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Krox20 and kreisler co-operate in the transcriptional control of segmental expression of Hoxb3 in the developing hindbrain

2002; Springer Nature; Volume: 21; Issue: 3 Linguagem: Inglês

10.1093/emboj/21.3.365

1460-2075

Miguel Manzanares, Jeannette Nardelli, Pascale Gilardi‐Hebenstreit, Heather Marshall, François Giudicelli, María Teresa Martínez‐Pastor, Robb Krumlauf, Patrick Charnay,

Hedgehog Signaling Pathway Studies

Article1 February 2002free access Krox20 and kreisler co-operate in the transcriptional control of segmental expression of Hoxb3 in the developing hindbrain Miguel Manzanares Miguel Manzanares Division of Developmental Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA GB Present address: Department of Developmental Neurobiology, Insituto Cajal, CSIC, Av. Doctor Arce 37, E-28002 Madrid, Spain Search for more papers by this author Jeannette Nardelli Jeannette Nardelli Unité 368 de I'Institut National de la Santé et de la Recherche Médicale, Ecole Normale Supérieure, 46 rue d'Ulm, F-75230 Paris, Cedex 05, France Present address: UMR 7000 du Centre National de la Recherche Scientifique, CHU Pitié-Salpêtrière, 105 bd de l'Hôpital, 75013 Paris, France Search for more papers by this author Pascale Gilardi-Hebenstreit Pascale Gilardi-Hebenstreit Unité 368 de I'Institut National de la Santé et de la Recherche Médicale, Ecole Normale Supérieure, 46 rue d'Ulm, F-75230 Paris, Cedex 05, France Search for more papers by this author Heather Marshall Heather Marshall Division of Developmental Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA GB Present address: Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110 USA Search for more papers by this author François Giudicelli François Giudicelli Unité 368 de I'Institut National de la Santé et de la Recherche Médicale, Ecole Normale Supérieure, 46 rue d'Ulm, F-75230 Paris, Cedex 05, France Search for more papers by this author María Teresa Martínez-Pastor María Teresa Martínez-Pastor Division of Developmental Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA GB Search for more papers by this author Robb Krumlauf Robb Krumlauf Division of Developmental Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA GB Present address: Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110 USA Search for more papers by this author Patrick Charnay Corresponding Author Patrick Charnay Unité 368 de I'Institut National de la Santé et de la Recherche Médicale, Ecole Normale Supérieure, 46 rue d'Ulm, F-75230 Paris, Cedex 05, France Search for more papers by this author Miguel Manzanares Miguel Manzanares Division of Developmental Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA GB Present address: Department of Developmental Neurobiology, Insituto Cajal, CSIC, Av. Doctor Arce 37, E-28002 Madrid, Spain Search for more papers by this author Jeannette Nardelli Jeannette Nardelli Unité 368 de I'Institut National de la Santé et de la Recherche Médicale, Ecole Normale Supérieure, 46 rue d'Ulm, F-75230 Paris, Cedex 05, France Present address: UMR 7000 du Centre National de la Recherche Scientifique, CHU Pitié-Salpêtrière, 105 bd de l'Hôpital, 75013 Paris, France Search for more papers by this author Pascale Gilardi-Hebenstreit Pascale Gilardi-Hebenstreit Unité 368 de I'Institut National de la Santé et de la Recherche Médicale, Ecole Normale Supérieure, 46 rue d'Ulm, F-75230 Paris, Cedex 05, France Search for more papers by this author Heather Marshall Heather Marshall Division of Developmental Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA GB Present address: Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110 USA Search for more papers by this author François Giudicelli François Giudicelli Unité 368 de I'Institut National de la Santé et de la Recherche Médicale, Ecole Normale Supérieure, 46 rue d'Ulm, F-75230 Paris, Cedex 05, France Search for more papers by this author María Teresa Martínez-Pastor María Teresa Martínez-Pastor Division of Developmental Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA GB Search for more papers by this author Robb Krumlauf Robb Krumlauf Division of Developmental Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA GB Present address: Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110 USA Search for more papers by this author Patrick Charnay Corresponding Author Patrick Charnay Unité 368 de I'Institut National de la Santé et de la Recherche Médicale, Ecole Normale Supérieure, 46 rue d'Ulm, F-75230 Paris, Cedex 05, France Search for more papers by this author Author Information Miguel Manzanares1,2, Jeannette Nardelli3,4, Pascale Gilardi-Hebenstreit3, Heather Marshall1,5, François Giudicelli3, María Teresa Martínez-Pastor1, Robb Krumlauf1,5 and Patrick Charnay 3 1Division of Developmental Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA GB 2Present address: Department of Developmental Neurobiology, Insituto Cajal, CSIC, Av. Doctor Arce 37, E-28002 Madrid, Spain 3Unité 368 de I'Institut National de la Santé et de la Recherche Médicale, Ecole Normale Supérieure, 46 rue d'Ulm, F-75230 Paris, Cedex 05, France 4Present address: UMR 7000 du Centre National de la Recherche Scientifique, CHU Pitié-Salpêtrière, 105 bd de l'Hôpital, 75013 Paris, France 5Present address: Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110 USA ‡M.Manzanares and J.Nardelli contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:365-376https://doi.org/10.1093/emboj/21.3.365 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In the segmented vertebrate hindbrain, the Hoxa3 and Hoxb3 genes are expressed at high relative levels in the rhombomeres (r) 5 and 6, and 5, respectively. The single enhancer elements responsible for these activities have been identified previously and shown to constitute direct targets of the transcription factor kreisler, which is expressed in r5 and r6. Here, we have analysed the contribution of the transcription factor Krox20, present in r3 and r5. Genetic analyses demonstrated that Krox20 is required for activity of the Hoxb3 r5 enhancer, but not of the Hoxa3 r5/6 enhancer. Mutational analysis of the Hoxb3 r5 enhancer, together with ectopic expression experiments, revealed that Krox20 binds to the enhancer and synergizes with kreisler to promote Hoxb3 transcription, restricting enhancer activity to their domain of overlap, r5. These analyses also suggested contributions from an Ets-related factor and from putative factors likely to heterodimerize with kreisler. The integration of multiple independent inputs present in overlapping domains by a single enhancer is likely to constitute a general mechanism for the patterning of subterritories during vertebrate development. Introduction The vertebrate hindbrain is organized into segmental compartments, termed rhombomeres, that provide a basic ground plan for generating regional diversity of structures during craniofacial development (Lumsden, 1990). Several different gene classes have been shown to be associated with the generation and maintenance of these segmental territories and with the specification of their antero-posterior (A–P) identity. Among these, the transcription factors Krox20, kreisler, Hox and retinoic acid receptors display rhombomere-restricted patterns of expression that have been shown to be functionally important in the segmental processes (Lumsden and Krumlauf, 1996; Schneider-Maunoury et al., 1998; Trainor et al., 2000). Through a variety of approaches, a picture of the interactions existing between these genes and of the regulatory hierarchy of events governing hindbrain segmentation is beginning to emerge (Maconochie et al., 1996; Trainor and Krumlauf, 2000; Trainor et al., 2000). Krox20 encodes a zinc finger transcription factor, expressed in r3 and r5, that is essential for development and maintenance of these segments (Wilkinson et al., 1989; Schneider-Maunoury et al., 1993, 1997; Swiatek and Gridley, 1993; Giudicelli et al., 2001). Krox20 has also been shown to play an essential role in the specification of odd- versus even-numbered rhombomere identity by controlling the expression of a number of downstream regulatory genes (Seitanidou et al., 1997; Mechta-Grigoriou et al., 2000; Giudicelli et al., 2001; Voiculescu et al., 2001). In particular, Krox20 directly regulates the transcription of Hoxa2 and Hoxb2 in rhombomeres (r) 3 and 5 through distinct combinations of Krox20-binding sites in their 5′-flanking regions (Sham et al., 1993; Nonchev et al., 1996b). These Krox20-binding sites are also found in association with conserved sites for other factors that differentially contribute to the segmental regulation of these Hox genes (Vesque et al., 1996; Maconochie et al., 2001). Hence, this Krox20-dependent regulatory mechanism is highly conserved during vertebrate evolution, as evidenced by the similarity in both patterns of expression and the organization of regulatory elements (Nieto et al., 1991; Nonchev et al., 1996a; Vesque et al., 1996). Finally, Krox20 also directly regulates the expression of the EphA4 tyrosine kinase receptor gene in r3 and r5, contributing to the control of segregation or mixing between cells from adjacent segments (Theil et al., 1998; Voiculescu et al., 2001). Another gene that plays an important role in the early formation of segments is kreisler (kr), which encodes a Maf/basic leucine zipper (b-ZIP) protein (Krml1) that is expressed in r5 and r6 (Cordes and Barsh, 1994). Mutational analyses have shown that it is necessary for the formation of r5 in mouse and for the proper development of r5 and r6 into mature segments in zebrafish (Frohman et al., 1993; McKay et al., 1994; Moens et al., 1996, 1998; Manzanares et al., 1999b). kreisler also plays a later role in controlling rhombomere identity through the Hox genes, since conserved kreisler-binding sites in segmental enhancers from Hoxa3 and Hoxb3 are essential for initiating rhombomere-restricted expression of these genes (Manzanares et al., 1997, 1999a, 2001). Hox genes themselves play multiple roles in regulating segmental processes with a major input into A–P specification, but they are also involved in the formation of segmental territories, cell mixing, generation of cranial neural crest and dorso-ventral specification (Chen and Ruley, 1998; Davenne et al., 1999; Gavalas et al., 2001). Detailed analyses of single and compound mutants have revealed complex phenotypes, with evidence for synergy within domains of overlap and defects outside of the domains of expression (Carpenter et al., 1993; Dolle et al., 1993; Mark et al., 1993; Gavalas et al., 1997, 1998, 2001; Helmbacher et al., 1998; Studer et al., 1998). This shows that the segmental control genes exert their influences at multiple steps through a combination of both direct and indirect interactions. Hox genes are major targets of the upstream pathways, such as Krox20 and kreisler, that regulate segmentation and morphogenesis (Krumlauf, 1994; Lumsden and Krumlauf, 1996; Maconochie et al., 1996; Trainor and Krumlauf, 2000). However, mechanistically, the way in which the information from such pathways is integrated at the level of Hox genes is still poorly understood, and represents an important unresolved issue. In particular, we need to know whether the critical cis-acting regulatory regions are modular in nature, with discrete elements that independently receive inputs from a variety of components, or whether the transcriptional output depends upon a complex balance of direct synergistic or antagonistic interactions in association with other factors (Mann and Affolter, 1998; Affolter and Mann, 2001). In this regard, segmental expression in r3 and r5 of the members of Hox paralogy groups 2 and 3 is of particular interest, as it represents an example where there are complex overlaps and differences between the expression and function of Krox20, kreisler and Hox genes. As detailed above, the group 2 genes (Hoxa2 and Hoxb2) are expressed in r3 and r5 through direct transcriptional activation by Krox20, although there are differences in the nature of the Krox20-dependent control of these two genes (Sham et al., 1993; Nonchev et al., 1996b; Maconochie et al., 2001). Similarly, the group 3 genes are directly up-regulated early in hindbrain segmentation by the action of kreisler (Manzanares et al., 1997, 1999b). However, they are differentially maintained in later stages through the presence of an auto/cross-regulatory loop that acts on Hoxa3 but not Hoxb3 (Manzanares et al., 1997, 2001). There are also differences in the pattern of initiation of the group 3 genes. While Hoxa3 is activated in both r5 and r6, reflecting the normal segmental domains of kreisler, the Hoxb3 enhancer is active only in r5, representing a subset of the kreisler domain. This indicates that in addition to kreisler, the segmental restriction of Hoxb3 expression to r5 must be mediated by other cis-elements and/or factors in the r5/r6 region, which modulate the activity of the Hoxb3 enhancer. It is possible that Krox20 could participate in this additional input to segmental restriction, based on the timing and overlapping patterns of expression of Krox20 and kreisler in r5 and genetic evidence suggesting that Krox20 function is required for proper Hoxb3 expression (Seitanidou et al., 1997). Therefore, in the present study, we have investigated the possibility that Krox20 and kreisler might act together on common Hox targets in r5. We have performed a detailed analysis of the regulatory elements involved in Hoxb3 and Hoxa3 transcriptional control by using Hox/lacZ reporter lines, targeted Krox20 mutants, mutational analysis in transgenic mice and in ovo electroporation in chick. We demonstrate that kreisler and Krox20 indeed co-operate to activate the Hoxb3 enhancer within their common domain of expression, i.e. r5, while the Hoxa3 enhancer is independent of Krox20 activity. Furthermore, our data reveal the presence and importance of other regulators that work in conjunction with kreisler and Krox20 to generate the complex information needed to restrict Hoxb3 expression in r5. Results Differences in the Krox20 dependence of Hoxb3 and Hoxa3 segmental enhancers The Hoxb3 and Hoxa3 genes are expressed at high relative levels in r5 and r5/r6, respectively, and at lower levels in more posterior regions of the neural tube (Hunt et al., 1991; Manzanares et al., 2001). We previously have identified cis-acting regulatory elements responsible for the segmentally restricted expression of group 3 Hox genes in the hindbrain, and shown their dependence on direct binding of the b-ZIP transcription factor kreisler that is expressed in r5 and r6 (Manzanares et al., 1999a). While the activity of the Hoxa3 element in r5 and r6 reflects that of kreisler, the response of the Hoxb3 element is restricted to r5 and must be modulated by additional factors. One candidate potentially involved in mediating this difference is the zinc finger transcription factor Krox20. To investigate genetically the involvement of Krox20 in the activity of these Hox enhancers, a lacZ reporter coupled to a minimal promoter under the control of the Hoxb3 r5 enhancer element or the Hoxa3 r5/r6 enhancer was introduced into a Krox20 null background (Swiatek and Gridley, 1993). In a wild-type background, reporter expression under the control of the Hoxb3 r5 enhancer is clearly visible as a single stripe in the region of the otic sulcus after 8.0 days post-coitum (d.p.c.), when rhombomere territories are being established (Figure 1A). Expression gradually increases until rhombomeres are morphologically visible, being restricted exclusively to r5 (Figure 1B and C). In contrast, in Krox20−/− embryos no expression was ever detected in the hindbrain (Figure 1F–H), even at stages when the prospective r5 territory was still present (Figure 1F and G; Schneider-Maunoury et al., 1993; Swiatek and Gridley, 1993). The other domains of expression of the transgene, such as the posterior lateral mesoderm, are not affected in the mutant. Expression of lacZ in the Hoxa3-r5/6 transgenic line is initiated at a similar time (Figure 1D), but in a broader domain that presumably corresponds to prospective r5 and r6 (Figure 1E). In Krox20−/− embryos, expression at 8.5 d.p.c. is identical to that of wild-type littermates (Figure 1I), but by 9.5 d.p.c. it is reduced to a single rhombomere width (Figure 1J). This latter effect is the result of the loss of the r5 territory in Krox20−/− embryos at this stage of development (Schneider-Maunoury et al., 1993, 1997; Swiatek and Gridley, 1993). Figure 1.Analysis of the genetic requirement of Krox20 for the segmental activity of the Hoxb3 and Hoxa3 enhancers in the mouse hindbrain. Stable transgenic lines carrying the lacZ gene under the control of the Hoxb3 r5 enhancer (A–C and F–H) or the Hoxa3 r5/r6 enhancer (D, E, I and J) were crossed into a Krox20 mutant background. Wild-type littermates (A–E) or Krox20 homozygous mutant (F–J) embryos were examined by X-gal staining at the indicated stages (d.p.c.). (A–C and F–H) Expression in r5 driven by the Hoxb3 element is eliminated in the homozygous mutant background. (D and I) In contrast, the activity of the Hoxa3 element is not affected in r5 or r6 at 8.5 d.p.c. (E and J) At the 9.5 d.p.c. stage, the r5 territory has disappeared in the Krox20 homozygous mutant, but the activity of the Hoxa3 enhancer is maintained in r6. Download figure Download PowerPoint These genetic experiments establish that the activity of the Hoxb3 r5 enhancer depends on the presence of a functional Krox20 protein even to activate early expression in r5. In contrast, expression directed by the Hoxa3 r5/6 enhancer does not require Krox20. These data demonstrate the existence of a fundamental difference in the mechanisms leading to establishment and restriction of the segmental pattern of expression of Hoxa3 and Hoxb3 in the mouse hindbrain. Synergistic activation of the Hoxb3 r5 enhancer by Krox20 and kreisler To determine whether the involvement of Krox20 in the activity of the Hoxb3 r5 enhancer requires co-operation with kreisler, we performed ectopic expression experiments using in ovo electroporation of the chick hindbrain (Itasaki et al., 1999). A construct consisting of the lacZ reporter driven by a minimal human β-globin promoter and carrying the Hoxb3 r5 enhancer was co-electoporated together with an empty expression vector or with Krox20 and/or kreisler expression constructs. Electroporation was performed at stages HH10–12, and β-galactosidase activity was assayed 24 h later by X-gal staining. When the reporter was co-electroporated with the empty expression vector (pAdRSV; Giudicelli et al., 2001), only a few cells weakly positive for X-gal were observed and they were all restricted to r5 (Figure 2A). Co-electroporation of the reporter construct with a Krox20 expression plasmid (pAdRSVKrox20; Giudicelli et al., 2001) led to a significant activation of the reporter construct in r5 and r6 (Figure 2B). Co-electroporation of the reporter with a kreisler expression construct (pAdRSVkreisler) led to strong and reproducible activation of the reporter construct in r3 and to a lower extent in r5, together with weaker and more variable activation in other regions of the electroporated neural tube (Figure 2C). Finally, co-electroporation with both the Krox20 and kreisler expression constructs led to massive activation of the reporter construct throughout the electroporated neural tube (Figure 2D). Activation of the lacZ reporter in these experiments was dependent on the presence of the Hoxb3 enhancer, since similar co-electroporations of a control reporter construct without enhancer did not lead to any activation (data not shown). Figure 2.Krox20 and kreisler co-operate on the Hoxb3 r5 enhancer. Chick embryos were electroporated into the left side of the neural tube with a lacZ reporter construct. β-galactosidase activity was detected subsequently by X-gal staining. In the reporter, lacZ is placed under the control of the human β-globin minimal promoter and of the Hoxb3 r5 enhancer. (A) Co-electroporation of the reporter construct with an empty expression vector (pAdRSV). Weak lacZ expression is detected in a few cells restricted to r5. (B) Co-electroporation with a Krox20 expression vector. Activation of the reporter is observed in r5 and r6. (C) Co-electroporation of the reporter with a kreisler expression vector. Strong activation of the reporter is observed reproducibly in r3 and to a lower extent in r5. X-gal-positive cells are also seen occasionally in other areas of the electroporated region. (D) Co-electroporation with both the Krox20 and kreisler expression vectors. Massive induction of the reporter is observed throughout the electroporated region. Download figure Download PowerPoint In conclusion, these data indicate that when either Krox20 or kreisler are expressed ectopically in the chick hindbrain, they lead to preferential activation of the Hoxb3 enhancer in the territories where the other partner is endogenously expressed: r5/6 upon expression of Krox20, and r3 and r5 upon expression of kreisler. Furthermore, co-expression of both genes leads to ubiquitous activation of the enhancer. These data clearly demonstrate that Krox20 and kreisler can co-operate synergistically to activate the Hoxb3 r5 enhancer. Furthermore, they suggest that, in this assay, ectopic expression of Krox20 and kreisler is sufficient to promote efficient activation of the enhancer in a large part of the neural tube, including the entire hindbrain. Krox20 binds in vitro to two distinct sites within the Hoxb3 r5 enhancer In light of the above data, we investigated the possibility that Krox20 was directly controlling the activity of the Hoxb3 r5 enhancer. For this purpose, a 400 bp NcoI–HindIII fragment containing the enhancer activity was analysed for its capacity to bind Krox20 in a gel retardation assay. In the presence of bacterially produced Krox20, two retarded complexes were observed (Figure 3A). The formation of these complexes was prevented by addition of an oligonucleotide containing a high affinity Krox20-binding site (5′-GCGTGGGCG-3′), but not by addition of a related oligonucleotide, containing a mutation in this sequence known to abolish Krox20 binding (5′-GCGTCGGCG-3′, Figure 3A, lines 3 and 4; Nardelli et al., 1991). These data suggested the existence of two Krox20-binding sites within the enhancer fragment, and this was confirmed by a dimethylsulfate (DMS) interference analysis of the fragment, which revealed two protected regions (Figure 3B). Examination of the nucleotide sequence of these regions indicated that each contained a sequence with similarity to a consensus Krox20-binding site (Chavrier et al., 1990; Nardelli et al., 1991; Swirnoff and Milbrandt, 1995). Hence, these sites were named KroxA (5′-CTGTAGGAG-3′) and KroxB (5′-ATGTAGGTG-3′) (Figure 3B). Figure 3.The Hoxb3 r5 enhancer contains two Krox20-binding sites. (A) In vitro binding of Krox20 to the 400 bp NcoI–HindIII fragment carrying the Hoxb3 r5 enhancer, analysed by electrophoretic mobility shift assay. Lane 1, control protein extract from bacteria transfected with the empty expression vector. Lanes 2–4, protein extracts from Krox20-expressing bacteria. Two retarded complexes are observed in lane 2 (B1 and B2), suggesting the existence of two Krox20-binding sites. In lanes 3 and 4, protein–DNA incubation was performed in the presence of a 50-fold molar excess of competitor oligonucleotides containing a high affinity Krox20-binding site or a mutated version of this site, respectively. Complex formation is competed specifically by the Krox20 oligonucleotide (lane 3). P, free probe. (B) DMS interference analysis performed on a 120 bp Sfe–HindIII subfragment containing the two Krox20-binding sites. Lane 1, G + A scale; lanes 2–5, G scales of the probe before electrophoresis (lane 2), of the free probe (lane 3) and of the probe extracted from complex B1 (lane 4), and from complex B2 (lane 5). This analysis reveals two regions where guanine methylation prevents complex formation. The stronger interference observed in the case of B2 suggests that this corresponds to a ternary complex in which both sites are occupied by Krox20. Two sequences with homology to the Krox20 consensus binding site (KroxA and KroxB) are centred within the regions of interference. Download figure Download PowerPoint The Hoxb3 r5 enhancer contains two blocks of conserved sequences between mouse and chick, one of 19 bp that includes the kreisler-binding site Kr1, and another of 46 bp that contains the kreisler-binding site Kr2 and an Ets-related activation site (ERAS) (Figure 4; Manzanares et al., 1997). Deletion of any of the kreisler-binding sites or extensive mutation of the ERAS resulted in the loss of expression in r5 (Manzanares et al., 1997). Both of the Krox20-binding sites identified by the in vitro assay are located within the 46 bp sequence (Figure 4A) and partially overlap with the ERAS (KroxA) and the Kr2 sites (KroxB). Thus, the mutations tested previously [Kr2Δ and ERAS-m1; see Figure 4A and Manzanares et al. (1997)] could have also interfered with Krox20 input on the Hoxb3 r5 enhancer. Therefore, we constructed a series of new point mutations within the 46 bp fragment to assess independently the binding and function of kreisler, Krox20 and Ets proteins on the enhancer, and correlate these with their in vivo roles in the segmental expression of Hoxb3 (Figure 4A and B). Figure 4.Mutational analysis of the Hoxb3 r5 enhancer. (A) The top line shows the 2.2 kb BamHI–HindIII genomic fragment in the context of which all transgenic analyses of mutated versions were carried out. The advantage of this fragment is that, apart from the r5 element, it includes other enhancers responsible for posterior expression (Manzanares et al., 1997) that may be used as an internal control for transgenesis and lacZ activity. Under this is shown the sequence of the 46 bp conserved block from the Hoxb3 r5 enhancer where the kreisler-binding site Kr2 and the Ets-related activation site (ERAS) previously had been mapped (Manzanares et al., 1997), and where Krox20-binding sites KroxA and KroxB have been located. Sites are boxed, highlighting the overlaps between Kr2 and KroxB on one hand, and between ERAS and KroxA on the other. Below are indicated the nucleotide changes introduced in the different mutated versions. B, BamHI; H, HindIII. (B) In vitro and in vivo behaviour of the different mutated versions of the Hoxb3 r5 enhancer. Results for Kr1Δ, Kr2Δ and ERAS-m1 were described in Manzanares et al. (1997). +, positive binding or transgene expression; −, lack of binding or transgene expression; +/−, reduced transgene expression; ++, increased binding; nt, not tested; N, number of transgenic embryos showing a reproducible pattern. Download figure Download PowerPoint The KroxA site is necessary for activity of the Hoxb3 r5 element The original ERAS-m1 mutation modified four out of nine base pairs of the KroxA site (Figure 4A), so it was likely that this mutation would affect binding of both Krox20 and Ets proteins, and this was confirmed experimentally (Figure 4B and data not shown). To separate the Ets and Krox activities, two additional mutations within the non-overlapping portions of the ERAS and KroxA sites (ERAS-m2 and KroxA-m1) were therefore generated in this region (Figure 4A). Direct in vitro binding studies of Krox20 were then performed on a 23 bp oligonucleotide spanning the ERAS and KroxA sites (see Materials and methods). They revealed the formation of a unique retarded complex (Figure 5A), presumably corresponding to binding of Krox20 to the KroxA site. Consistent with this interpretation, introduction of the KroxA-m1 mutation abolished binding, while the ERAS-m2 mutation had no effect (Figures 4B and 5A). Competition experiments performed with oligonucleotides confirmed these results. Oligonucleotides carrying a high affinity Krox20-binding site (5′-GCGTGGGCG-3′; Figure 5B, lanes 2–4), the wild-type version of the KroxA site (Figure 5B, lanes 7–9) or the ERAS-m2 version of the 23 bp oligonucleotide (Figure 5B, lanes 13–15) effectively competed binding of Krox20 protein to the wild-type 23 bp oligonucleotide. In contrast, oligonucleotides carrying a mutated version of the high affinity site (5′-GCGTCGGCG-3′; Figure 5B, lanes 5 and 6) or the m1 version of the KroxA site (Figure 5B, lanes 10–12) did not compete efficiently. Figure 5.In vitro analysis of the binding of Krox20 and Ets1 to the KroxA/ERAS region of the Hoxb3 r5 enhancer. (A) Analysis of Krox20 binding by direct gel mobility shift assay. The 23 bp oligonucleotide probes (see Materials and methods) spanning the KroxA and ERAS sites and corresponding to the wild-type (WT), KroxA-m1 and ERAS-m2 mutated sequences were incubated with control (C) or Krox20-containing (K20) bacterial extracts. The KroxA-m1 mutation prevents Krox20 binding, whereas the ERAS-m2 has no effect. (B) Competition gel mobility shift assay performed as in (A), but in the presence of 20- (lanes 2, 7, 10 and 13), 50- (lanes 3, 5, 8, 11 and 14) or 250-fold (lanes 4, 6, 9, 12 and 15) molar excess of competitor oligonucleotides carrying a high affinity Krox20-binding site (lanes 2–4), a mutated version of this site (lanes 5 and 6), the KroxA/ERAS region (lanes 7–9), and this region with the KroxA-m1 mutation (lanes 10–12) and