Can Transcription Factors Function As Cell–Cell Signalling Molecules?

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. Science 294, 2181–2186 (2001). and for several homeoproteins, such as developmental decisions. Many of them also 62. Hutchinson, C. Lamins: building blocks or regulators Engrailed2,3,Hoxa5, Hoxb4, Hoxc8, Emx1, function in the control of adult physiology. of gene expression. Nature Rev. Mol. Cell Biol. 3, 848–858 (2002). Emx2, Otx2 and Pax6 (G. Mainguy, A. Maizel, For example, Engrailed 1 and Engrailed 2 63. Burke, B. & Stewart, C. L. Life at the edge: the nuclear A.P.and A.J., unpublished observations). On (EN1 and EN2; collectively known as envelope and human disease. Nature Rev. Mol. Cell Biol. 3, 575–585 (2002). the basis of the conservation of the internal- Engrailed) are expressed in adult aminergic 64. Hoffmann, K. et al. 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

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. This was also shown by the internal- Pax6 (G. Mainguy, A. Maizel, A.J. and A.P., studies. The secretion sequence (in green) is part ization of a Penetratin peptide composed of unpublished observations). 12 of the nuclear export sequence (in yellow) and its D-enantiomers ,which cannot, therefore, rec- deletion blocks nuclear export. The internalization ognize a protein receptor of opposite chirality Potential secretion mechanisms. Subcellular sequence (in red) has been used as a vector to (composed of L-amino acids). Current mod- localization studies showed that, in cultured introduce cargoes into live cells and is therefore also known as Penetratin. It is important to note els of internalization involve peptide binding cells, a pool of cEN2 was associated with mem- that the secretion and internalization sequences to the cell surface through charge interactions brane fractions and, in part, was protected are distinct and that their presence within the and membrane destabilization after W48 against proteolysis17.By contrast, the non-sec- homeodomain makes it impossible to define insertion in the bilayer. Destabilization might retable variant (∆1 deletion) is accessible to pro- mutations that would block intercellular transfer assist in the formation of inverted micelles13, teases3,which indicates that there might be an without modifying transcription properties. which would allow peptides to translocate exportable pool of cEN2 (~5% of the entire across the membrane and be released into the cytoplasm. Full-length homeoproteins are encoding such information, and because also internalized by live cells14 and, when Engrailed homeoproteins can transfer between cells tested, the same mutations (for example, the in vitro,we shall focus on homeoproteins to deletion of W48 in the homeodomain) block outline our model that transcription factors the internalization of the third helix, of the D Cytosol can function as true signalling proteins. In homeodomain or of the entire protein3,15.So, NLS support of this hypothesis is the well-estab- it is assumed that the same mechanisms are lished transfer of homeoproteins in higher responsible in all cases when homeoproteins 5 NES plants ,which occurs through intercytoplas- are internalized. ? B Nucleus mic bridges known as plasmodesmata. It is, NLS ? C however, noteworthy that the intercellular Secretion of homeoproteins from cells A transfer of Knotted-1 (KN1), a maize homeo- The evidence. The concept of a ‘messenger protein, through plasmodesmata is poorly protein’ (FIG. 2) requires that transcription fac- affected by conditions that strongly affect the tors — in this case, homeoproteins — be also passage of other types of protein6,7.So there exported into the extracellular medium. This Figure 2 | Intercellular transfer of Engrailed might be other mechanisms of transfer that is despite the absence of a classical signal pep- homeoprotein. At equilibrium, Engrailed is work in parallel. tide, which is also absent from other detected in the cytosol and nucleus. In principle, this dual distribution can result from an early hydrophilic proteins — in particular, inter- nucleocytoplasmic partition of newly synthesized Homeoprotein entry into cells leukin-1 (IL-1) and mammalian fibroblast Engrailed (A) or, alternatively, from Engrailed The evidence. The finding that the 60-amino- growth factors (FGFs). In co-cultures of pri- continuous shuttling between the nucleus and acid DNA-binding domain (homeodomain) mary neurons and COS-7 cells that express cytosol owing to its nuclear import (NLS) and of the Antennapedia homeoprotein translo- chick Engrailed 2 (cEN2), cEN2 is found — export (NES) sequences (B). Our data favour the cates across biological membranes was intact — in the 100,000 g supernatant (not latter hypothesis and indicate that nuclear export 8 might comprise an important step to gain access serendipitous .But, once observed, the phe- in the pellet) of the culture medium and to the secretory compartment. So, Engrailed 3 nomenon had to be investigated further. It was within the neurons .This indicates that the transport from the nucleus to the cytoplasm then shown that Antennapedia homeodomain protein has been secreted by the COS cells (binding to transporters, dissociation from DNA) internalization properties are conferred by its into the surrounding medium, and has then could be a site of regulation for its secretion. Only third helix (now referred to as Penetratin9; been taken up by the neurons. forms of Engrailed that can be secreted are directed into the lumen of vesicular compartments (FIG. 1)) and that this region absolutely requires Interestingly, the cellular export and (C) by a mechanism that is still undefined but that the tryptophan at position 48 (W48) in a basic import pathways seem to be distinct, as is distinct from the internalization mechanism (D). environment. Exogenously added fluorescein mutating W48, which is mandatory for inter- Engrailed internalization (D) is endocytosis isothiocyanate (FITC)-labelled homeodomain nalization, does not affect cEN2 export. independent, and requires the third helix of the and Penetratin peptides can be detected in the Systematic mutagenesis has been used to homeodomain as the driving domain.

Growing axon Target hydrophilic proteins seems unlikely. In fact, in genetically well-characterized organisms (such as bacteria), six different unconventional Cytosol secretion pathways have been identified, even though they usually do not co-exist in the a Stabilization a′ same organism.

b Collapse An active role for the nucleus? Intriguingly, a vast number of secreted hydrophilic proteins Nucleus find their way to the cell nucleus (VP22, galectin-3, FGFs, thioredoxin, high-mobility group box (HMGB) proteins and homeopro- Mature axon teins) and some of them are capable of nucleocytoplasmic exchange24.This exchange might control the amount of protein that is available a a′ for secretion, as alluded to below. The nucleo- a a′ mRNA cytoplasmic distribution of FGF2 (including its 18-kDa secreted isoform) is highly regu- b lated during development25.In culture, phos- Ribosome Presynaptic axon Nascent phorylation modulates (directly or indirectly) polypeptide the nucleocytoplasmic distribution of both b FGF2 (REF.26) and thioredoxin27. FGF1 accu- Postsynaptic element mulates in the nucleus before reaching the cytoplasm28.Transfer of nuclear non-histone Figure 3 | Hypothetical signalling with homeoproteins. The example taken here is signalling between HMGB1 protein to the cytoplasm precedes its either a navigating axon (growing axon) and an intermediate target, or the pre- and postsynaptic elements lysophosphatidylcholine-induced secretion29. of a functioning synapse (mature axon). The main advantage of this model is that, in addition to a classical Both annexin II30 and Engrailed16 contain an signalling activity (for example, a neurotransmitter-induced change in ionic conductance), it also confers active nuclear export signal. positional information. Indeed, because the combination of homeoproteins expressed within a cell is a signature of its position in the organism, the cells are reciprocally informed of their topological origins. The Furthermore, secretion often correlates exchange of transcription factors between the axon (growing or mature; blue) and the target cell (green) with nuclear, rather than cytoplasmic, local- can be interpreted at the level of translation (a, a′) or transcription (b). For example, homeoprotein transfer ization. The phorbol ester phorbol 12-myris- into the growth cone might regulate (up or down) the translation (a) of a messenger encoding a receptor or tate 13-acetate (PMA) promotes both an adhesion molecule, leading to stabilization or turning away (or collapse) of the growth cone. thioredoxin secretion31 and its nuclear local- Reciprocally, homeoprotein passage from the growth cone or nerve terminal into the target cell might ization27.Similarly, amino-terminal deletion regulate the translation (a′) of messengers (for example, sub-synaptic dendritic messengers in adult abolishes galectin-3 secretion and its nuclear synapses) or the transcription of specific genes (b) after transfer into the target-cell nucleus. Indeed, it 32 cannot be precluded that, following transfer, homeoproteins show unsuspected physiological activities (for accumulation .In the case of cEN2, the fact example, activation of second messengers). Green circles represent proteins originating from the growth that an active nuclear export sequence that cone or presynaptic axon, and the red circles represent proteins travelling in the opposite direction. takes the protein out of the nucleus is necessary for cEN2 secretion might be seen as a counter example. However, the addition of an cellular pool) that exists within vesicles. These secreted hydrophilic proteins — have been extra nuclear localization signal (NLS) does vesicles have ‘caveolae-like’ properties, such as detected in exosomes by mass spectroscopy18. not impair secretion (A. Maizel and A.J., high cholesterol and glycosphingolipid con- In some cases, for example for IL-1β19 and unpublished observations) and many modifi- tents, which confer resistance to Triton and FGF2 (REF.20),protein translocation channels cations that decrease cEN2 secretion correlate separation in sucrose density gradients17.The of the multidrug resistance (MDR) family with a pronounced cytoplasmic localization. same proportion of endogenous rat EN1 and seem to be used for unconventional secretion. For example, Engrailed phosphorylation EN2 that is expressed in the embryonic mes- However, the correlation between Engrailed blocks its secretion33 and favours its accumu- encephalon is resistant to proteolysis and pre- secretion and its localization in the lumen of lation in the cytoplasm (A. Maizel and A.J., sent in these caveolae-like vesicles in vivo. vesicles3 indicates the potential existence of an unpublished observations). So, although exit Although intercellular transfer has only been unidentified mechanism that directs from the nucleus is important to gain access directly visualized in vitro,the presence of EN1 Engrailed into secretory vesicles. to the secretory compartment, a passage and EN2 in a secretion compartment in vivo Unconventional secretion does not neces- through the nucleus seems necessary, there- gives strength to the idea that this phenome- sarily require cell-type-specific machineries, fore indicating an active role for the nucleus non is of physiological relevance. and so it might reflect an ancestral mode of in the secretion of secreted hydrophilic pro- So how might these homeoprotein-con- secretion. For instance, MDR-dependent teins. Whether this role is related to the taining vesicles arise? An interesting possibil- secretion is found in organisms ranging from nuclear accumulation of some endocytic ity is that homeoproteins are incorporated bacteria to humans. Another example is the proteins34 or to the nucleus–membrane into nascent vesicles as they form; exosomes, unconventional secretion of thioredoxin that shuttling of some proteins (for example, the with their inside–out membrane orientation is observed in bacteria21 (Helicobacter pylori), scaffold proteins Ste5 (REF. 35) or regulator of (their cytosolic face is inside), are ideal candi- plants22 and vertebrates23.Taken together, a G-protein signalling 4 (REF. 36)) remains to be dates. In fact, galectin-3 and annexin II — two single unconventional pathway for all secreted investigated.

Regulation of intercellular transfer transcription factors from pathogen to host Transcription and translation. It seems evi- The data described above show that cEN2 also happens during infection by pathogenic dent that homeoproteins that have been and many other homeoproteins can pass bacteria40.Also, although it is not considered transferred from one cell to another should between cells by two original and distinct to be a transcription factor, the T-DNA-bind- regulate transcription in the nucleus of the mechanisms of secretion and internaliza- ing protein VirE2 of Agrobacterium is directly receiving cell. This is likely, but non- tion. Secretion involves a category of vesicles involved in the penetration of the host plasma autonomous target genes might differ from that contain the protein within their lumi- membrane by the T-DNA, and its subsequent autonomous ones — for example, the pro- nal compartment, and EN1 and EN2 are nuclear import41. teins might become modified during trans- found in such vesicles in vivo. Finally, the port, or they might take up a different role in sequences that are necessary for unconven- Functions in higher plants. Movement pro- tional export and import both reside within teins that undergo intercellular transfer are a VE Epi VE Epi VE cell Epi cell the homeodomain and are conserved commonplace in plants and some of these among homeoproteins. How might this proteins are transcription factors. This ? intercellular transfer be regulated? The explains why paracrine activity of transcrip- Otx2 Otx2 Otx2 AVE AVE ANE absence of chiral receptors and a mode of tion factors, including homeoproteins, has entry that is primarily based on the interaction been proposed in higher plants. In specific of the proteins with charged phospholipids cases, homeoproteins might even be used as almost certainly preclude any regulation of chaperones for protein transfer, as implied by Otx2 Otx2 intercellular transfer at the entry site. By the observation that a single mutation antag- contrast, the secretion of homeoproteins is onizes the intercellular transfer of a plant- b MHB probably highly regulated. virus movement protein and its binding to a Neural tube (E 8,5) Engrailed is a phosphoprotein in vivo and homeodomain protein42. is phosphorylated by casein kinase 2 (CK2). Because movement proteins traffic Anterior Posterior In CK2–cEN2 co-transfection experiments, through plasmodesmata, it is thought that c CK2 blocks cEN2 secretion by phosphorylat- homeoproteins use the same pathway. MHB ing a short serine-rich domain that is However, the homeodomain of the shuttling Wild-type upstream of the homeodomain33.Secretion of plant homeoprotein KN1 can be transferred proteins in which serine residues within the between animal cells (which lack plasmodes- d serine-rich domain were mutated to alanine mata), and the only mutation — located in residues was insensitive to CK2. By contrast, the homeodomain — that is known to 5 Otx2 the replacement of the same serine residues inhibit KN1 intercellular transfer in plants Gbx2 by glutamate residues — mimicking phos- also inhibits the intercellular transfer of the phorylated serine residues — produced a KN1 homeodomain between animal cells e MHB Otx1–/– Otx2+/– protein that was unable to be secreted. (M. Tasseto, A. Maizel, A.P.and A.J., unpub- Other putative modes to regulate secretion lished observations). This indicates a possi- f MHB control are the regulation of the nucleocyto- ble homology between metaphytes and En1Otx2+/– plasmic distribution or the fusion of caveo- metazoans with respect to homeoprotein lae-like vesicles with the plasma membrane. transfer. In fact, it would be curious if, even Figure 4 | A model for homeoproteins as The latter process is known to be regulated, in though animal proteins have efficient infectious proteins. a | Signalling and inductive particular, through protein kinase C activa- import and export signals, animals are activity between two layers is presented here in tion37, and therefore any mechanism regulat- unique in not using this powerful signalling the case of Otx2. Otx2 that is first expressed in ing the fusion of these vesicles with the pathway. the anterior visceral endoderm (AVE; left) is plasma membrane might also regulate homeo- transported into the anterior epiblast (Epi), where it protein exocytosis. So, homeoprotein secre- In search of paracrine functions activates Otx2 transcription (middle panels), which tion is probably a highly regulated process Most secreted hydrophilic proteins have is necessary for the formation of the anterior neural ectoderm (ANE). This auto-induction could that can only happen in specific physiological extracellular functions that involve interac- be dependent of, or amplified by, co-factors. and developmental situations. tions with plasma-membrane receptors Following its induction in the epiblast, Otx2 (these could be on the cells that secrete them, spreads by cell–cell transfer into the posterior part Transfer in other phyla as well as on different cells). However, for of the neuroepithelium (b) until it encounters a Behaviours that are indicative of transcrip- some of them, such as TAT43 and FGFs44,45, nuclear environment (such as the presence of tion-factor transfer have been observed in internalization is known to be required for Gbx2) that represses its replication. The latter model is hypothetical and supported by the several phyla. This generality might indicate them to elicit their full range of biological following observations: first, homeoproteins can that the phenomenon is important. effects. Homeoproteins, however, seem to be be secreted and internalized; second, in the wild- atypical secreted hydrophilic proteins that so type (c), the mid–hindbrain border (MHB) forms Yeast and bacterial functions. In many lower far have no characterized extracellular func- where Otx2 and Gbx2 meet; third, Gbx2 and Otx2 eukaryotes, some homeoprotein functions are tions in metazoans. It is therefore anticipated are auto-activators and reciprocal inhibitors (d); related to intercellular communication. that their paracrine targets are intracellular. fourth, downregulation of Otx expression shifts the expression of Gbx2 and the position of the MHB Following cell fusion that accompanies mat- Because homeoproteins regulate transcrip- into a more anterior position (e); and, finally, ing, the homeoprotein content of each con- tion and, in some instances, translation, these conversely forcing the expression of Otx2 in the tributing cell determines the behaviour of the two modes of action are most likely to affect En1 domain represses Gbx2 and shifts the MHB resultant diploid cell38,39.Direct transfer of paracrine action. position into a more posterior position (f).

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 4 | OCTOBER 2003 | 817 © 2003 Nature PublishingGroup PERSPECTIVES a different cellular context. But additional Tangential induction within an epithelium homeoproteins are transferred between cells functions can also be envisaged. As men- has been proposed as a patterning mecha- but when, where and why they are. tioned, homeoproteins might regulate mes- nism54.We speculate that homeoprotein tan- To test the proposed hypotheses, it will be senger RNA stability and/or translation gential transfer might be involved in border necessary to block the intercellular transfer of (FIG. 3a).Direct evidence that a homeoprotein formation within the neuroepithelium, homeoproteins in vivo without modifying can regulate translation is the repression, in thereby participating in the formation of the their intracrine activity. A main objective Drosophila,ofcaudal mRNA translation by compartments of the future brain. For exam- would be to identify and mutate sequences Bicoid46,47.The Bicoid mRNA-binding ple, the isthmus is an important embryonic that confer direct paracrine properties and to domain has been identified48 and its conser- brain structure that forms where the expres- insert the mutated gene into the normal locus vation among homeoproteins raises the pos- sion of Otx2 meets that of Gbx2 (REF.55; FIG. 4b), to produce an ‘intracrine-only’ mouse. As sibility that other homeoproteins bind RNA. and genetically decreasing Otx2 or Gbx2 lev- shown in FIG. 1, the two sequences that are Indirect evidence is that Bicoid-dependent els anteriorizes or posteriorizes (respectively) necessary for secretion and internalization repression of translation involves its bind- the position of the isthmus. The same holds are in the homeodomain. This is very inter- ing to the eukaryotic translation initiation true for Emx2 and Pax6 in the compartmen- esting with respect to homeodomain conser- factor 4E (eIF4E)49,a property that is shared talization of the cortex56,ofGsh2 and Pax6 for vation, but makes it impossible to introduce by proline-rich homeobox proteins and the dorso–ventral patterning of the telen- mutations that would not modify the roles of Hox11 (REF.50).Most importantly, the domain cephalon57,or of Engrailed and Pax6 for the these proteins as transcriptional regulators. of interaction with eIF4E is present in 200 mesencephalon–diencephalon border58.It is So, the strategy that is being used at pre- other homeoproteins50. striking that each homeoprotein in a pair acti- sent is to design mini-genes that encode Homeoproteins are known to have a role vates its own transcription and antagonizes homeoprotein-binding polypeptides and to in shaping neuronal arbors8,15,51,52.The devel- that of its ‘partner’. force their expression in the extracellular opment of the nervous system involves the In the hypothetical models outlined in FIG. 4, milieu at the appropriate developmental peri- formation of many pathways and synapses homeoproteins can be considered as ‘infec- ods. Such anti-homeoprotein agents are being with an extremely precise topology. In addi- tious entities’ — they invade the neuroep- produced and it is our hope that their expres- tion, the functioning of adult neuronal net- ithelium until they encounter a nuclear sion under the control of the appropriate reg- works requires that each synapse be informed environment that is unsuitable for their own ulatory sequences will allow us to prevent or of its position within the network. So, eco- induction, partly because of high expression retard homeoprotein transfer without inter- nomical mechanisms for position coding are of the antagonist homeoprotein (FIG. 4b).In fering with their intracrine functions. of primary importance in the developing and fact, the protein does not have to travel far — Alain Prochiantz and Alain Joliot are both at adult nervous systems. Because homeopro- just one cell — but propagates rapidly by Centre National de la Recherche Scientifique, teins contribute to positional information, inducing and amplifying its own transcrip- Unité Mixte de Recherche 8542, homeoprotein transfer would indeed be a tion. All this happens as if the genes them- Ecole Normale Supérieure, very economical way to combine signal trans- selves were diffusible, a term that evokes the 46 rue d’Ulm, 75230 Paris Cedex 05, France. e-mails: [email protected]; duction and topological information (FIG. 3). definition of morphogens by Alan Turing in [email protected] We speculate that homeoproteins might 1953 (REF.59): affect axonal navigation during development “The substances will be called mor- doi:10.1038/nrm1227 and modify synaptic properties within adult phogens, the word being intended to convey 1. Ensoli, B. et al. Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat neuronal networks by regulating transcrip- the idea of a form producer. It is not intended protein on cell growth and viral transactivation. J. Virol. tion and/or translation in the receiving com- to have any very exact meaning, but is simply 67, 277–287 (1993). 2. Prochiantz, A. Messenger proteins: homeoproteins, partment (FIG. 3).For example, the levels of the kind of substance concerned in this the- TAT and others. Curr. Opin. Cell Biol. 12, 400–406 cell-surface receptors or cell-adhesion mole- ory…The genes themselves may also be con- (2000). 3. Joliot, A. et al. Identification of a signal sequence cules might be changed, which, in turn, would sidered to be morphogens. But they form necessary for the unconventional secretion of affect axon guidance. However, we do not rather a special class. They are quite indif- Engrailed homeoprotein. Curr. 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