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• W2124304811 abstract "We have characterized the transcription unit of a murine Hox gene in the fourth paralogous group,Hoxd4. We have identified two Hoxd4transcription start sites by S1 analysis. The upstream promoter (P2) is 5.2 kilobase pairs upstream from the coding region, while the downstream promoter (P1) is 1.1 kilobase pairs distant. Both promoters bear a cluster of start sites. Multiple transcripts were identified by Northern blot, originating from both promoters and multiple polyadenylation signals. Expression of P1 transcripts in the neural tube shows an anterior border at the rhombomere 6/7 boundary, corresponding to previous reports (Gaunt, S. J., Krumlauf, R., and Duboule, D. (1989) Development 107, 131–141; Morrison, A., Moroni, M. C., Ariza-McNaughton, L., Krumlauf, R., and Mavilio, F. (1996) Development 122, 1895–1907). A more posterior boundary in the central nervous system was observed for P2 transcripts. We observed strong expression up to somite 6 and weak expression in somite 5, correlating with the phenotype of Hoxd4 null mutant mice (Horan, G. S. B., Nagy Kovàcs, E., Behringer, R. R., and Featherstone, M. S. (1995) Dev. Biol. 169, 359–372). In response to retinoic acid, expression from P1 in the hindbrain was anteriorized after 4 or 24 h of treatment. P2 transcripts seemed to be less responsive and/or to have an indirect response to retinoic acid. The long 5′-untranslated region found in all Hoxd4 transcripts suggests that translation does not occur by a classical ribosome scanning mechanism. We have characterized the transcription unit of a murine Hox gene in the fourth paralogous group,Hoxd4. We have identified two Hoxd4transcription start sites by S1 analysis. The upstream promoter (P2) is 5.2 kilobase pairs upstream from the coding region, while the downstream promoter (P1) is 1.1 kilobase pairs distant. Both promoters bear a cluster of start sites. Multiple transcripts were identified by Northern blot, originating from both promoters and multiple polyadenylation signals. Expression of P1 transcripts in the neural tube shows an anterior border at the rhombomere 6/7 boundary, corresponding to previous reports (Gaunt, S. J., Krumlauf, R., and Duboule, D. (1989) Development 107, 131–141; Morrison, A., Moroni, M. C., Ariza-McNaughton, L., Krumlauf, R., and Mavilio, F. (1996) Development 122, 1895–1907). A more posterior boundary in the central nervous system was observed for P2 transcripts. We observed strong expression up to somite 6 and weak expression in somite 5, correlating with the phenotype of Hoxd4 null mutant mice (Horan, G. S. B., Nagy Kovàcs, E., Behringer, R. R., and Featherstone, M. S. (1995) Dev. Biol. 169, 359–372). In response to retinoic acid, expression from P1 in the hindbrain was anteriorized after 4 or 24 h of treatment. P2 transcripts seemed to be less responsive and/or to have an indirect response to retinoic acid. The long 5′-untranslated region found in all Hoxd4 transcripts suggests that translation does not occur by a classical ribosome scanning mechanism. Hox genes encode homeodomain-containing transcription factors that specify positional identity along the anteroposterior and appendicular axes of the developing embryo (4Krumlauf R. Cell. 1994; 78: 191-201Abstract Full Text PDF PubMed Scopus (1749) Google Scholar). Hox homologs can be found from Hydra to humans in the animal kingdom. This high degree of conservation throughout evolution suggests that these genes are indispensable participants in embryonic development. Mammals have 39 Hox genes, organized in four clusters, namely Hox A, B, C, and D(4Krumlauf R. Cell. 1994; 78: 191-201Abstract Full Text PDF PubMed Scopus (1749) Google Scholar, 5Zeltser L. Desplan C. Heintz N. Development. 1996; 122: 2475-2484PubMed Google Scholar). These multiple clusters are the result of duplication events from the original cluster. Therefore, corresponding genes are distributed in the same order along each cluster and are called paralogs. During embryogenesis, Hox genes are sequentially expressed both in time and space, according to their position in the cluster. The genes at the 3′-end are the first to be expressed and have the most anterior borders of expression. The genes more to the 5′-end are expressed later in time and have more posterior domains of expression (4Krumlauf R. Cell. 1994; 78: 191-201Abstract Full Text PDF PubMed Scopus (1749) Google Scholar). To safeguard their correct expression and function, Hoxgenes must be under precise regulation. Misexpression caused by either loss or gain of function has shown that the mutant mice present transformations and malformations that can affect central nervous system organization, somite derivatives, limbs, and other structures (4Krumlauf R. Cell. 1994; 78: 191-201Abstract Full Text PDF PubMed Scopus (1749) Google Scholar, 6St-Jacques B. McMahon A.P. Curr. Opin. Genet. Dev. 1996; 6: 439-444Crossref PubMed Scopus (30) Google Scholar). Murine Hoxd4 is expressed in the embryo from day 8.5 onward, declining by day 12.5 (1Gaunt S.J. Krumlauf R. Duboule D. Development. 1989; 107: 131-141PubMed Google Scholar). Its expression is detected in the spinal cord and prevertebra (1Gaunt S.J. Krumlauf R. Duboule D. Development. 1989; 107: 131-141PubMed Google Scholar, 7Featherstone M.S. Baron A. Gaunt S.J. Mattei M.-G. Duboule D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4760-4764Crossref PubMed Scopus (120) Google Scholar). Hoxd4 knockout results suggest that this transcription factor functions in the specification of vertebral identity along the cervical region (3Horan G.S.B. Nagy Kovács E. Behringer R.R. Featherstone M.S. Dev. Biol. 1995; 169: 359-372Crossref PubMed Scopus (118) Google Scholar). The Hoxd4 gene bears an autoregulatory element and a retinoic acid response element in its 5′-flanking region, and both elements are functional in vitro in transfected P19 cells (8Pöpperl H. Featherstone M.S. EMBO J. 1992; 11: 3673-3680Crossref PubMed Scopus (89) Google Scholar, 9Pöpperl H. Featherstone M.S. Mol. Cell. Biol. 1993; 13: 257-265Crossref PubMed Scopus (133) Google Scholar). The retinoic acid response element is highly conserved in the human homolog (10Moroni M.C. Vigano M.A. Mavilio F. Mech. Dev. 1993; 44: 139-154Crossref PubMed Scopus (58) Google Scholar), and it is required to partially recapitulate the endogenous expression of Hoxd4 in the central nervous system (CNS) 1The abbreviations used are: CNS, central nervous system; RA, retinoic acid; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; kb, kilobase pair(s); bp, base pair(s); UTR, untranslated region. of transgenic mice (2Morrison A. Moroni M.C. Ariza-McNaughton L. Krumlauf R. Mavilio F. Development. 1996; 122: 1895-1907PubMed Google Scholar). Although two regulatory elements have been mapped, a more basic feature of the murine Hoxd4 transcription unit remains unknown, the transcription start site(s). To better understand the regulation of murine Hoxd4, we have characterized its transcription unit. We have mapped two promoters, each one composed of a cluster of start sites. A complex splicing pattern gives rise to transcripts originating from the upstream start sites. We demonstrate that both promoters are active in the embryo and that a number of transcripts are expressed as a result of alternative promoter and/or polyadenylation signal usage. The anterior expression boundary of the upstream promoter is posterior to that of the downstream promoter and thus follows the colinearity rule. In addition, the two promoters respond differentially to RA induction. First strand synthesis and tailing were performed under the conditions recommended by Life Technologies, Inc. 5′-RACE kit, except for the reverse transcriptase reaction, which was incubated at 42 and 37 °C for 30 min each (11Lipkowitz S. Gobel V. Varterasian M.L. Nakahara K. Tchorz K. Kirsch I.R. J. Biol. Chem. 1992; 267: 21065-21071Abstract Full Text PDF PubMed Google Scholar). 1 μg of kidney or 11.5-day-old embryo total RNA was used for first strand cDNA synthesis. The RNA was annealed to antisense primer a (5′-CTCGCCTAGGTAGCCACCCC-3′) ore (5′-GAGATGGCGGCTTAATTGCC-3′) (Figs. 1 and 2). The cDNA was amplified using an antisense oligonucleotide from Hoxd4, primer b (5′-GCAAATATTCCTCGCACGGA-3′) or primer f(5′-TCCTAGAATTCGAGCAATTTACCT-3′), and an anchor primer complementary to the oligo(dC) tail (5′-CUACUACUACUAGGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3′) supplied by the manufacturer. PCR conditions were 0.4 μm primer, 0.2 mm dNTP mix, 1.25 units of Taq polymerase (Boehringer Mannheim), and PCR reaction buffer (10 mm Tris, pH 8.4, 50 mm KCl, 1.5 mm MgCl2, 0.01% gelatin, 0.01% Nonidet P-40, and 0.01% Tween 20). Cycling parameters were one long cycle (95 °C for 5 min, 60 °C for 2 min, and 72 °C for 40 min) followed by 35 cycles of 95 °C for 45 s, 60 °C for 45 s, and 72 °C for 3 min (11Lipkowitz S. Gobel V. Varterasian M.L. Nakahara K. Tchorz K. Kirsch I.R. J. Biol. Chem. 1992; 267: 21065-21071Abstract Full Text PDF PubMed Google Scholar). For the second round of amplification, 5 μl (110) of the first PCR reaction was used as template, and the long cycle was omitted. Nested antisense oligonucleotide c (5′-TCCTTACTCACCATCGCCTG-3′) org (5′-TTTCGGATCCCGCTGCTGCTGCTTCTGCTG-3′) and a shorter version of the anchor primer (5′-CUACUACUACUAGGCCACGCGRCGACTAGTAC-3′) were used in the second PCR. For both rounds of PCR, the enzyme was added to the reaction after the denaturation step.Figure 2Hoxd4 transcripts are composed of multiple exons. Panel a, representation of the S1 analysis and the results of the second 5′-RACE. The upper thick linedepicts Hoxd4 genomic sequences. S1, S1 end point of protection. e, primer used for the reverse transcriptase reaction; f and g, nested primers used for PCR.Boxes represent the Hoxd4 coding region; thestippled box represents the homeobox. K,KpnI; E, EcoRI. The cDNA clone obtained from 5′-RACE is shown to scale below the genomic map. It is composed of part of exon 1, exons 2 and 3, and a portion of exon 4 bounded by primer g. Note that primers eand f fall within exon 4 sequences that do not appear in this final 5′-RACE product. The sequence given at the bottomshows the consensus 3′-acceptor splice site flanking the S1 end point of protection adjacent to exon 4 (C, position −1817).Panel b, comparison between the consensus sequence for 5′-donor and 3′-acceptor splice sites and Hoxd4 intron exon/boundaries. The human HOXD4 (hHOXD4) sequence corresponds to the boundaries of exons 4 and 5 in the mouse (mHoxd4) but does not conserve the splice sites (see Fig.3 c for the relative positions of exons 4 and 5).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Specific PCR products (as assessed by Southern blot; data not shown) were gel-purified and cloned into EcoRV-linearized pBluescript KS (Stratagene) or T-tailed pBluescript KS. T-tailed vector was made by linearizing pBluescript KS with EcoRV and incubating with Taq polymerase in the presence of 2 mm dTTP for 2 h at 70 °C. The T-tailed vector was purified by phenol extraction and ethanol precipitation. RACE clones were sequenced by the standard Sanger method (Pharmacia T7 sequencing kit). S1 analysis was performed exactly as described (12Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 1. Wiley Interscience, Boston1989: 4.6.1-4.6.13Google Scholar). For single-stranded DNA probes, antisense oligonucleotideh (5′-TGGTAGAGAAGCTTAGAGG-3′) or d(5′-GGCTGTACAATTTCACCAGGCAAAGTCGATCATCCTGG-3′) was kinase-labeled, extended with Klenow using genomic murine Hoxd4 as template, and linearized with HindII or KpnI, respectively. 5 × 104 cpm of probe was hybridized to 50 μg of 11.5-day-old embryo total RNA. The BamHI-AccI probe was a 2.1-kb fragment double-stranded, labeled at theAccI end. An RNase protection assay was performed to verify if the 5′-end of the RACE clone (shown in Fig. 1 b) corresponded to a transcription start site (data not shown). Probe synthesis and hybridization were carried out as described (13Nédellec P. Dveksler G.S. Daniels E. Turbide C. Chow B. Basile A.A. Holmes K.V. Beauchemin N. J. Virol. 1994; 68: 4525-4537Crossref PubMed Google Scholar). RNase T2 (Life Technologies, Inc.) was used for digestion (14Saccomanno C.F. Bordonaro M. Chen J.S. Nordstrom J.L. BioTechniques. 1992; 13: 847-849Google Scholar), and protected fragments were resolved in a 6% denaturing acrylamide gel. The probe was made by subcloning a 493-bp EcoRI-KpnI fragment from the Hoxd4 5′ region into pBluescribe (Stratagene), which was called EK (Fig. 4 a). The template was linearized with EcoRI, and the antisense probe was synthesized using T3 RNA polymerase (Boehringer Mannheim). 5 × 104 cpm of probe was hybridized to 30 μg of 11.5-day-old embryo total RNA. A control reaction using tRNA was run in parallel. Northern blot was performed essentially as described by Chow et al. (15Chow L.M.L. Ratcliffe M.J.H. Veillette A. Mol. Cell. Biol. 1992; 12: 1226-1233Crossref PubMed Scopus (17) Google Scholar). 5 μg of poly(A)+ RNA (isolated using the Dynabeads system, Dynal) from 11.5-day-old mouse embryo was used. The 32P-labeled probe was added at 1 × 106 cpm/ml. Probe H-X is homologous to the 3′-untranslated region (UTR), and it is aHindIII-XbaI fragment about 400 bp long. Probe A is a 323-bp-long PCR fragment, from −1811 to −1489 from the coding region. Probe B is a PstI-EcoRI fragment within the intronic region just upstream of exon 5. Probe C is a 282-bp-long PCR fragment, from −402 to −121 from the coding region. To avoid cross-hybridization across paralog group 4, we designed probe C outside a region of high homology among the members of this group (from −120 up to the coding region). The blot hybridized with probe C was run alongside commercial size markers (Promega). Whole mount in situ hybridization was performed essentially as described (16Wilkinson D.G. Nieto M.A. Methods Enzymol. 1993; 225: 361-372Crossref PubMed Scopus (724) Google Scholar), incorporating one extra step (17Conlon R.A. Rossant J. Development. 1992; 116: 357-368Crossref PubMed Google Scholar). Just before prehybridization, embryos were treated with 0.1% borohydride in PBT (phosphate-buffered saline with 0.1% Tween 20) for 20 min at room temperature followed by three PBT washes. The alkaline phosphatase-conjugated anti-digoxigenin antibody (Boehringer Mannheim) was preadsorbed with embryo powder prepared as described (16Wilkinson D.G. Nieto M.A. Methods Enzymol. 1993; 225: 361-372Crossref PubMed Scopus (724) Google Scholar) and used at a 1:5000 dilution. Embryos were incubated with antibody overnight at 4 °C; washed the next day, with five changes of the washing solution; and left overnight in TBST (25 mm Tris-HCl, pH 7.5, 0.14 m NaCl, 2.7 mm KCl, 0.1% Tween 20, 2 mm levamisole). Color reaction was performed as described (16Wilkinson D.G. Nieto M.A. Methods Enzymol. 1993; 225: 361-372Crossref PubMed Scopus (724) Google Scholar). Single-stranded RNA probes containing digoxigenin were synthesized from linearized template DNA exactly as directed by the manufacturer (DIG RNA labeling kit, Boehringer Mannheim). Probe BgH is a 950-bp-longBglII-HindIII fragment of murine Hoxd4starting in the homeobox, cloned into pGem-1 (Promega) (1Gaunt S.J. Krumlauf R. Duboule D. Development. 1989; 107: 131-141PubMed Google Scholar). The template was linearized with EcoRI, and the antisense strand was synthesized with SP6 RNA polymerase (Boehringer Mannheim). Probe Sma is a 470-bp Sma fragment starting 370 bp upstream of theHoxd4 coding region, cloned into pBluescribe. Probe EK is a 493-bp EcoRI-KpnI fragment 5′ of the downstream start site, cloned into pBluescribe. Both templates were linearized with EcoRI, and the probes were synthesized using T3 RNA polymerase (Boehringer Mannheim). The Krox20 probe was generously provided by Dr. D. Wilkinson. Pregnant CD-1 female mice were administered RA essentially as described by Conlon and Rossant (17Conlon R.A. Rossant J. Development. 1992; 116: 357-368Crossref PubMed Google Scholar). A stock solution of 25 mg/ml all-trans-retinoic acid (Sigma) in Me2SO was diluted 10 times in corn oil just before use, and 0.2 ml was delivered by gavage for a final dose of approximately 20 mg/kg of maternal body weight. Control mice were administered the same mixture without RA. For treatments of 4 h (short treatment), mothers were treated at 8 a.m. on embryonic day 8.5, and the embryos were recovered at noon of the same day. For treatments of 24 h (long treatment), mothers were treated at noon on embryonic day 8.5, and the embryos were recovered at noon on the following day. The day of appearance of the vaginal plug was taken as 0.5 days postcoitus. For all treatments, we analyzed between 10 and 25 embryos. To map the transcriptional start site of Hoxd4, we performed an initial S1 analysis with mouse kidney RNA. Using aBamHI-AccI fragment as a probe, we observed two end points of protection (indicated as x and y in Fig. 1 a). This suggested that murine Hoxd4 may have two promoters, as does its human homolog (18Cianetti L. Cristofaro A.D. Zappavigna V. Bottero L. Boccoli G. Testa U. Russo G. Boncinelli E. Peschle C. Nucleic Acids Res. 1990; 18: 4361-4368Crossref PubMed Scopus (36) Google Scholar). We designed a 5′-RACE strategy based on the S1 end point of protection y(Fig. 1 b). The first RACE product was obtained using oligonucleotide a as a primer for the reverse transcriptase reaction and using primers b and c for each round of PCR, respectively (Fig. 1 b). Hoxd4-specific fragments (evaluated by Southern blot; data not shown) were cloned into pBluescript and sequenced. One clone, 140 bp long, showed homology to mouse Hoxd4 sequence; however, the homology was not contiguous. The 5′-end of the clone started at position −1372 from the coding region, and at position −1300 the homology was interrupted. The sequence continued from position −402, and the 3′-end of the clone corresponded to primer c (Fig. 1 b). Analysis of the genomic sequence flanking the point of discontinuity showed homology to the conserved consensus for 5′-donor and 3′-acceptor splice sites. An intron between positions −1300 and −402 within the 5′-UTR was thus delimited. Interestingly, neither splice site is conserved in the human HOXD4 sequence, which demonstrates a divergence between the two species (Fig.2 b). RNase protection analysis showed a protection of the probe by sequences upstream of the 5′-end of the RACE product. This demonstrated that the RACE clone 5′-end did not correspond to a transcription start site (data not shown). S1 analyses were performed on sequences further upstream in an attempt to map the start site. An S1 analysis product was finely mapped to a cytidine at position −1817 from the coding region, which is preceded by PyAG, the known consensus for a 3′-splice acceptor site (see Fig. 2 a). To test the possibility of another splice event, we designed a second 5′-RACE strategy (Fig.2 a). For the second RACE product, primer e was used for the reverse transcriptase reaction, and nested primersf and g were used for the two rounds of PCR, respectively. Specific fragments (evaluated by Southern blot; data not shown) were cloned into a T-tailed vector and sequenced. Eleven clones contained a sequence whose 3′-end corresponded to primer gbut became discontiguous at position −1817, at the S1 end point of protection. The diverged 5′-end of these products was composed of three juxtaposed sequences of 85, 79, and 36 bp with homology toHoxd4 genomic sequences in a region between 4 and 5 kb upstream of the open reading frame. We compared the genomic flanking residues of these three segments and observed a very good homology with splice site consensus sequences (Fig. 2 b). Therefore, this cDNA clone was composed of four exons, which we named 1, 2, 3, and 4. Overall, the two cDNA clones demonstrated that the transcript starting from an upstream promoter undergoes several splice events (Fig. 4 a) and therefore is composed of multiple exons. We had yet to confirm whether the 5′-end of the second RACE clone corresponded to a transcription start site. For that purpose, we did an S1 analysis using a probe made by extension from primer h, within exon 1, and extending up to a HindII site (Fig.3, a and c). We obtained 10 end points of protection covering 70 nucleotides. These were further confirmed on a second S1 analysis using a longer probe linearized at the Pst site and gave one more protection (data not shown). This gave a total of 11 start sites spanning a region of 120 nucleotides. In this manner, we mapped a distal promoter (P2) 5.2 kb upstream from the coding region, whose transcripts are processed by the splicing machinery to yield a 5′-UTR of 1.1 kb (Fig.4 a). The original S1 end point of protection x (Fig. 1 a) was confirmed by an S1 analysis using a probe 183 nucleotides long, extended from oligonucleotide d and linearized with KpnI (Fig.3, b and c). This S1 product was analyzed beside a sequencing reaction, and the promoter (P1) was finely mapped. P1 is composed of a cluster of four start sites, ATGG, at positions −1137 to −1140 from the coding region (Fig. 3 b). This maps very close to a human HOXD4 start site (18Cianetti L. Cristofaro A.D. Zappavigna V. Bottero L. Boccoli G. Testa U. Russo G. Boncinelli E. Peschle C. Nucleic Acids Res. 1990; 18: 4361-4368Crossref PubMed Scopus (36) Google Scholar) and suggests a conservation of regulatory elements between the two species. In the human HOXD4 gene, there is a downstream promoter 21 bp 5′ from the coding region (18Cianetti L. Cristofaro A.D. Zappavigna V. Bottero L. Boccoli G. Testa U. Russo G. Boncinelli E. Peschle C. Nucleic Acids Res. 1990; 18: 4361-4368Crossref PubMed Scopus (36) Google Scholar). To determine whether an equivalent start site was present in the murine gene, we performed an RNase protection assay spanning that region. However, we were unable to detect any transcripts originating from this area (data not shown). Northern blot analysis had shown that multiple transcripts of Hoxd4 are present in the mouse embryo (7Featherstone M.S. Baron A. Gaunt S.J. Mattei M.-G. Duboule D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4760-4764Crossref PubMed Scopus (120) Google Scholar). We wished to correlate these multiple transcripts with the usage of the two promoters identified. For this purpose, we did Northern blots using probes derived from the 5′-UTR (Fig. 4 a). Probe A should only detect transcripts originating at P2. Probe B should detect transcripts originating from P1 and potential unspliced transcripts originating from P2 (if there are any). Probe C detects transcripts originating at both promoters. With all three probes, we detected three major bands of similar sizes, which are 2.6, 4.2, and 5.6 kb, based on commercial molecular size markers run alongside blot C (Fig.4 b and data not shown). The 2.6-kb band corresponds to the predicted size of transcripts originating from either P1 or P2 (assuming that all introns have been spliced out of the P2 transcript). The two known poly(A) signals are 270 and 572 nucleotides downstream of the stop codon. 2M. S. Featherstone, unpublished results. We believe that the broad 2.6-kb band comprises transcripts terminating at both of these sites. In the human HOXD4 homolog, there are poly(A) signals 3 kb downstream of the stop codon (18Cianetti L. Cristofaro A.D. Zappavigna V. Bottero L. Boccoli G. Testa U. Russo G. Boncinelli E. Peschle C. Nucleic Acids Res. 1990; 18: 4361-4368Crossref PubMed Scopus (36) Google Scholar). This suggests the possibility of additional poly(A) signals further downstream in the mouseHoxd4 homolog as well. Using aHindIII-Xba fragment as a probe (Fig.4 a), we detected two bands corresponding to the larger size range, at 4.2 and 5.6 kb (data not shown). Based on this result, we propose two additional polyadenylation signals further downstream. Alternatively, there could be only one additional poly(A) signal, and the transcripts would be subject to additional post-transcriptional processing at the 3′-end. Either option would result in two more variations in the 3′-UTR. Therefore, we conclude that both promoters are used in the mouse embryo and that the variety of molecular weights detected in the Northern blots is due to the presence and usage of various poly(A) signals. Having demonstrated that at least twoHoxd4 promoters are used in the mouse, we wanted to examine how these promoters are regulated in the mouse embryo, for which we used whole mount in situ hybridization. We first hybridized 9.5-day embryos with probe BgH (Fig. 4 a). Expression in the CNS confirmed the anterior boundary at the border of rhombomeres 6 and 7 (Fig. 5, a andb), as earlier described (1Gaunt S.J. Krumlauf R. Duboule D. Development. 1989; 107: 131-141PubMed Google Scholar). In the somitic mesoderm, expression was strong in somite 6 and posterior and was weak in somite 5 (Fig. 5 b). There was strong expression in the fore limb bud, as previously shown (19Dollé P. Izpisúa-Belmonte J.-C. Falkenstein H. Renucci A. Duboule D. Nature. 1989; 342: 767-772Crossref PubMed Scopus (474) Google Scholar) and along the tail bud. In addition, we detected expression in the fourth branchial arch (Fig.5 a). We used different probes to compare the expression of the P1 and P2 promoters. Probe EK detects only transcripts originating at P2 (Fig.4 a). Probe Sma detects transcripts originating at both promoters (Fig. 4 a). Thus, expression domains uniquely detected by the P1-P2 Sma probe reflect the specific activity of the P1 promoter. In both 8.5- and 9.5-day-old embryos, the staining was different for the two probes, especially in the CNS, thus demonstrating differential activity of the two promoters (Fig. 5, c and d). The staining with the P1-P2 Sma probe reproduced the pattern observed with probe BgH, which is consistent with the predicted ability of both probes to detect all transcripts from the Hoxd4 locus (Fig.5 c, embryo on bottom; d, embryo onleft). However, transcripts detected with the P2-specific EK probe have an anterior border in the CNS that is more posterior (Fig.5 c, embryo on top; d, embryo onright). This result shows that the more anterior expression domain in the CNS is due to P1 activation but not P2. In the other domains, including the entire somitic column, the activity of both promoters overlaps (Fig. 5, c and d). Both mouse and human Hoxd4 transcripts are induced in cell culture upon treatment with RA. A retinoic acid response element is conserved in the genes of both species (2Morrison A. Moroni M.C. Ariza-McNaughton L. Krumlauf R. Mavilio F. Development. 1996; 122: 1895-1907PubMed Google Scholar, 9Pöpperl H. Featherstone M.S. Mol. Cell. Biol. 1993; 13: 257-265Crossref PubMed Scopus (133) Google Scholar, 10Moroni M.C. Vigano M.A. Mavilio F. Mech. Dev. 1993; 44: 139-154Crossref PubMed Scopus (58) Google Scholar), located between the P1 and P2 promoters as mapped here (Fig. 4 a). We wished to examine how each promoter would respond to retinoic acid in vivo. After a 4-h RA treatment on day 8.5, embryos hybridized with the P1-P2 Sma probe showed an anterior shift of Hoxd4 expression in the hindbrain. This anteriorization is especially evident in embryos labeled simultaneously for Hoxd4 and Krox20 (Fig.6 a). In the RA-treated embryos, the Hoxd4 boundary is pushed anteriorly toward r5 (Fig. 6 a, embryo on the right). Embryos stained with P2-specific EK probe showed no difference in expression after 4 h of RA treatment (data not shown). The 24-h RA treatment also led to anteriorization in the CNS of transcripts detected with the P1-P2 Sma probe. The anterior border is now halfway overlapping with the otic vesicle at the rhombomere 5 and 6 boundary, which is one rhombomere more anterior than the normal border (Fig. 6 b, embryo on the right). Embryos stained with the P2- specific EK probe showed anteriorization in one limited domain, ventral to the neural tube (Fig. 6 c, embryo on theright). Overall, these results suggest that embryonic Hoxd4expression is responsive to RA treatment. This response is primarily from P1, which is affected after 4 h of treatment. Thus, P1 is more sensitive to RA and probably subject to a direct effect of RA. P2 showed a delayed and limited response; therefore, we concluded that it is less sensitive to RA and responds indirectly to its action. We have analyzed various aspects of the murine Hoxd4transcription unit. First, we mapped two transcription start sites that drive Hoxd4 expression and dissected the ensemble of transcripts present in 11.5-day-old embryos. Second, we characterized the differential usage of these promoters in the mouse at 8.5 and 9.5 days postcoitus and their response to exogenous retinoic acid. Both the upstream and the downstream promoters of murine Hoxd4 are composed of a cluster of start sites and do not bear a recognizable TATA box. TATA-less promoters were originally found in so-called “house-keeping” genes but later became a common feature especially among genes that are differentially expressed during development, such as oncogenes, growth factors, growth factor receptors, and transcription factors (20Azizkhan J.C. Jensen D.E. Pierce A.J. Wade M. Crit. Rev. Eukaryotic Gene Expression. 1993; 3: 229-254PubMed Google Scholar). In the absence of a TATA box, another class of binding sites might be present, the initiator motif (21Smale S.T. Baltimore D. Cell. 1989; 57: 103-113Abstract Full Text PDF PubMed Scopus (1151) Google Scholar). There are different classes of initiatior elements, and they can bear binding sites for specific DNA-binding proteins (22Zawel L. Reinberg D. Prog. Nuc" @default.
• W2124304811 created "2016-06-24" @default.
• W2124304811 creator A5002112758 @default.
• W2124304811 creator A5032765558 @default.
• W2124304811 creator A5070812463 @default.
• W2124304811 date "1997-11-01" @default.
• W2124304811 modified "2023-10-18" @default.
• W2124304811 title "Characterization and Retinoic Acid Responsiveness of the Murine Hoxd4 Transcription Unit" @default.
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