PERSPECTIVES 38. Hewish, D. R. & Burgoyne, L. A. Chromatin 66. Waterham, H. R. et al. Autosomal recessive manuscript. Several anonymous referees made significant substructure. The digestion of chromatin at regularly HEM/Greenberg skeletal dysplasia is caused contributions towards the improvement of this essay. The spaced sites by a nuclear deoxyribonuclease. by 3β−hydroxysterol ∆14-reductase deficiency authors dedicate this review to the memory of H. G. Davis Biochem. Biophys. Res. Comm. 52, 504–510 (1973). due to mutations in the lamin B receptor (formerly at the Department of Biophysics, King’s College, 39. Oudet, P., Gross-Bellard, M. & Chambon, P. Electron gene. Am. J. Hum. Genet. 72, 1013–1017 London), an excellent microscopist and our good friend. microscopic and biochemical evidence that chromatin (2003). structure is a repeating unit. Cell 4, 281–300 (1975). 67. Grandville, J. J. Un Autre Monde (H. Fournier, Paris, 40. Wolffe, A. Chromatin Structure and Function 1844). Online links (Academic Press, San Diego, 1998). 68. DuPraw, E. J. Quantitative constraints in the 41. Kornberg, R. & Lorch, Y. Twenty-five years of the arrangement of human DNA. Cold Spring Harb. DATABASES nucleosome, fundamental particle of the eukaryotic Symp. Quant. Biol. 38, 87–98 (1974). The following terms in this article are linked online to: chromosome. Cell 98, 285–294 (1999). LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ 42. Thomas, G. J., Prescott, B. & Olins, D. E. Secondary Acknowledgements H2A | H2B | H3 | H4 | HMGN1 | HMGN2 | lamin A | lamin B1 | structure of histones and DNA in chromatin. Science The authors express their gratitude to Bowdoin College and lamin B2 | LBR 197, 385–388 (1977). to the German Cancer Research Center (Heidelberg) for pro- OMIM: http://www.ncbi.nlm.nih.gov/Omim/ 43. Mardian, J. K., Paton, A. E., Bunick, G. J. & Olins, D. E. viding stimulating intellectual and scientific environments. H. Emery–Dreifuss muscular dystrophy | Nucleosome cores have two specific binding sites for Herrmann and P. Lichter, our generous hosts at the German Pelger–Huët anomaly nonhistone chromosomal proteins HMG 14 and HMG Cancer Research Center, supplied helpful comments on the Access to this interactive links box is free online. 17. Science 209, 1534–1536 (1980). 44. Sandeen, G., Wood, W. I. & Felsenfeld, G. The interaction of high mobility proteins HMG14 and 17 with nucleosomes. Nucl. Acids Res. 8, 3757–3778 (1980). 45. Olins, D. E. et al. Electron microscope tomography: transcription in three dimensions. Science 220, 498–500 (1983). OPINION 46. Olins, A. L., Olins, D. E. & Bazett-Jones, D. P. Balbiani ring hnRNP substructure visualized by selective staining and electron spectroscopic imaging. J. Cell Biol. 117, 483–491 (1992). 47. Olins, D. E. & Olins, A. L. The replication band of Can transcription factors function as ciliated protozoa. Int. Rev. Cytol. 153, 137–170 (1994). 48. Richmond, T. J., Finch, J. T., Rushton, B., Rhodes, D. & Klug, A. Structure of the nucleosome core particle at cell–cell signalling molecules? 7 Å resolution. Nature 311, 532–537 (1984). 49. Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F. & Richmond, T. J. Crystal structure of the nucleosome core particle at 2.8 Å resolution. Nature 389, 251–260 (1997). Alain Prochiantz and Alain Joliot 50. Uberbacher, E. C. & Bunick, G. J. X-ray structure of the nucleosome core particle. J. Biomol. Struct. Dyn. Recent data support the view that covered in the fly on the basis of mutations 2, 1033–1055 (1985). 51. Harp, J. M., Hanson, B. L., Tim, D. E. & Bunick, G. J. transcription factors — in particular, that affect the spatial identity of segments and Asymmetries in the nucleosome core particle at 2.5 Å homeoproteins — can be transferred from appendages (for example, antennae can be resolution. Acta Crystallogr. (Section D Biol. Crystallogr.) 56, 1513–1534 (2000). cell to cell and have direct non-cell- transformed into legs). Within a single struc- 52. Harp, J. M., Hanson, B. L. & Bunick, G. J. The autonomous (and therefore paracrine) ture, such as the spinal cord, specific combi- Structure of the Nucleosome Core Particle (Elsevier Science B. V., Amsterdam) (in the press). activities. This intercellular transfer, based nations and concentrations of homeoproteins 53. Arents, G., Burlingame, R. W., Wang, B. C., Love, W. E. on atypical internalization and secretion, has define the anterior–posterior and dorso–ven- & Moudrianakis, E. N. The nucleosomal core histone octamer at 3.1 Å resolution: a tripartite protein important biotechnological consequences. tral positions of cells. Furthermore, the assembly and a left-handed superhelix. Proc. Natl But the real excitement stems from the homeoprotein Engrailed can define the mid- Acad. Sci. USA 88, 10148–10152 (1991). 54. Richmond, T. J. & Davey, C. A. The structure of DNA physiological and developmental brain and the position of cells within the in the nucleosome core. Nature 423, 145–150 implications of this mode of signal anterior–posterior axis of the midbrain. It is (2003). 55. Woodcock, C. L. F. & Dimitrov, S. Higher-order transduction. widely thought that homeoprotein function structure of chromatin and chromosomes. Curr. Opin. involves the regulation of genes that encode Genet. Dev. 11, 130–135 (2001). 56. Horn, P. J. & Peterson, C. L. Chromatin higher order Transcription factors are present in the signalling molecules such as surface receptors folding: wrapping up transcription. Science 297, nucleus, and sometimes in the cytoplasm, but or growth factors. By contrast, direct 1824–1827 (2002). 57. de la Serna, I. L. & Imbalzano, A. N. Unfolding on the whole they are not thought to travel paracrine homeoprotein activity is not gener- heterochromatin for replication. Nature Genet. 32, between cells. This is because of their ally envisaged, although in theory it repre- 560–562 (2002). 58. Turner, B. M. Decoding the nucleosome. Cell 75, 5–8 hydrophilic properties and the absence of a sents a parsimonious way for neighbouring (1993). signal peptide. But there are exceptions and, cells to coordinate positional information. So 59. Jenuwein, T. & Allis, C. D. Translating the histone code. Science 293, 1074–1080 (2001). in fact, some transcription factors travel the ability of homeoproteins to transfer 60. Fry, C. J. & Peterson, C. L. Unlocking the gates to between cells because they contain protein between cells is extremely exciting. There are gene expression. Science 295, 1847–1848 (2002). 61. Heun, P., Laroche, T., Shimada, K., Furrer, P. & domains that allow them to do so. This is the more than 400 of these proteins in mice and Gasser, S. M. Chromosome dynamics in the yeast case for the HIV transcription factor TAT1 humans, and they are involved in all the main interphase nucleus. 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Mutations in the gene encoding the ization and secretion signals that have been nuclei that control motor behaviour, mood lamin B receptor produce an altered nuclear 4 morphology in granulocytes (Pelger–Huët anomaly). identified in Engrailed (see below), it is antici- and addiction . Nature Genet. 31, 410–414 (2002). pated that this property is shared by most Because the transfer of positional infor- 65. Shultz, L. D. et al. Mutations at the mouse ichthyosis locus are within the lamin B receptor gene: a single homeoproteins. mation is a general phenomenon that occurs gene model for human Pelger–Huët anomaly. Hum. Homeoproteins are known to contribute during development and throughout adult- Mol. Genet. 12, 61–69 (2003). to cellular positioning. They were actually dis- hood, because homeoproteins contribute to 814 | OCTOBER 2003 | VOLUME 4 www.nature.com/reviews/molcellbio © 2003 Nature PublishingGroup PERSPECTIVES nucleus of live cells. Internalization is inhibited identify a short sequence (∆1 sequence; FIG. 1) Homeodomain by the replacement of W48 by a phenylalanine. that is necessary for secretion. This sequence, Secretion sequence This, and the visualization of internalized which spans part of helices 2 and 3 of the AQELGLNESQ homeodomains and homeoproteins in non- homeodomain, is also required for efficient fixed cells8,precludes internalization being a cEN2 nuclear export16,which indicates that post-fixation artefact (as has recently been sug- secreted cEN2 might originate from the gested for several basic peptides and pro- nucleus (FIG. 1;see below). As already men- Internalization sequence teins10). The property of internalization into tioned, the homeodomain has a highly (Penetratin) RQIKIWFQNRRMKWKK live cells is shared by all homeodomains that conserved structure and this nuclear Nuclear export sequence have been tested, probably because the third export/secretion domain is also highly con- QSLAQELGLNESQIKI helix is highly conserved. served, indicating that intercellular transfer might be a conserved property of homeopro- Figure 1 | Functional domains for Potential entry mechanisms. Biophysical data teins. Accordingly, intercellular passage has homeoprotein intercellular transfer. Within the indicate that Penetratin fully translocates into been confirmed for almost all homeoproteins homeodomain, three domains that are required artificial lipid vesicles11,which confirms that that have so far been tested, including Hoxa5, for secretion, internalization and nuclear export have been characterized by loss-of-function chiral receptors are not required for internal- Hoxb4, Hoxc8, cEN2, Emx1, Emx2, Otx2 and (deletion) or gain-of-function (synthetic peptides) ization.