Nancy Kleckner *^{, @}, Denise Zickler ¹, Gareth H. Jones ² , Job Dekker *, ¶, Ruth Padmore *, Jim Henle *, and
John Hutchinson **^{, @}

*Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138; ¹ Laboratoire de Génétique, Institut de Génétique et Microbiologie, Université Paris-Sud, Centre d'Orsay, Orsay Cedex 91405 France;
² School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom;
¶Program in Gene Function and Expression and Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 360 Plantation Street, Worcester, MA 01605; and
**Department of Engineering and Applied Sciences, Harvard University, 29 Oxford Street, Cambridge, MA 02138

^@ To whom correspondence may be addressed.
E-mail:    kleckner@fas.harvard.edu   or   hutchinson@husm.harvard.edu

We propose that chromosome function is governed by internal mechanical forces generated by programmed tendencies for expansion of the DNA/chromatin fiber against constraining features. 

Spatial patterning effects suggest that physical forces are present and play important roles within chromosomes. How might such forces arise? And how might they exert their effects?

DNA/Chromatin Expansional Force.

A segment of chromatin free in solution will tend to occupy a particular "envelope volume" (the volume of the smallest shell containing all of the chromatin). If the state of the chromatin changes appropriately, it may tend to occupy a larger envelope volume. In solution, such expansion can occur freely and without effect. However,
inside the cell, such expansion may be constrained, or "caged," either by external features (e.g., other chromatin) or by an internal meshwork of intersegment interlinks (e.g., nucleosome–nucleosome contacts plus interactions involving chromatin/chromosome structure molecules). In both cases, a tendency for expansion will cause the chromatin to push on the constraining components, which will push back. These pushing forces can do chromosomal work. Physically, chromatin expansion could result from any change in the DNA/chromatin fiber that causes it to become stiffer and/or more extended, i.e., to have a longer contour length. In eukaryotic cells, increased contour lengths are produced by histone modification, loss of nonhistone architectural components, or histone elimination.

Predicted Effects.

Compression stress along the DNA/chromatin fiber. Expansion of the DNA/chromatin fiber as constrained by a closepacked internal "meshwork" would cause it to come under compression stress along its length. Such stress would tend to destabilize the DNA duplex, causing discrete buckling or shearing/denaturation (Fig. 3A). It would also tend to promote conformational changes that place the fiber in a more compact shape, e.g., smooth bending and writhing as well as local buckling (Fig. 3B). Such tendencies could promote basic processes that involve access to Watson–Crick base pairs (initiation of transcription or DNA replication or DNA recombination) and organizational processes (wrapping of DNA around the nucleosome core, chromatin fiber folding, or development of programmed chromatin loops; Fig. 3B Bottom). Additionally, a single- or double-strand DNA interruption would lead to local and domainal relaxation, which could then be used to signal the presence of the lesion (e.g., refs. 7 and 10).

Fig. 3. Stresses that could be generated by chromatin expansion against contraining features.

(A and B) Compression stress along the DNA/chromatin fiber can destabilize DNA (A) or promote a more compact fiber configuration (B).
(C) Interchromatin pushing produces opposing forces for separation and interface compression.
(D) Linear/loop axis array (Left) and prophase configuration with cooriented sister arrays (Right).
(E) Changes in axis status predicted from pushing forces between adjacent chromatin loops.

If two touching chromatin masses expand, they will push against one another at their interface ("chromatin pressure"; ref. 11). Two opposing tendencies are created: the two masses will tend to push one another apart,
thus providing a force for separation; concomitantly, they will tend to push into one another, thus providing a force for conjunction and intermingling (Fig. 3C). Either or both tendencies may come into play according to the state of the DNA/chromatin fiber. The separation force could drive, e.g., separation of sister chromatids or homologs, declustering of heterochromatic regions, and separation of unrelated chromosomes into distinct territories or, analogously, separation of chromatin from the nuclear envelope. The conjunction force could promote, e.g., intermingling of chromosome territories and linkages between different chromatin regions or chromatin and the nuclear envelope. Expansion forces will also cause different chromatin regions to push themselves through the nucleus into the "least-stressed" configuration, thus influencing local or global chromosome disposition (e.g., ref. 12).

Axis destabilization.

From prophase onward, the chromatin of each chromosome is organized into a linear array of loops, the bases of which are elaborated by proteins to form a geometric and structural axis (Fig. 3D). If adjacent loops expand,
they cannot move apart because of tethering to their underlying axis; instead, the axis itself will come under stress (Fig. 3E). An immediate effect will be a tendency for axis extension and, ultimately, fracturing. Axes will also tend to twist, thus placing adjacent loops out of phase, where they have more room to expand. Finally, chromatin will tend to expand differentially in the periphery of the loops, where there is more room, rather than along the axis. This effect will cause the axis to bend, with stretching forces arising immediately above the axis and compression stress arising along the axis itself. Axial compression stress, in turn, can promote axis bending, writhing, or buckling. These various effects can explain axial coiling of late-stage chromosomes (Fig. 4A); twisting, bending, buckling, and fracturing seen along meiotic prophase chromosomes (e.g., Fig. 4 B–F); destabilization of axis components; and axis disassembly.

Fig. 4. Observed changes in axis conformation.

(A) Muntjac mitotic anaphase chromosomes with coiled axes (13).
(B–F) Meiosis.
(B) Coiled homologs of lily at diakinesis (14).
(C) Axis fracturing and sister separation in late leptotene in Sordaria (15).
(D) Twisting of a single leptotene homolog axis in Neotiella (16).
(E) In Allium, coaligned homolog axes linked by bridges (Left) undergo apparent buckling plus ensuing nucleation of synaptonemal complex at late leptotene (1, 17).
(F) Twisting synaptonemal complex (18).

[Reproduced with permission from, respectively:
(A) The Journal of Cell Biology, 1995, 131, pp. 7–17, by copyright permission of The Rockefeller University Press; (B) ref. 14 (Copyright 1991, NRC Research Press); (C) ref. 15 (Copyright 1999, Elsevier); (D) ref. 16 by
courtesy of the Carlsberg Laboratory (Copyright 1985, The Carlsberg Laboratory, Copenhagen); (E) ref. 17 (Copyright 2004, Elsevier); and (F) ref. 18 (Copyright 1978, NRC Research Press).]

Chromatin expansion could drive and direct topoisomerase-mediated changes in DNA supercoiling and
catenation/decatenation (11). Mechanical forces within chromatin/chromosomes could promote conformational changes in associated proteins or RNAs, altering structural or catalytic properties. Macromolecules that respond in this way would be molecular "stress sensors." Onset of anaphase is known to be governed by regulatory sensing of tension on centromere/kinetochore regions arising from microtubule-mediated bipolar spindle forces (19). Diverse molecules might analogously sense mechanical stresses generated internally within the chromosomes, transducing that information to chromosomal or nonchromosomal components. 

Expanded Roles for DNA and Chromosomal Molecules.

If molecular and chemical changes within chromosomes are governed by mechanical forces, the DNA
and its associated chromosomal macromolecules acquire expanded roles in relation to those forces. DNA base sequence/structure, covalent DNA modifications (e.g., methylation, which favors melting), histone modifications, architectural proteins, and chromatin remodeling activities would be key determinants of basic expansion/contraction state. Other chromosomal components, including nucleosomes, bulk chromatin structure proteins, RNAs, and proteins with specialized functions, would be constrainers, transducers, or targeters of expansional stresses and/or sensors of expansional stress or stress relief.

Intrinsic Chromatin Stress Cycling Versus the Cell Cycle Engine.

Global chromatin expansion/contraction cycling could be mediated by the cell cycle engine. Alternatively, chromosomes might have an intrinsic capacity to cycle between expanded and contracted conditions, e.g., via stress-sensitive chromatin modifying proteins. Information might then flow from the chromosomes to the cell cycle engine. Such "reverse information flow" could explain diverse powerful effects of altered histone modification on cell cycle progression and modulation of G1/S progression by chromosome structure proteins (15, 37). Intrinsic chromatin stress cycling could also explain situations where chromosome output varies cyclically irrespective of the cell cycle engine (38–40). Chromosome stress cycling might even have been the original cell cycle engine, mediated by DNA gyrase in eubacteria as a primordial cell cycle and then replaced by more complex mechanisms.

Diseases.

If mechanical stress status governs chromosome function, conditions of chromosome dysfunction should be cases of "aberrant stress status." Chromosomal molecules implicated in disease disorders could thus be assigned specific roles in modulating, constraining, sensing, or transducing mechanical stresses.

Beyond the Chromosomes.

Chromosomes are in physical contact with nuclear architectural components and the nuclear
envelope, which contact cytoplasmic and cytoskeletal components, which contact the cell surface, which contacts other cells. Information might therefore travel by direct mechanical linkage from the chromosomes outward and from external components inward to the chromosomes. Moreover, the types of effects described above for chromosomes might be involved in other cellular processes.

Conclusion:

We propose that basic chromosome function is governed by internally generated mechanical forces generated by tendencies for DNA/chromatin expansion. An attractive feature of this model is that the DNA plays a governing role not only via its information content but also via its intrinsic mechanical properties.

Acknowledgements:

We gratefully acknowledge the help of many colleagues. This research is supported by grants from the National Institutes of Health (GM44794 and GM25326, to N.K.), the Centre National de la Recherche Scientifique (UMR8126, to D.Z.); the National Science Foundation (DMR 0213805, to J.H.); and the United Kingdom–Biotechnology and Biological Sciences Research Council (6/G5097, to G.H.J.); and by European Molecular Biology Organization Fellowship ALTF 223-1998, a Medical Foundation Fellowship, and National Institutes of Health Grant HG03143 (to J.D.).

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Additional References:

1. Hovsepian JA, and Frenster JH, "Euchromatin as an Extensile Force within Mammalian Cell Nuclei".

2. Frenster JH, and Hovsepian JA, "Activator RNA Exchange during Interphase Chromatin Reprogramming".

3. Kreth G, Finsterle J, von Hase J, Cremer M, and Cremer C, "Radial Arrangement of Chromosome Territories in Human Cell Nuclei: A Computer Model Approach Based on Gene Density Indicates a Probabilistic Global Positioning Code".

4. Chambeyron S,  and Bickmore WA, "Chromatin decondensation and nuclear reorganization of the HoxB locus upon induction of transcription".

5. Parada LA, McQueen PG,  and Misteli T, "Tissue-specific spatial organization of genomes".

6. Frenster JH, and Hovsepian JA, "Ultrastructure  of Closed Loops within Euchromatin of Isolated Lymphocyte Nuclei".

Further Topics in:  Euchromatin,  active DNA, and  RNA  ribo-regulators:

Links to Euchromatin Activator RNA Reviews:
Links to Euchromatin Activator RNA Research:
Links to Ultrastructural Probes of DNase I-Sensitive Sites:
Links to RNA as a Therapeutic Agent:
Links to Hodgkin Lymphoma Immuno-Pathology:
Links to Activated T-Lymphocyte Immunotherapy:
Links to Medical Systems Biology:
Links to Selective Gene Transcription:
Links to RNA-Induced Epigenetics:
Links to RNA-Induced Embryogenesis:
Links to RNA and Biological Causality:
Links to Reprogramming and Neoplasia:

"Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA".

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