution system in combination with atomic force and fluorescence microscopic analyses to determine how Topo II affects chromosomal structure. Topo II bound to bare DNA and clamped the two DNA strands together, even in the absence of ATP. In addition, Topo II promoted chromatin compaction in a manner dependent on histone H1 but independent of ATP. Histone H1-induced 30-nm chromatin fibers were converted into a large complex by Topo II. Fluorescence microscopic analysis of the Brownian motion of chromatin stained with 4′,6-diamidino-2-phenylindole showed that the reconstituted chromatin became larger following the addition of Topo II in the presence but not the absence of histone H1. Based on these findings, we propose that chromatin packing is triggered by histone H1-dependent, Topo II-mediated clamping of DNA strands

シケンカンナイ サイコウチク ジッケン オヨビ ナノスケール カンサツ ニ ヨリ アキラカニ サレタ クロマチン コウゾウ コウチク メカニズム

京都大学0048新制・論文博士博士(理学)乙第11771号論理博第1465号新制||理||1443(附属図書館)23826UT51-2006-C693(主査)教授 杉山 弘, 教授 三木 邦夫, 教授 吉川 研一学位規則第4条第2項該当Doctor of ScienceKyoto UniversityDA

The hierarchical structure of chromatin : Nucleosomal array reconstitution with ring and linear DNA(Knots and soft-matter physics: Topology of polymers and related topics in physics, mathematics and biology)

この論文は国立情報学研究所の電子図書館事業により電子化されました。真核生物で見られる階層的な折りたたみの基本構造としてヌクレオソーム(長さ50nmのDNAがコアヒストンと呼ばれる直径7nmのタンパクに巻きついた構造)が知られている。DNAの長さ、ねじれ、大域的な構造(環状等)がヌクレオソーム形成に与える影響は未だ十分に理解されていない。本研究ではDNAの両端がつながっている環状構造がヌクレオソーム形成に与える影響を明らかにするために、全長1マイクロメートルの線状・環状DNAを用いて再構成を行い、その効率とヌクレオソーム間相互作用の検討を行った。その結果、環状のDNAを用いた場合の方が環状構造に由来して再構成の効率が高くなることが明らかになった

Hydrodynamic radius of circular DNA is larger than that of linear DNA

It is generally accepted that linear polymers are larger, such as with regard to the end-to-end distance and gyration radius, than the corresponding circular polymers. We measured the Brownian motion of individual linear and circular 106-kbp DNA molecules using fluorescence microscopy. Unexpectedly, the hydrodynamic radius of linear DNA was markedly smaller than that of circular DNA

Removal of histone tails from nucleosome dissects the physical mechanisms of salt-induced aggregation, linker histone H1-induced compaction, and 30-nm fiber formation of the nucleosome array

In order to reveal the roles of histone tails in the formation of higher-order chromatin structures, we employed atomic force microscopy (AFM), and an in vitro reconstitution system to examine the properties of reconstituted chromatin composed of tail-less histones and a long DNA (106-kb plasmid) template. The tail-less nucleosomes did not aggregate at high salt concentrations or with an excess amount of core histones, in contrast with the behavior of nucleosomal arrays composed of nucleosomes containing normal, N-terminal tails. Analysis of our nucleosome distributions reveals that the attractive interaction between tail-less nucleosomes is weakened. Addition of linker histone H1 into the tail-less nucleosomal array failed to promote the formation of 30 nm chromatin fibers that are usually formed in the normal nucleosomal array. These results demonstrate that the attractive interaction between nucleosomes via histone tails plays a critical role in the formation of the uniform 30-nm chromatin fiber

Schematic illustrations of the molecular mechanisms of chromatin compaction by histone H1 and Topo II

<p><b>Copyright information:</b></p><p>Taken from "Topoisomerase II, scaffold component, promotes chromatin compaction in a linker-histone H1-dependent manner"</p><p></p><p>Nucleic Acids Research 2007;35(8):2787-2799.</p><p>Published online 11 Apr 2007</p><p>PMCID:PMC1885653.</p><p>© 2007 The Author(s)</p> () Stepwise packing of reconstituted chromatin. First, DNA forms a nucleosomal fiber with core histones, and histone H1 induces the formation of 30-nm fibers. Second, Topo II induces compaction when the 30-nm fiber is formed in the presence of histone H1. () Molecular mechanism of Topo II-induced chromatin compaction. Topo II clamping of two DNA strands induces a small amount of aggregation of beads-on-a-string nucleosomal fibers but promotes large-scale aggregation of 30-nm fibers

Analysis of the Brownian motion of DAPI-stained reconstituted chromatin in solution

<p><b>Copyright information:</b></p><p>Taken from "Topoisomerase II, scaffold component, promotes chromatin compaction in a linker-histone H1-dependent manner"</p><p></p><p>Nucleic Acids Research 2007;35(8):2787-2799.</p><p>Published online 11 Apr 2007</p><p>PMCID:PMC1885653.</p><p>© 2007 The Author(s)</p> ( and ) Differences between H1-containing reconstituted chromatin in the presence (B) and absence of Topo II (A). Top, fluorescent microscope images; bottom, trails of Brownian motion during 3 s. () Mean square displacement (MSD) of the H1-containing chromatin without and with Topo II. (–) Histograms of hydrodynamic radii of the chromatin complexes without (D–F) and with H1 (G–I). Reconstituted chromatin containing 80 ng of DNA was treated with 2 ng (E and H) or 20 ng of Topo II (F and I)

In the absence of ATP, Topo II induces small aggregates, but large aggregates are induced in the presence of histone H1

<p><b>Copyright information:</b></p><p>Taken from "Topoisomerase II, scaffold component, promotes chromatin compaction in a linker-histone H1-dependent manner"</p><p></p><p>Nucleic Acids Research 2007;35(8):2787-2799.</p><p>Published online 11 Apr 2007</p><p>PMCID:PMC1885653.</p><p>© 2007 The Author(s)</p> () Nucleosomal fibers were reconstituted from the 26-kb plasmid and core histones by the salt dialysis method and then incubated without Topo II and in the absence (A) or presence (D) of histone H1. ( and ) The reconstituted nucleosomal fibers containing (E and F) or lacking (B and C) histone H1 were treated with Topo II in the absence of ATP and MgCl. Nucleosomal fiber containing 80 ng DNA was mixed with 2 ng (B and E) or 20 ng (C and F) of Topo II. The mixed weight ratio of Topo II versus DNA is indicated on the left side of the images. In each condition, at least three 12.5 × 12.5-μm areas were scanned, and 10–30 of the reconstituted chromatin complexes were scanned a second time at a higher magnification