- Email: [email protected]
HOX gene activation by retinoic acid
[]~EVIEWS 31 Graziadei, P.P. and Monti-Graziadei, G.A. (1978) in Neural Plasticity (Cotman, C.W., ed.), pp. 131-153,
39 Kelly, F. and Condamine, H. (1982) Biochim. Biophys. Acta 651, 105-141
Raven Press 32 Ingraham, H.A. etal. (1990) Annu. Rev. Physiol. 52, 773-791 33 Li, S. et al. (1990) Nature 347, 528-533 34 Sch61er, H.R. et al. (1989) EMBO,L 8, 2551-2557 35 Sch61er, H.R., Ciesiolka, T. and Gruss, P. (1991) Ce1166, 291-304 36 Fibi, M. et al. (1988) Development 102, 349-359 37 Murphy, S.P. el al. (1988) Proc. Natl Acad. Sci. USA 85, 5587-5591 38 Curatola, A.M. and Basilico, C. (1990) Mol. Cell. BioL 10, 2475-2484
40 Rosner, M.H., De Santo, R.J., Arnheiter, H. and Staudt, L.M. (1991) Cel164, 1103-1110 41 Verrijzer, C.P., Kal, A.J. and van der VileR, P.C. (1990) ~ B O J . 9, 1883-1888 42 Schreiher, E. et al. (1990) Nucleic Acids Res. 18, 5495-5503
I t is now well established that genes containing a h o m e o b o x are regulatory genes coding for nuclear proteins that probably act as transcription factors 1. The h o m e o d o m a i n encoded by the h o m e o b o x is simply a protein domain capable of recognizing and binding to specific DNA sequences 2. Through the recognition properties of their homeodomain, homeoproteins are believed to regulate the expression of batteries of target genes. Among h o m e o b o x genes of vertebrates, those belonging to the Hox family show a number of interesting characteristics. First, they occur in clusters that have been confined in compact genomic regions since the remote evolutionary past. The first evidence for such clustering came from the study of Drosophila homeotic genes 3, located in two genomic regions called the Antennapedia (ANT-C) and Bithorax (BX-C) complexes. Subsequently, evidence has accumulated that these two complexes present in flies may have arisen from a single complex, called HOM-C, early in insect radiation s. The ancestral cluster corresponding to the HOM-C of insects has undergone duplications in the lineage leading to vertebrates ~',7. Higher vertebrates have four such homologous clusters, termed HOX loci in humans ~ and Hox loci in the mouse 9. Within these clusters, all Hox genes share the same transcriptional orientation 8. Hox genes were originally identified on the basis of the primary sequence of their homeodomains m. In fact, most Hox genes encode proteins containing an homeodomain closely related to the archetypal Antennapedia (Antp) homeodomain, often referred to as a class I homeodomain. However, since the resemblance is very slight for Hox genes at the ends of the four clusters (Fig. 1), it is more convenient to define Hox genes on the basis both of the primary sequence of the encoded products and of their physical location on chromosomes. Second, Hox genes appear to cooperate in providing positional information along the anteroposterior axis 9 of animals with bilateral symmetry, and perhaps even of all metazoans. This was originally recognized in flies, where homeotic genes specify the fate of segmental units within the metameric body plan3. Evidence is accumulating that they may provide positional information along other axes as well, for example in developing limb buds 11.
H.R. SCHOLER IS IN THE MAX-PLANCg INSTITUTE OFI BIOPHYSICAL CHEMISTRY,DEPARTMENTOF MOLECULARCELLI BIOLOGY, 1)-3400 GOTTINGEN, FRG; PRESENT ADDRESX" EMBL MEYERHOFSTRASSEI, D-6900 HEIDELBERG,FRG.
HOXgeneactivation by retin0ic acid EDOARDO BONCINELLI,ANTONIO S1MEONE, DARIO ACAMPORAAND FULVIOMAVIHO Vertebrate homeohox genes of the Hox family are, like Drosophila homeotic genes, organized in gene clusters and show a strict correspondence, or coUinearity, between the order of the genes (3' to 5') within the chromosomal cluster and that of their expression domains (anterior to posterior) in the embryo. Recent data obtained from embryonal carcinoma cells induced to differentiate by retinoic acid cast some light on the molecular mechanisms underlying the collinear expression of the Hox genes. Third, the genomic organization of Hox genes is reflected in their expression pattern. All the Hox genes are developmentally regulated in mouse 9, frog 12 and human 13 embryos and are expressed in partially overlapping domains. The anterior boundaries of the expression domains of the various genes of a given cluster uniformly follow a 5'-posterior/Y-anterior role along the embryonic anteroposterior body axis 1~,1s (reviewed in Ref. 4). This phenomenon was first observed for the homeotic genes in the BX-C of Drosophila and has been termed collinearity 3. A similar collinear expression pattern seems to apply to the genes of the four human HOX loci in differentiating embryonal carcinoma (EC) cells.
Human HOX gene organization So far, 38 h u m a n H O X g e n e s have b e e n identified 16. T h e y are distributed a m o n g four h o m o l o gous c h r o m o s o m a l loci, H O X I to HOX4, on chrom o s o m e s 7, 17, 12 a n d 2, respectively. Each cluster contains at least nine g e n e s organized in a h o m o l o gous linear arrangement. The 38 genes can be
Nomenclature: Hox: Vertebrate homeobox genes (species not defined). Hox: Mot,se homeobox genes. HOX: Human homeobox genes.
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expression in embryonic tissues has been complemented by studies in ELm differentiating embryonal carcinoma S---VT ....... V-E--VIN HOXIJ GRKK-VP--K V-LK---R-Y AT-KFI-KDK -RR-SATTNS---VT ....... V-E--WS HOX3G GRKK-VP--K V-LK ..... Y AASKFI-KEK -RR-SATTNKs~(EC) cells, both in mouse (reviewed KLKDT S---VT ....... V-D--IVS HOX4I GRKR-VP--K L-LK---N-Y AI-KFINKDK -RR-SA-TNin Ref. 9) and humans. These cells RE~AL SDQ-V .......... K-RLLL HOX3F SRK~-KP-SK L-LA---G-LV-EFI--Q-R-LSDB-NRE{~L provide a convenient model for the SDQ-V .......... K-RWL HOX4H ARKK-KP--K Q-IA---N-LV-EFIN-QK -K-LSNR-NDR~Y study of the molecular basis of -D--V .......... E--I-R HOXII TRKK-CP--K --IR---R-F-SV-INKEK -LQLSRM-N-D--V .......... E--LSR DRILY HOX3H TRKK-CP-SK F-IR---R-F--V-INKEK -LQLSRM-Ndevelopmental gene regulation, in -D--V .......... E - - L - ~ % DRLQ~ Hox4g SRKK-CP--K --IR---R-F--V-INKEK -LQLSRM-NENRIR response to specific stimuli. -D--V .......... L--M-R HOXIH GRKK-CP--K H ......... L--M .... E- -L--SRSVH-D--V .......... L--M-R HOX31 GRKK-CP--K H ......... L--M .... E- -L--SETINE~R~R The expression of human HOX -D--V .......... L--MSR ENRIR HOX4D GRKK-CP--K H ......... L--M .... E- -L--SRSVNDR~D genes has been extensively studied .... V .......... M--I-HOXIG TRKK-CP--K H ......... L--M .... D- -Y-V-RL-NS---V .......... M--M-HOX2E SREK-CP--K .......... L--M .... D- -H-V-RL-NEQ-~E in the human EC cell line NT2/D1 -- -V .......... M--M-E-TOE HOX3B TRRK-CP--K .......... L--M .... D- -Y-V-RV-NE-CPE (Refs 16, 22). Cells of this line may .... V .......... M--MSHOX4C TRKK-CP--K .......... L--M .... D- -Y-V-RI-NK o ~ be induced to differentiate when . . . . V . . . . . . . . . . . . . . . N HOX2D -R ...... S ........... L--P .... K .... VS---G.... V ............... N HOX3A -RS ..... S ........... L--P .... E .... VS---GKDKF-EDKL- cultured in a medium containing .... V ............... N HOX~E -R ...... S- F ......... L--P .... K .... VS---Aretinoic acid (RA). Differentiation is HOXIA ........................................................... H- DE-PI HOX2C ..................... Y ........................................ A-PG characterized by the appearance of GR ................................. N ......................... LINST HOXIB several cell types, including neurons, GR ................... Y ..................................... S- LLSAS HOX2B HOX3C -R .... I-S .......................... N ....................... S N LTSTL 7-28 days after induction. We will L-SMS discuss the results with reference to HOXIC G--A-TA ................................. S ................. D-L-SMS HOX2A G--A-TA ................................. S ................. D-M-S~E the scheme in Fig. 2. HOX3D G--S-TS ............................ NN--N ................. DSL P N T K (1) Northern blot analysis reveals HOXID P--S-TA--Q-V ....................... T--S---V ............. DHHOX2F P--S-TA --- Q-V ........ Y ......... V ........ S ................. DHLPNrE that none of the 38 HOX genes is LPNTK HOX3E P--S-AA--Q-V ........ Y .............. S--S ................ DHR HOX4B P--S-TA--Q-V ....................... T--S ................. DHLPNTK significantly expressed in untreated MOXIE S--R-TA--S A-LV ............ V-P-V-M-NL-N ................ Y--DQG--~L NT2/D1 stem cells. Whereas 24 of A--LA HOX2G S--A-TA--S A-LV ............ C-P-V-M-NL-NS .............. Y--DQA-A*L these genes are activated within a HOX4A S--V-TA--S A-LV ............ C-P-V-M-NL-N ............... Y--DQC-E~Q week in differentiating cells treated HOXIK SR-L-TA--N T-L .......... K--C-P-V---AL-D ..... V-V ........ H-RQTQ KOX2H AB-L-TA--N T-L .......... K--C-P-V---AL-D ..... V-V ........ R-RQTQ ~REPwith 10- s M RA, the expression of HOXIF PNAV-TNF-T K-LT ......... K .... A- -V---AS-QN-T-V .......... Q--REEGLLD 14 HOX genes remains undetectable EG-RV HOX21 PSGL-TNF-T R-LT ......... K--S-A-V~--AT-E - N-T-V .......... Q--RER HOX4G SSAI-TNFST K-LT ......... K .... A ...... NC-HNDT-V .......... Q--RER EGLLA even after this treatment. These 14 genes are located at the 5'-end FIGB of their loci: four in HOX1, five List of human HOX homeodomains. The one-letter amino acid code is used. in HOX3 and five in HOX4. The Sequences are compared with a consensus homeodomain represented by the boundary between responding and Antennapedia homeodomain (top line). Dashes indicate amino acid identity with the nonresponding genes roughly corconsensus. Five amino acid residues followingthe homeodomain are also shown. responds to the HOX genes of ]he homeodomains can be grouped into 13 subfamilies (separated by line spaces). group 5. (2) The expression pattern of the nine HOX2 genes was further studied in detail n. This divided into 13 subfamilies, primarily on the basis locus contains only responding genes belonging to of the peptide sequence of the encoded homeoproreins (Fig. 1). Genes belonging to the same subfamily groups 5-13 (Fig. 2). HOX2 genes located in the 3' half of the cluster are induced at peak levels by 10-8 M o c c u p y corresponding positions in the four loci that can be easily aligned with the fly complexes s,16 RA, whereas a concentration of 10-s M RA is required (Fig. 2). The hypothesis that human and mouse Hox to activate 5' genes fully. The time-course of individual loci are true homologues of the insect homeotic gene expression was monitored by RNase protection assays at RA concentrations that activate all the genes gene complexes 4 has recently received further sup(10-5 M) or only the 3' genes of the cluster (10 -7 M). At port from a series of gene swap experiments 10-5 M RA, HOX2H and HOX21 were rapidly induced, (reviewed in Ref. 17). The structural similarities between Hox genes in vertebrates and homeotic while the other genes were activated sequentially in the 3' to 5' direction from HOX2G to HOX2E (Fig. 3). At genes in insects seem to translate into similar regu10-7 M RA, only the five 3'-most genes, HOX2I to latory specificities. The evolutionary implications of HOX2A, were induced, with slower kinetics but in the such a conserved genomic organization appear to same order. be even more intriguing, as we have recently ident(3) This analysis has been extended 16 to all 38 ified two human homologues of the fly evenHOX genes (Fig. 3) in NT2/D1 cells and in a second skipped (eve) segmentation gene at the 5'-end of the EC line, Tera-2/clone 13. The general pattern of seHOX1 and HOX4 loci, albeit in the opposite orienquential activation of responding genes and the separtation to the HOX genes (Ref. 18 and E. Boncinelli ation of two collinear sets of responding and nonet aL, unpublished) (Fig. 2). These genes, termed responding genes was observed in both EC cell lines. EVX1 and EVX2, respectively, are homologous to In all four HOX loci, responding genes are sequentially Evx 1 and Evx 2 identified in the mousem. activated by 10-5 M RA in a 3' to 5' order. For the nonresponding genes at the 5'-end of the HOX1, HOX3 HOX gene expression in EC cells Since the pioneering studies of Gruss and collab- and HOX4 loci, the more sensitive RNase protection assay introduced a further distinction between (i) truly orators 2° (see also Ref. 21), analysis of Hox gene 10 20 30 40 50 60 RKRGROTYTR YOTLVIVRVV HFNRYLTRRR R I g I A M A L C L TVROIKTWVO NRRMRWEERNK TRGEP
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FIG[] Schematic representation of 40 homeodomains (circles) in the four human HOX clusters. Genes in any given cluster have been mapped on overlapping genomic clones and shown to be linked. The transcriptional orientation of all 38 HOX genes is from left to right. This alignment identifies 13 Hox homeodomain groups, indicated at the bottom. Solid dots indicate homeodomains predicted in the scheme but so far unidentified and probably missing. Below the circles are the names of known murine Hox homologues. Homeodomains from Drosophila BX-C and ANT-C genes are indicated above the scheme (abd-A, abdominal-A; Ubx, Ultrabithoro~, Scr, Sex combs reduced; zen, zerknf~llt; Abd-B, Abdominal-B; Dfd, Deformed; pb, proboscipedia; lab, labial). Each fly homeodomain is placed above the group of human homeodomains to which it is most closely related in sequence. The correspondence of fly homeodomains in parentheses is unclear. Homology groups 5 and 10 are highlighted. Hatching reflects HOX gene activation in NT2/D1 cells induced to differentiate by 10-~ M RA. Ascending hatching (e.g. HOX1A) indicates genes activated with time upon RA addition; descending hatching (e.g. HOX3G) represents downregulation. HOX4C is constitutively expressed at a very low level. Open circles indicate genes whose expression is always undetectable in these cells. The scheme also includes the EVX1and EVX2 homeodomains; the transcriptional orientation of the corresponding genes is from right to left, opposite to that of all HOXgenes. silent genes, belonging to groups 1-4 in HOX1 and 2-5 in HOX3, and (ii) genes expressed at low levels in uninduced stem cells and downregulated upon RA induction, such as HOX3G and those belonging to groups 1-4 in the HOX4 cluster (Fig. 2). HOX4C represents a separate class as it is expressed at a very low level throughout. In conclusion, human HOX genes are differentially activated by RA in EC cells according to their location within the four clusters, in a time- and RA concentration-dependent fashion. This order corresponds to the expression patterns in developing axial systems such as the axis skeleton and the central nervous system of humans and mice, where 3' genes are expressed more rostratly and 5' genes more caudally 923. The observed differential response is not a peculiarity of a single EC cell line but rather reflects a phenomenon of general physiological relevance. Similar, but not identical, results have been reported for several Hox genes in the murine F9 EC line 9,24.
Identification of subsets of HOX genes These data suggest a threefold subdivision of the four human HOX loci, with the two borders roughly corresponding to groups 5 and 10 (Fig. 2). The boundaries between RA-responding and nonresponding genes are approximately at the level of group 5. The genes of the first five groups can also be discriminated from those of the other groups located downstream on the basis of other independent lines of evidence. (1) All the genes of the first five groups correspond to only one Drosophila gene (Abdominal-B), albeit with varying degrees of homologys,n. (2) These genes lack the conserved YPWM homeopeptide 25 present just upstream from the homeodomain in the products of all other HOX genes. (3) The HOX2 locus apparently does not contain genes corresponding to the first four groups. (4) Genes belonging to the first five groups in HOX1 and HOX3 appear to be coordinately switched on and off, respectively, in blocks during differentiation of blood cells committed to the myelomonocytic lineage 26.
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Expression of HOX genes in differentiating NT2/D1 cells. The graphs show the changes in RNA levels for responding and downregulated HOX genes; 100% represents plateau RNA levels, which are comparable for different genes although they may be reached at different times. A second boundary may be drawn approximately at the level of the four genes belonging to group 10. Even at high RA concentrations, HOX2 genes of groups 5-9 are not activated in Tera-2/clone 13 cells 16. In the mouse Hox 2 complex this boundary separates genes expressed in the embryonic hindbrain from those expressed more caudally, within the spinal cord (Refs 13, 23; reviewed in Ref. 27). Published evidence and preliminary data suggest that the situation may be similar for the 3' genes of H o x I and Hox 4 as well. Anterior expression boundaries of genes of the five 3'-most groups are almost coincident, at variance with what has been observed for Hox genes of other groups9. It is not clear which H O X gene of groups 6-9 corresponds to which fly homeotic gene (Fig. 2). These genes may derive from independent intralocus gene duplication events in the two evolutionary lineages.
Implicationsfor the studyof development These data obtained in EC cells in vitro may provide clues to a better understanding of the establishment and maintenance of positional information in embryos. RA levels regulated in space and/or time may at least partly underlie a differential activation of Hox genes in vivo that is consistent with the observed region-restricted expression pattern. Indeed, RA has been implicated as a natural morphogen in chicken development, where it contributes to the specification of the limb anteroposterior axis (Refs 28, 29; reviewed TIG
in Ref. 30) and possibly in frogs, where alteration of embryonic RA levels dramatically affects specific structures of the developing central nervous system3L Differential exposure to the morphogen may determine which genes are to be locally activated in cells of different embryonic regions, thus providing positional information for the developing embryo. Many potential embryonic sources of RA have been identified and a role for RA in connection with homeobox gene function in diverse aspects of vertebrate development has repeatedly been considered30. In this light, the posterior transformation of the central nervous system in frog embryos treated with RA could be interpreted as a phenomenon correlated with overexpression and/or ectopic expression of Hox genes. The observed respecification implies a considerable reduction in the size of the forebrain and midbrain, where Hox genes are normally not expressed, with a concomitant expansion of the spinal cord and hindbrain, where they are expressed during normal embryogenesis. Circumstantial evidence exists in the literature TM that the equation 3'-anterior-early/5'-posterior-late may also be valid for the expression of Hox genes in embryogenesis. In the developing mouse limb, 3' H o x 4 genes are expressed earlier and more proximally and 5' genes are expressed later and more distally 11. Moreover, a recent extensive analysis of the expression of these genes in chick wing development led to the conclusion that cells cannot express upstream
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FIGI~I Transcriptional organization of a region of the human HOX3 locus. Transcripts containing one of the three homeoboxes (solid boxes), HOX3C HOX3D or HOX3E, may derive from a master promoter (MP) or from one of three individual proximal promoters (IP1-3). members of this cluster without first expressing downstream members32. This temporal sequence of expression could in turn account for the lack of real homeotic transformations noted in vertebrates. Establishment versus maintenance The molecular mechanisms underlying the differential activation of HOX genes seen in EC cells have been further investigated because there have been controversial reports in the literature I6. The RNA accumulation seen in differentiating NT2/D1 cells appears to be regulated primarily at the transcriptional level, as revealed both by run-on transcription assays on isolated nuclei and comparison of nuclear and cytoplasmic RNA levels during induction. Induction of the early responding genes does not require de novo protein synthesis. Moreover, treatment with inhibitors of protein synthesis alone caused a rapid appearance of RNA transcripts from most responding HOX genes, with loss of the 3' to 5' pattern of sequential activation, while leaving the expression levels of silent and downregulated genes unaffected. Synthesis of short half-life proteins is apparently required to maintain the timing and polarity of expression of responding HOX genes in these cells. The complex control network operating on HOX gene clusters may include active repression, and activation of 5' responding HOX genes in vivo could involve downregulation of these repressor proteins. In vitro, this programmed downregulation may be mimicked by cycloheximide treatment.
If the observed HOX gene activation takes place at the transcriptional level, the promoters implicated in this activation need to be identified, This is not simple because expression of Hox genes appears to originate from a multiplicity of promoters (Refs 33, 34; reviewed in Ref. 12). Most of them are individual proximal promoters, located upstream from the coding region of every Hox gene. Additional promoters, called master promoters ~2 or major upstream promoters 33, have been shown to be active at least in Hox 3. In certain human embryonic and adult tissues, transcription from a major upstream promoter yields an RNA containing at least three contiguous HOX3 homeoboxes (HOX3C, 3D and 3E)33 (Fig. 4). At least two mature mRNAs contain the HOX3C homeobox: one derived from its individual proximal promoter and the other obtained by alternative splicing of the primary transcript originating from the master promoter. Exactly the same situation applies to mRNAs containing the HOX3D or the HOX3E homeobox. Indeed, the two mRNAs containing the HOX3C homeobox have been shown to encode two different proteins sharing a carboxy-terminal domain of 153 residues including the homeodomain. The homeoprotein encoded by the mRNA from the proximal promoter (mRNA 2 in Fig. 4) has an extra 82 amino acid domain at the amino terminus 3334. Both northern blot analysis and RNase protection assays revealed that this HOX3 major upstream promoter is not induced in differentiating NT2/D1 cells 16. The HOX3C, 3D and 3E mRNAs induced in these cells
TIG OCTOBER1991 VOL.7 NO. 10
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[~EVIEWS by RA are transcribed from individual promoters proximal to the corresponding coding regions. If RA-treated EC cells actually mimic the first appearance of H O X gene transcripts in development, we conclude that H O X 3 genes are first expressed as transcripts starting from the individual proximal promoters. Results of experiments studying expression of Hox genes as transgenes agree with this conclusion. An individual H o x gene, together with its proximal promoter, introduced into a transgenic mouse can reproduce the early expression pattern, but fails to duplicate properly all the spatial and temporal changes in pattern at later stages of development35.36. Reconstruction of both the early and late pattern probably depends on maintaining the clustered organization. It is conceivable that alternative or additional biological stimuli, such as specific peptide growth factors or cell-cell signals, are required for the activation of the HOX3 master promoter in EC cells. Another possible interpretation of these data is that RA treatment does not initiate HOX gene expression in EC cells but perturbs a previous pattern of expression. Indeed, it has been shown that HOX21, 2H, 1F and some HOX4 genes are already expressed in these cells. In addition, 5' genes such as HOX2E are actively repressed in NT2/D1 stem cells. Finally, these cells also differentiate in response to other drugs, but only RA activates H O X genes. Fly HOM genes are regulated in two different ways in the establishment phase: when their transcripts appear for the first time in embryogenesis, and in the maintenance phase, when their expression pattern established in a particular body region is clonally transmitted from cell generation to cell generation 4. The gene products required to activate the genes for the first time are no longer available in the second phase and a mechanism of mutual regulation of the HOM genes themselves probably takes over. It has been suggested that vertebrate homeobox genes also go through two phases of establishment and maintenance (reviewed in Ref. 37). There is also some debate about whether the genomic organization of Hox genes is so highly conserved because it plays a major role in the maintenance phase of the expression of these genes or in the establishment phase, or in both n37. Hence, the question of the last two paragraphs becomes: are individual proximal promoters active in the establishment phase, while the master promoters are turned on subsequently, or is the reverse true? Several examples have been reported of RA addition in vivo changing the pattern of developing structures, including regenerating urodele limbs (reviewed in Ref. 38). It is conceivable that this effect is mediated, at least in part, through a disruption of the Hox gene expression pattern already established in a given developing structure. One might further suggest that this disruption results from the activation of some or all individual proximal promoters in the Hox loci. In conclusion, the analysis of many different biological systems supports the idea that homeoproteins are involved in early patterning events through the translation of primary morphogenetic signals into a complex network of positional information.
Acknowledgements We thank Robb Kmmlauf and Vincenzo Nigro for helpful suggestions. The experimental work was supported by grants from Progetti Finalizzati CNR 'Biotecnologia e Biostrumentazione' and 'Ingegneria Genetica', the Third AIDS Project of the Ministero della Sanita and the Italian Association for Cancer Research (MRC).
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Levine,M. and Hoey, T. (1988) Cell 55,537-540 Gehring, W.J. et al. (1990) Trends Genet. 6, 323--329 Lewis, E.B. (1978) Nature 276, 565-570 Akam, M. (1989) Cell 57, 347-349 Stuart, J.J., Brown, S.J., Beeman, R.W. and Denell, R.E. (1991) Nature 350, 72-74 Hart, C.P., Fainsod, A. and Ruddle, F.H. (1987) Genomics 1, 182-195 Kappen, C., Schughart, K. and Ruddle, F.H. (1989) Proc. Natl Acad. Sci. USA 86, 5459-5463 Acampora, D. etal. (1989) Nucleic Acids Res. 17, 10385-10402 Kessel, M. and Gruss, P. (1990) Science 249, 374-379 Scott, M.P., Tamkun, J.W. and Hartzell, G.W., III (1989) Biochim. Biophys. Acta 989, 25-48 Doll6, P. et al. (1989) Nature 342, 767-772 Wright, C.V.E., Cho, K.W.Y., Oliver, G. and De Robertis, EM. (1989) Trends Biochem Sci. 14, 52-56 Giampaolo, A. et al. (1989) Differentiation 40, 191-197 Duboule, D. and Doll6, P. (1989) EMBOJ. 8, 1497-1505 Graham, A., Papalopulu, N. and Krumlauf, R. (1989) Cell 57, 367-378 Simeone, A. etal. (1991) Mech. Dev. 33, 215-228 Akam, M. (1991) Nature 349, 282 D'Esposito, M. et al. (1991) Genomics 10, 43-50 Bastian, H. and Gross, P. (1990) EMBOJ. 9, 1839--1852 Colberg-Poley, A.M., Voss, S.D., Chowdhury, K. and Gruss, P. (1985) Nature 314, 713-718 Hauser, C.A. etal. (1985) Cell43, 19-28 Simeone, A. et al. (1990) Nature 346, 763-766 Wilkinson, D.G. et al. (1989) Nature 341,405--409 Papalopulu, N. et al. (1990) in Advances in Neural Regeneration Research (Sell, F.J., ed.), pp. 291-307, Alan Liss Mavilio, F. et al. (1986) Nature 324, 664-668 Magli, C. et al. Proc. Natl Acad. Sci. USA (in press) Wilkinson, D.G. and Kmmlauf, R. (1990) Trends NeuroscL 13, 335-339 Thaller, C. and Eichele, G. (1987) Nature 327, 625-628 Tickle, C., Alberts, B., Wolpert, L. and Lee, J. (1982) Nature 296, 564-566 Brockes, J.P. (1989) Neuron 2, 1285-1294 Durston, A.J. etal. (1989) Nature340, 140-144 Izpisfia-Belmonte, J-C. et al. (1991) Nature 350, 585-589 Simeone, A. et al. (1988) Nucleic Acids Res. 16, 5379-5387 Cho, K.W.Y. et al. (1988) EMBOJ. 7, 2139-2149 Ptischel, A.W., Bailing, R. and Gross, P. (1990) Development 108, 435-442 Bieberich, C.J., Utset, M.F., Awgulewitsch, A. and Ruddle, F.H. (1990) Proc. Nail Acad. Sci. USA 87, 8462-8466 Gaunt, S.J and Singh, P.B. (1990) Trends Genet. 6, 208-212 Brockes, J.P. (1991) Nature350, 15
E. BONCINELLI, A. ~IMEONE AND D. ACAMPORA ARE IN THE INTERNATIONAL INSTITUTE OF GENETICSAND BIOPHYSIC& C N ~ VIA MARCONI 10, 80125 NAP~& ITALY AND F. MAVILIO IS IN I THE I37Tn~TO SCIENTIFICOH.S. RAFFAELE, VIA OLGETITNA 60, 132 MILAN, ITALY.
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39 Kelly, F. and Condamine, H. (1982) Biochim. Biophys. Acta 651, 105-141
Raven Press 32 Ingraham, H.A. etal. (1990) Annu. Rev. Physiol. 52, 773-791 33 Li, S. et al. (1990) Nature 347, 528-533 34 Sch61er, H.R. et al. (1989) EMBO,L 8, 2551-2557 35 Sch61er, H.R., Ciesiolka, T. and Gruss, P. (1991) Ce1166, 291-304 36 Fibi, M. et al. (1988) Development 102, 349-359 37 Murphy, S.P. el al. (1988) Proc. Natl Acad. Sci. USA 85, 5587-5591 38 Curatola, A.M. and Basilico, C. (1990) Mol. Cell. BioL 10, 2475-2484
40 Rosner, M.H., De Santo, R.J., Arnheiter, H. and Staudt, L.M. (1991) Cel164, 1103-1110 41 Verrijzer, C.P., Kal, A.J. and van der VileR, P.C. (1990) ~ B O J . 9, 1883-1888 42 Schreiher, E. et al. (1990) Nucleic Acids Res. 18, 5495-5503
I t is now well established that genes containing a h o m e o b o x are regulatory genes coding for nuclear proteins that probably act as transcription factors 1. The h o m e o d o m a i n encoded by the h o m e o b o x is simply a protein domain capable of recognizing and binding to specific DNA sequences 2. Through the recognition properties of their homeodomain, homeoproteins are believed to regulate the expression of batteries of target genes. Among h o m e o b o x genes of vertebrates, those belonging to the Hox family show a number of interesting characteristics. First, they occur in clusters that have been confined in compact genomic regions since the remote evolutionary past. The first evidence for such clustering came from the study of Drosophila homeotic genes 3, located in two genomic regions called the Antennapedia (ANT-C) and Bithorax (BX-C) complexes. Subsequently, evidence has accumulated that these two complexes present in flies may have arisen from a single complex, called HOM-C, early in insect radiation s. The ancestral cluster corresponding to the HOM-C of insects has undergone duplications in the lineage leading to vertebrates ~',7. Higher vertebrates have four such homologous clusters, termed HOX loci in humans ~ and Hox loci in the mouse 9. Within these clusters, all Hox genes share the same transcriptional orientation 8. Hox genes were originally identified on the basis of the primary sequence of their homeodomains m. In fact, most Hox genes encode proteins containing an homeodomain closely related to the archetypal Antennapedia (Antp) homeodomain, often referred to as a class I homeodomain. However, since the resemblance is very slight for Hox genes at the ends of the four clusters (Fig. 1), it is more convenient to define Hox genes on the basis both of the primary sequence of the encoded products and of their physical location on chromosomes. Second, Hox genes appear to cooperate in providing positional information along the anteroposterior axis 9 of animals with bilateral symmetry, and perhaps even of all metazoans. This was originally recognized in flies, where homeotic genes specify the fate of segmental units within the metameric body plan3. Evidence is accumulating that they may provide positional information along other axes as well, for example in developing limb buds 11.
H.R. SCHOLER IS IN THE MAX-PLANCg INSTITUTE OFI BIOPHYSICAL CHEMISTRY,DEPARTMENTOF MOLECULARCELLI BIOLOGY, 1)-3400 GOTTINGEN, FRG; PRESENT ADDRESX" EMBL MEYERHOFSTRASSEI, D-6900 HEIDELBERG,FRG.
HOXgeneactivation by retin0ic acid EDOARDO BONCINELLI,ANTONIO S1MEONE, DARIO ACAMPORAAND FULVIOMAVIHO Vertebrate homeohox genes of the Hox family are, like Drosophila homeotic genes, organized in gene clusters and show a strict correspondence, or coUinearity, between the order of the genes (3' to 5') within the chromosomal cluster and that of their expression domains (anterior to posterior) in the embryo. Recent data obtained from embryonal carcinoma cells induced to differentiate by retinoic acid cast some light on the molecular mechanisms underlying the collinear expression of the Hox genes. Third, the genomic organization of Hox genes is reflected in their expression pattern. All the Hox genes are developmentally regulated in mouse 9, frog 12 and human 13 embryos and are expressed in partially overlapping domains. The anterior boundaries of the expression domains of the various genes of a given cluster uniformly follow a 5'-posterior/Y-anterior role along the embryonic anteroposterior body axis 1~,1s (reviewed in Ref. 4). This phenomenon was first observed for the homeotic genes in the BX-C of Drosophila and has been termed collinearity 3. A similar collinear expression pattern seems to apply to the genes of the four human HOX loci in differentiating embryonal carcinoma (EC) cells.
Human HOX gene organization So far, 38 h u m a n H O X g e n e s have b e e n identified 16. T h e y are distributed a m o n g four h o m o l o gous c h r o m o s o m a l loci, H O X I to HOX4, on chrom o s o m e s 7, 17, 12 a n d 2, respectively. Each cluster contains at least nine g e n e s organized in a h o m o l o gous linear arrangement. The 38 genes can be
Nomenclature: Hox: Vertebrate homeobox genes (species not defined). Hox: Mot,se homeobox genes. HOX: Human homeobox genes.
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expression in embryonic tissues has been complemented by studies in ELm differentiating embryonal carcinoma S---VT ....... V-E--VIN HOXIJ GRKK-VP--K V-LK---R-Y AT-KFI-KDK -RR-SATTNS---VT ....... V-E--WS HOX3G GRKK-VP--K V-LK ..... Y AASKFI-KEK -RR-SATTNKs~(EC) cells, both in mouse (reviewed KLKDT S---VT ....... V-D--IVS HOX4I GRKR-VP--K L-LK---N-Y AI-KFINKDK -RR-SA-TNin Ref. 9) and humans. These cells RE~AL SDQ-V .......... K-RLLL HOX3F SRK~-KP-SK L-LA---G-LV-EFI--Q-R-LSDB-NRE{~L provide a convenient model for the SDQ-V .......... K-RWL HOX4H ARKK-KP--K Q-IA---N-LV-EFIN-QK -K-LSNR-NDR~Y study of the molecular basis of -D--V .......... E--I-R HOXII TRKK-CP--K --IR---R-F-SV-INKEK -LQLSRM-N-D--V .......... E--LSR DRILY HOX3H TRKK-CP-SK F-IR---R-F--V-INKEK -LQLSRM-Ndevelopmental gene regulation, in -D--V .......... E - - L - ~ % DRLQ~ Hox4g SRKK-CP--K --IR---R-F--V-INKEK -LQLSRM-NENRIR response to specific stimuli. -D--V .......... L--M-R HOXIH GRKK-CP--K H ......... L--M .... E- -L--SRSVH-D--V .......... L--M-R HOX31 GRKK-CP--K H ......... L--M .... E- -L--SETINE~R~R The expression of human HOX -D--V .......... L--MSR ENRIR HOX4D GRKK-CP--K H ......... L--M .... E- -L--SRSVNDR~D genes has been extensively studied .... V .......... M--I-HOXIG TRKK-CP--K H ......... L--M .... D- -Y-V-RL-NS---V .......... M--M-HOX2E SREK-CP--K .......... L--M .... D- -H-V-RL-NEQ-~E in the human EC cell line NT2/D1 -- -V .......... M--M-E-TOE HOX3B TRRK-CP--K .......... L--M .... D- -Y-V-RV-NE-CPE (Refs 16, 22). Cells of this line may .... V .......... M--MSHOX4C TRKK-CP--K .......... L--M .... D- -Y-V-RI-NK o ~ be induced to differentiate when . . . . V . . . . . . . . . . . . . . . N HOX2D -R ...... S ........... L--P .... K .... VS---G.... V ............... N HOX3A -RS ..... S ........... L--P .... E .... VS---GKDKF-EDKL- cultured in a medium containing .... V ............... N HOX~E -R ...... S- F ......... L--P .... K .... VS---Aretinoic acid (RA). Differentiation is HOXIA ........................................................... H- DE-PI HOX2C ..................... Y ........................................ A-PG characterized by the appearance of GR ................................. N ......................... LINST HOXIB several cell types, including neurons, GR ................... Y ..................................... S- LLSAS HOX2B HOX3C -R .... I-S .......................... N ....................... S N LTSTL 7-28 days after induction. We will L-SMS discuss the results with reference to HOXIC G--A-TA ................................. S ................. D-L-SMS HOX2A G--A-TA ................................. S ................. D-M-S~E the scheme in Fig. 2. HOX3D G--S-TS ............................ NN--N ................. DSL P N T K (1) Northern blot analysis reveals HOXID P--S-TA--Q-V ....................... T--S---V ............. DHHOX2F P--S-TA --- Q-V ........ Y ......... V ........ S ................. DHLPNrE that none of the 38 HOX genes is LPNTK HOX3E P--S-AA--Q-V ........ Y .............. S--S ................ DHR HOX4B P--S-TA--Q-V ....................... T--S ................. DHLPNTK significantly expressed in untreated MOXIE S--R-TA--S A-LV ............ V-P-V-M-NL-N ................ Y--DQG--~L NT2/D1 stem cells. Whereas 24 of A--LA HOX2G S--A-TA--S A-LV ............ C-P-V-M-NL-NS .............. Y--DQA-A*L these genes are activated within a HOX4A S--V-TA--S A-LV ............ C-P-V-M-NL-N ............... Y--DQC-E~Q week in differentiating cells treated HOXIK SR-L-TA--N T-L .......... K--C-P-V---AL-D ..... V-V ........ H-RQTQ KOX2H AB-L-TA--N T-L .......... K--C-P-V---AL-D ..... V-V ........ R-RQTQ ~REPwith 10- s M RA, the expression of HOXIF PNAV-TNF-T K-LT ......... K .... A- -V---AS-QN-T-V .......... Q--REEGLLD 14 HOX genes remains undetectable EG-RV HOX21 PSGL-TNF-T R-LT ......... K--S-A-V~--AT-E - N-T-V .......... Q--RER HOX4G SSAI-TNFST K-LT ......... K .... A ...... NC-HNDT-V .......... Q--RER EGLLA even after this treatment. These 14 genes are located at the 5'-end FIGB of their loci: four in HOX1, five List of human HOX homeodomains. The one-letter amino acid code is used. in HOX3 and five in HOX4. The Sequences are compared with a consensus homeodomain represented by the boundary between responding and Antennapedia homeodomain (top line). Dashes indicate amino acid identity with the nonresponding genes roughly corconsensus. Five amino acid residues followingthe homeodomain are also shown. responds to the HOX genes of ]he homeodomains can be grouped into 13 subfamilies (separated by line spaces). group 5. (2) The expression pattern of the nine HOX2 genes was further studied in detail n. This divided into 13 subfamilies, primarily on the basis locus contains only responding genes belonging to of the peptide sequence of the encoded homeoproreins (Fig. 1). Genes belonging to the same subfamily groups 5-13 (Fig. 2). HOX2 genes located in the 3' half of the cluster are induced at peak levels by 10-8 M o c c u p y corresponding positions in the four loci that can be easily aligned with the fly complexes s,16 RA, whereas a concentration of 10-s M RA is required (Fig. 2). The hypothesis that human and mouse Hox to activate 5' genes fully. The time-course of individual loci are true homologues of the insect homeotic gene expression was monitored by RNase protection assays at RA concentrations that activate all the genes gene complexes 4 has recently received further sup(10-5 M) or only the 3' genes of the cluster (10 -7 M). At port from a series of gene swap experiments 10-5 M RA, HOX2H and HOX21 were rapidly induced, (reviewed in Ref. 17). The structural similarities between Hox genes in vertebrates and homeotic while the other genes were activated sequentially in the 3' to 5' direction from HOX2G to HOX2E (Fig. 3). At genes in insects seem to translate into similar regu10-7 M RA, only the five 3'-most genes, HOX2I to latory specificities. The evolutionary implications of HOX2A, were induced, with slower kinetics but in the such a conserved genomic organization appear to same order. be even more intriguing, as we have recently ident(3) This analysis has been extended 16 to all 38 ified two human homologues of the fly evenHOX genes (Fig. 3) in NT2/D1 cells and in a second skipped (eve) segmentation gene at the 5'-end of the EC line, Tera-2/clone 13. The general pattern of seHOX1 and HOX4 loci, albeit in the opposite orienquential activation of responding genes and the separtation to the HOX genes (Ref. 18 and E. Boncinelli ation of two collinear sets of responding and nonet aL, unpublished) (Fig. 2). These genes, termed responding genes was observed in both EC cell lines. EVX1 and EVX2, respectively, are homologous to In all four HOX loci, responding genes are sequentially Evx 1 and Evx 2 identified in the mousem. activated by 10-5 M RA in a 3' to 5' order. For the nonresponding genes at the 5'-end of the HOX1, HOX3 HOX gene expression in EC cells Since the pioneering studies of Gruss and collab- and HOX4 loci, the more sensitive RNase protection assay introduced a further distinction between (i) truly orators 2° (see also Ref. 21), analysis of Hox gene 10 20 30 40 50 60 RKRGROTYTR YOTLVIVRVV HFNRYLTRRR R I g I A M A L C L TVROIKTWVO NRRMRWEERNK TRGEP
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FIG[] Schematic representation of 40 homeodomains (circles) in the four human HOX clusters. Genes in any given cluster have been mapped on overlapping genomic clones and shown to be linked. The transcriptional orientation of all 38 HOX genes is from left to right. This alignment identifies 13 Hox homeodomain groups, indicated at the bottom. Solid dots indicate homeodomains predicted in the scheme but so far unidentified and probably missing. Below the circles are the names of known murine Hox homologues. Homeodomains from Drosophila BX-C and ANT-C genes are indicated above the scheme (abd-A, abdominal-A; Ubx, Ultrabithoro~, Scr, Sex combs reduced; zen, zerknf~llt; Abd-B, Abdominal-B; Dfd, Deformed; pb, proboscipedia; lab, labial). Each fly homeodomain is placed above the group of human homeodomains to which it is most closely related in sequence. The correspondence of fly homeodomains in parentheses is unclear. Homology groups 5 and 10 are highlighted. Hatching reflects HOX gene activation in NT2/D1 cells induced to differentiate by 10-~ M RA. Ascending hatching (e.g. HOX1A) indicates genes activated with time upon RA addition; descending hatching (e.g. HOX3G) represents downregulation. HOX4C is constitutively expressed at a very low level. Open circles indicate genes whose expression is always undetectable in these cells. The scheme also includes the EVX1and EVX2 homeodomains; the transcriptional orientation of the corresponding genes is from right to left, opposite to that of all HOXgenes. silent genes, belonging to groups 1-4 in HOX1 and 2-5 in HOX3, and (ii) genes expressed at low levels in uninduced stem cells and downregulated upon RA induction, such as HOX3G and those belonging to groups 1-4 in the HOX4 cluster (Fig. 2). HOX4C represents a separate class as it is expressed at a very low level throughout. In conclusion, human HOX genes are differentially activated by RA in EC cells according to their location within the four clusters, in a time- and RA concentration-dependent fashion. This order corresponds to the expression patterns in developing axial systems such as the axis skeleton and the central nervous system of humans and mice, where 3' genes are expressed more rostratly and 5' genes more caudally 923. The observed differential response is not a peculiarity of a single EC cell line but rather reflects a phenomenon of general physiological relevance. Similar, but not identical, results have been reported for several Hox genes in the murine F9 EC line 9,24.
Identification of subsets of HOX genes These data suggest a threefold subdivision of the four human HOX loci, with the two borders roughly corresponding to groups 5 and 10 (Fig. 2). The boundaries between RA-responding and nonresponding genes are approximately at the level of group 5. The genes of the first five groups can also be discriminated from those of the other groups located downstream on the basis of other independent lines of evidence. (1) All the genes of the first five groups correspond to only one Drosophila gene (Abdominal-B), albeit with varying degrees of homologys,n. (2) These genes lack the conserved YPWM homeopeptide 25 present just upstream from the homeodomain in the products of all other HOX genes. (3) The HOX2 locus apparently does not contain genes corresponding to the first four groups. (4) Genes belonging to the first five groups in HOX1 and HOX3 appear to be coordinately switched on and off, respectively, in blocks during differentiation of blood cells committed to the myelomonocytic lineage 26.
TIG OCTOBER1991 VOL. 7 NO. 10
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Expression of HOX genes in differentiating NT2/D1 cells. The graphs show the changes in RNA levels for responding and downregulated HOX genes; 100% represents plateau RNA levels, which are comparable for different genes although they may be reached at different times. A second boundary may be drawn approximately at the level of the four genes belonging to group 10. Even at high RA concentrations, HOX2 genes of groups 5-9 are not activated in Tera-2/clone 13 cells 16. In the mouse Hox 2 complex this boundary separates genes expressed in the embryonic hindbrain from those expressed more caudally, within the spinal cord (Refs 13, 23; reviewed in Ref. 27). Published evidence and preliminary data suggest that the situation may be similar for the 3' genes of H o x I and Hox 4 as well. Anterior expression boundaries of genes of the five 3'-most groups are almost coincident, at variance with what has been observed for Hox genes of other groups9. It is not clear which H O X gene of groups 6-9 corresponds to which fly homeotic gene (Fig. 2). These genes may derive from independent intralocus gene duplication events in the two evolutionary lineages.
Implicationsfor the studyof development These data obtained in EC cells in vitro may provide clues to a better understanding of the establishment and maintenance of positional information in embryos. RA levels regulated in space and/or time may at least partly underlie a differential activation of Hox genes in vivo that is consistent with the observed region-restricted expression pattern. Indeed, RA has been implicated as a natural morphogen in chicken development, where it contributes to the specification of the limb anteroposterior axis (Refs 28, 29; reviewed TIG
in Ref. 30) and possibly in frogs, where alteration of embryonic RA levels dramatically affects specific structures of the developing central nervous system3L Differential exposure to the morphogen may determine which genes are to be locally activated in cells of different embryonic regions, thus providing positional information for the developing embryo. Many potential embryonic sources of RA have been identified and a role for RA in connection with homeobox gene function in diverse aspects of vertebrate development has repeatedly been considered30. In this light, the posterior transformation of the central nervous system in frog embryos treated with RA could be interpreted as a phenomenon correlated with overexpression and/or ectopic expression of Hox genes. The observed respecification implies a considerable reduction in the size of the forebrain and midbrain, where Hox genes are normally not expressed, with a concomitant expansion of the spinal cord and hindbrain, where they are expressed during normal embryogenesis. Circumstantial evidence exists in the literature TM that the equation 3'-anterior-early/5'-posterior-late may also be valid for the expression of Hox genes in embryogenesis. In the developing mouse limb, 3' H o x 4 genes are expressed earlier and more proximally and 5' genes are expressed later and more distally 11. Moreover, a recent extensive analysis of the expression of these genes in chick wing development led to the conclusion that cells cannot express upstream
ocrouee 1991 voL 7 ~o. 10
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FIGI~I Transcriptional organization of a region of the human HOX3 locus. Transcripts containing one of the three homeoboxes (solid boxes), HOX3C HOX3D or HOX3E, may derive from a master promoter (MP) or from one of three individual proximal promoters (IP1-3). members of this cluster without first expressing downstream members32. This temporal sequence of expression could in turn account for the lack of real homeotic transformations noted in vertebrates. Establishment versus maintenance The molecular mechanisms underlying the differential activation of HOX genes seen in EC cells have been further investigated because there have been controversial reports in the literature I6. The RNA accumulation seen in differentiating NT2/D1 cells appears to be regulated primarily at the transcriptional level, as revealed both by run-on transcription assays on isolated nuclei and comparison of nuclear and cytoplasmic RNA levels during induction. Induction of the early responding genes does not require de novo protein synthesis. Moreover, treatment with inhibitors of protein synthesis alone caused a rapid appearance of RNA transcripts from most responding HOX genes, with loss of the 3' to 5' pattern of sequential activation, while leaving the expression levels of silent and downregulated genes unaffected. Synthesis of short half-life proteins is apparently required to maintain the timing and polarity of expression of responding HOX genes in these cells. The complex control network operating on HOX gene clusters may include active repression, and activation of 5' responding HOX genes in vivo could involve downregulation of these repressor proteins. In vitro, this programmed downregulation may be mimicked by cycloheximide treatment.
If the observed HOX gene activation takes place at the transcriptional level, the promoters implicated in this activation need to be identified, This is not simple because expression of Hox genes appears to originate from a multiplicity of promoters (Refs 33, 34; reviewed in Ref. 12). Most of them are individual proximal promoters, located upstream from the coding region of every Hox gene. Additional promoters, called master promoters ~2 or major upstream promoters 33, have been shown to be active at least in Hox 3. In certain human embryonic and adult tissues, transcription from a major upstream promoter yields an RNA containing at least three contiguous HOX3 homeoboxes (HOX3C, 3D and 3E)33 (Fig. 4). At least two mature mRNAs contain the HOX3C homeobox: one derived from its individual proximal promoter and the other obtained by alternative splicing of the primary transcript originating from the master promoter. Exactly the same situation applies to mRNAs containing the HOX3D or the HOX3E homeobox. Indeed, the two mRNAs containing the HOX3C homeobox have been shown to encode two different proteins sharing a carboxy-terminal domain of 153 residues including the homeodomain. The homeoprotein encoded by the mRNA from the proximal promoter (mRNA 2 in Fig. 4) has an extra 82 amino acid domain at the amino terminus 3334. Both northern blot analysis and RNase protection assays revealed that this HOX3 major upstream promoter is not induced in differentiating NT2/D1 cells 16. The HOX3C, 3D and 3E mRNAs induced in these cells
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[~EVIEWS by RA are transcribed from individual promoters proximal to the corresponding coding regions. If RA-treated EC cells actually mimic the first appearance of H O X gene transcripts in development, we conclude that H O X 3 genes are first expressed as transcripts starting from the individual proximal promoters. Results of experiments studying expression of Hox genes as transgenes agree with this conclusion. An individual H o x gene, together with its proximal promoter, introduced into a transgenic mouse can reproduce the early expression pattern, but fails to duplicate properly all the spatial and temporal changes in pattern at later stages of development35.36. Reconstruction of both the early and late pattern probably depends on maintaining the clustered organization. It is conceivable that alternative or additional biological stimuli, such as specific peptide growth factors or cell-cell signals, are required for the activation of the HOX3 master promoter in EC cells. Another possible interpretation of these data is that RA treatment does not initiate HOX gene expression in EC cells but perturbs a previous pattern of expression. Indeed, it has been shown that HOX21, 2H, 1F and some HOX4 genes are already expressed in these cells. In addition, 5' genes such as HOX2E are actively repressed in NT2/D1 stem cells. Finally, these cells also differentiate in response to other drugs, but only RA activates H O X genes. Fly HOM genes are regulated in two different ways in the establishment phase: when their transcripts appear for the first time in embryogenesis, and in the maintenance phase, when their expression pattern established in a particular body region is clonally transmitted from cell generation to cell generation 4. The gene products required to activate the genes for the first time are no longer available in the second phase and a mechanism of mutual regulation of the HOM genes themselves probably takes over. It has been suggested that vertebrate homeobox genes also go through two phases of establishment and maintenance (reviewed in Ref. 37). There is also some debate about whether the genomic organization of Hox genes is so highly conserved because it plays a major role in the maintenance phase of the expression of these genes or in the establishment phase, or in both n37. Hence, the question of the last two paragraphs becomes: are individual proximal promoters active in the establishment phase, while the master promoters are turned on subsequently, or is the reverse true? Several examples have been reported of RA addition in vivo changing the pattern of developing structures, including regenerating urodele limbs (reviewed in Ref. 38). It is conceivable that this effect is mediated, at least in part, through a disruption of the Hox gene expression pattern already established in a given developing structure. One might further suggest that this disruption results from the activation of some or all individual proximal promoters in the Hox loci. In conclusion, the analysis of many different biological systems supports the idea that homeoproteins are involved in early patterning events through the translation of primary morphogenetic signals into a complex network of positional information.
Acknowledgements We thank Robb Kmmlauf and Vincenzo Nigro for helpful suggestions. The experimental work was supported by grants from Progetti Finalizzati CNR 'Biotecnologia e Biostrumentazione' and 'Ingegneria Genetica', the Third AIDS Project of the Ministero della Sanita and the Italian Association for Cancer Research (MRC).
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E. BONCINELLI, A. ~IMEONE AND D. ACAMPORA ARE IN THE INTERNATIONAL INSTITUTE OF GENETICSAND BIOPHYSIC& C N ~ VIA MARCONI 10, 80125 NAP~& ITALY AND F. MAVILIO IS IN I THE I37Tn~TO SCIENTIFICOH.S. RAFFAELE, VIA OLGETITNA 60, 132 MILAN, ITALY.
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