Proc. Nadl. Acad. Sci. USA Vol. 91, pp. 5335-5339, June 1994 Developmental Biology Inhibition of retinoic acid-induced activation of 3' human HOXB by antisense oligonucleotides affects sequential activation of genes located upstream in the four HOX clusters (/embryonal carclnoma/development/ expresslon/retinolc acid) ANTONIO FAIELLA*, VINCENZO ZAPPAVIGNA*, FULVIO MAVILIO*, AND EDOARDO BONCINELLI*tt *DIBIT, S. Raffaele Scientific Institute, Via Olgettina 60, 20132 Milan; and tCentro Infrastrutture Cellulari, Consiglio Nazionale delle Ricerche, Via Vanvitelli 32, 20129 Milan, Italy Communicated by Ronald M. Evans, February 28, 1994

ABSTRACT Most homeobox genes belonging to the Hox activity, Hensen's node, and the floor plate ofthe neural tube family are sequentially activated in embryonal carcinoma cells (5). Systemic or local administration of excess RA to devel- upon treatment with retinoic acid. Genes located at the 3' end oping vertebrate embryos has profound effects on axial ofeach one ofthe four Hox clusters are activated first, whereas patterning and specification ofregional identities in a number upstream Hox genes are activated progressively later. This of structures. This is invariably accompanied by repatterning activation has been extensively studied for human HOX genes of expression domains, thus suggesting a specific in the NT2/D1 cell line and shown to take place at the role for Hox gene products as molecular targets of the transcriptional level. Tounderstand the molecular mechanisms morphogenetic signals provided by retinoids (1, 4-7). of sequential HOX gene activation in these cells, we tried to The direct link between retinoids and Hox gene regulation modulate the expression of 3' HOX genes through the use of has been studied in human and mouse embryonal carcinoma antisense oligonucleotides added to the culture medium. We (EC) cells (2). Most Hox genes from all four clusters are chose the HOXB locus. A 5- to 15-fold reduction of the activated upon induction with RA in a concentration- expression of HOXBI and HOXB3 was sufficient to produce a dependent manner and in a sequential order: 3' HOXB genes signicant inhibition of the activation of the upstream HOXB respond early, whereas upstream genes respond progres- genes, as well as of their paralogs in the HOXA, HOXC, and sively later (8-10). This sequential activation is closely HOXD clusters. Conversely, no effect was detectable on down- related to that observed during embryonic development and stream HOX genes. The extent of this inhibition increased for has been called temporal colinearity (11, 12). progressively more-5' genes. The stability ofthe corresponding We have previously reported (2, 8, 9) the differential mRNAs appeared to be unaffected, supporting the idea that the activation of the 38 human HOX genes (13) in the human EC observed effect might be mediated at the tr tiona level. cell line NT2/D1 upon treatment with RA. This analysis These data suggest a cascade model ofprogressive activation of allowed us to define two major sets of HOX genes. This Hox genes, with a 3'-to-5' polarity. division again follows a colinearity scheme: genes ofparalogy groups 10-13, located at the 5' end of the HOX loci, do not Homeobox genes encode transcription factors containing a respond to RA treatment or are downregulated upon treat- highly conserved, sequence-specific DNA-binding motif ment, whereas HOX genes downstream from paralogy group termed the homeodomain (1). Homeobox genes belonging to 10 are responding genes. Most genes located at the 5' end of the Hox family encode containing a homeodomain HOXloci are not expressed in either untreated or treated EC closely related to the archetypal Drosophila Antennapedia cells; among these 5' genes, one HOXC (HOXC13) gene and homeodomain and appear to be homologous to the homeotic four HOXD (HOXD)O-13) genes are weakly expressed in genes ofthe fruit fly. Mouse and human Hox homeobox genes untreated cells and downregulated upon treatment with RA. are clustered in a homologous arrangement of 13 paralogy Responding 3' HOXgenes are activated by RA in a sequential groups in four restricted genomic regions, termed Hox loci, order, colinear with their 3'-to-5' arrangement in the cluster: Hoxa-Hoxdin the mouse and HOXA-HOXD in man, located 3' HOX genes respond early to the treatment, whereas on four (1, 2). The genes present in a given Hox upstream genes respond progressively later. This activation cluster are all transcribed in the same 5'-to-3' direction (2). appears to be regulated primarily at the transcriptional level, Hox genes are developmentally regulated according to tem- suggesting a cascade model ofactivatingHOXgene products. porally and spatially restricted patterns, particularly in the It was impossible to prove or disprove this model by using central nervous system, the axial skeleton, and the limbs (1). inhibitors ofprotein synthesis, since the observed timing and Their expression along the embryonic anterior-posterior polarity of gene activation are disrupted in the absence of body axis uniformly follows a 5'-posterior/3'-anterior rule. synthesis (2, 9). We now report on the use of This phenomenon, first observed for the homeotic genes of antisense oligodeoxynucleotides (ODNs) (14, 15) against 3' Drosophila and termed colinearity (3), still represents a HOXgenes in order to prevent their activation and show that formidable biological problem. this affects the subsequent activation of HOX genes located Hox genes are specifically regulated, both in vivo and in upstream in the four loci. cell culture, by retinoic acid (RA), a naturally occurring derivative of vitamin A with pleiotropic effects on develop- MATERIALS AND METHODS ment and cell differentiation (4, 5). Retinoids are present at Cell Culture and Antisense ODN Treatment. Human EC active concentrations in embryonic structures with a proven cells of the NT2/D1 cell line were cultured in high-glucose role in pattern formation, such as the zone of polarizing Dulbecco's modified Eagle's medium (DMEM) supple-

The publication costs ofthis article were defrayed in part by page charge Abbreviations: EC, embryonal carcinoma; ODN, oligodeoxynucle- payment. This article must therefore be hereby marked "advertisement" otide; RA, retinoic acid. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 5335 Downloaded by guest on September 25, 2021 5336 Developmental Biology: Faiella et al. Proc. Natl. Acad Sci. USA 91 (1994) mented with 10%o fetal bovine serum and 20 mM Hepes buffer looked for an ODN able to decrease significantly the expres- (all from GIBCO) in a humidified atmosphere of 5% CO2 in sion level of HOXB3, the third gene of the HOXB locus air. NT2/D1 cells were induced with all-trans-RA (10 ,uM; startingfrom its 3' extremity. We assayed several ODNs (Fig. Sigma) as described (9). Induced cells (3-6 x 106) were 1) centered around the first AUG codon. A treatment of this treated in 75-cm2 tissue culture flasks with the following type has repeatedly been successful in interfering with the antisense ODNs at 40 pM: 5'-CAGTGGGTGGCAACTTGG- translation of the corresponding protein (15). As a conse- 3'; 5'-TCGCTGGGTGAGGCCTGG-3'; 5'-GCCTGGG- quence ofthe pairing ofthe ODN and the mRNA, a decrease CAGTGGGTGGCAACTTGG-3'; 5'-TTTCTGCATCGC- in the abundance ofthe latter has often been observed. Since TGGGTGAGG-3'; 5'-CTGCATCGCTGGGTGAGG-3'; and an antibody against the HOXB3 gene product was not avail- 5'-GTCGTAGTAGGTGGCTTTCTGCATC-3'; all directed able, we decided to measure the abundance of the corre- against the region encoding the N terminus of the HOXB3 sponding RNA by means ofRNase protection mapping. One protein and/or its flanking regions (Fig. 1). Only the last one of these oligonucleotides produced a 5-fold decrease in was effective in reducing HOXB3 mRNA. HOXB3 mRNA after 24-48 hr ofRA treatment (Figs. 2a and NT2/D1 cells were similarly treated with the following 3a). We termed this ODN the anti-HOXB3 oligonucleotide antisense ODNs directed against the region encoding the N (Fig. 1). Similarly, we proceeded with HOXBI, the most 3' terminus of the HOXB1 protein: 5'-CTCTAACAAGGAGT- gene of the same HOXB locus and found an anti-HOXBI TCATC-3'; 5'-ATTATAGTCCATGCGTCA-3'; 5'-AGT- ODN (Fig. 1) able to decrease by a factor of 15 the HOXB1 TCATCCTATTATAGTCCATGCG-3'; 5'-CTCTAACAAG- mRNA abundance after 24 hr of RA treatment (Figs. 2b and GAGTTCATCCTATTA-3'; 5'-TCATCCTATTATAGTC- 3a). The complementary sense ODNs did not produce any CATGCGTCA-3'; 5'-GAAGGAGTTCATCCTATTAT- effect (data not shown). AGTCCATGCGTC-3'; 5'-GGTACTCTAAGAAGGAGT- We then studied the expression ofHOXB genes in NT2/D1 TCATCCTA-3'; and 5'-TCTAAGAAGGAGTTCATCC- cells cultured in the continuous presence of RA and anti- TATTATAGTCCATGCG-3'. Of these the last one was the HOXB3 ODN (Figs. 2a and 3 a and b). This locus does not most effective. For longer incubation periods (120 and 144 hr) contain genes ofparalogy groups 10-13 and every gene ofthis the medium was replaced after 72 hr with fresh medium locus is activated by RA treatment (2). With the addition of containing the same concentrations of RA and ODNs. Tests the anti-HOXB3 ODN, activation of upstream genes, were initially performed to assess the toxicity and stability of HOXB4-HOXB8, was reduced while the expression oftwo 3' the ODNs in culture by using in parallel conventional and genes of the same locus, HOXBI and HOXB2, appeared not phosphorothioated ODNs (15). No obvious toxic effects on to be affected (Figs. 2a and 3a). The extent of this downreg- the cells were observed even at a final concentration of 100 ulation was progressively higher for progressively more-5' ,pM. No significant degradation of either conventional or HOXBgenes, suggesting a cumulative effect derivedfrom the modified ODNs was observed in these cells, even after the inhibition of the gene products of the various HOXE genes longest incubation period. located downstream in the locus. Of course, the effect on RNA Analysis. Cytoplasmic RNA was extracted from upstream HOXB genes was observed later than that on confluent cells in 75-cm2 tissue culture flasks at various times downstream genes (Figs. 2a and 3b), because activation of after RA and antisense ODN treatment and hybridized over- these upstream genes takes place later. night in 30- to 50-pg aliquots to radiolabeled antisense RNA In conclusion, with the addition of the anti-HOXB3 ODN probes (described in ref. 9), corresponding to the analyzed we observed a polar effect: the activation of upstream genes HOX genes or f-actin RNA probe at 550C as previously was reduced while the expression oftwo 3' genes ofthe same described (9). Mixtures were then digested with RNases A locus, HOXBI and HOXB2, was not affected. The polarity of and T1 (1 hr at 320C) and proteinase K (all from Promega), the observed effect was confirmed by the analysis ofHOXE extracted with phenol/chlorophorm, and precipitated in eth- in the presence of the anti-HOXBJ ODN anol. Electrophoresis was carried out in urea/6% polyacryl- (Figs. 2b and 3 a and c). In this case, all upstream HOX8 amide sequencing gels, and the gels were dried and exposed genes were progressively downregulated. This experiment for 8-48 hr at -700C to Kodak X-AR5 film. Quantitation of also showed that the observed effect was not a peculiarity of the relative amounts of protected mRNA was performed by specific HOXB genes. standard densitometry scanning of at least three autoradio- We extended the analysis of the effect of the anti-HOX83 graphs representing independent experiments. ODN to genes of the other three HOX clusters (Figs. 2c and 3a). We observed a certain degree of inhibition of genes RESULTS AND DISCUSSION located upstream from paralogy group 3, to which HOXB3 Forour analysis we chose 3' genes ofthe HOXBlocus, whose belongs, in other HOX loci as well (Fig. 3a). Genes of the simpler structure has been well characterized (1, 2). We first downstream groups 1 and 2 were not affected. Thus, we MetGlnLysAlaThrTyrTyrAspAsn ... TTTCCAAGTTGCCCTCAACCCACTCACGCGATGCAGAAAGCcAccTAcTACGACAAC... HOXB3 anti HOXB3

MetAspTyrAsnArgMetAsnSerPheLeuGluTyrProLeuCysAsnArgGlyProSer ...TGACGCATGGACTATAATAGGATGAACTCCTTCTTAGAGTACCCACTCTGTAACCGGGGACCCAGC... HOXB1 anti HOXBW

FIG. 1. Location and sequence of ODNs assayed for their ability to interfere with the expression ofHOXB1 and HOXB3. Those exhibiting a maximal effect are shown as thick lines. Downloaded by guest on September 25, 2021 11.01vevelopmental Biology: Faiella et aL Proc. Nati. Acad. Sci. USA 91 (1994) 5337 C anti HOXBI a c anti HOXB3 b hrs 12 24 48 12 24 48 hrs 24 36 48 72 24 36 48 72 "A.l HOXB1 4wr_~ ___m HOXB1 1 act HOXB2 -_ _ hrs 12 24 48 12 24 48 13 act HOXB2

hrs 36 48 72 120 36 48 72 120 13 act HOXB3 hrs 36 48 72 36 48 72

AM _ or_ HOXB3 _ 13 act *e~IR -4 f3 act hrs 36 48 72 36 48 72 hrs 36 48 72 36 48 72 w.. '.0 HOXB4 _ _.. HOXB4 ;::: : HOXB5 Ismo. 13 act _lh,-Am. B13 act C C anti HOXB3

hrs 3648 120144 3648 120144 hrs 48 72 120 48 72 120 HOXB6 9 _1 HOXC13

A e HOXB7 13 act -IPW 61.M B act hrs 72 120 144 72 120144 hrs 36 48 120144 36 48 120144 _ _1111 HOXB8 aU --t HOXD13

_I~ 1_ act 13 act

FIG. 2. Expression of HOX genes in differentiating NT2/D1 cells in the presence of 10 t&M RA (control, C) and RA plus the indicated antisense ODN for various times (12-144 hr as indicated above the lanes). RNase protection analysis of total RNA (15 pg per lane) is shown. Expression ofhuman ,-actin (a-act) mRNA is shown as an internal control. (a) HOXB genes in the presence ofthe anti-HOXB3 ODN. (b) Four HOXB genes in the presence of the anti-HOXBI ODN. (c) The most5' HOXC (HOXC13) and HOXD (HOXD13) genes in the presence of the anti-HOXB3 ODN.

observed a polar effect that extended to other HOX loci. We measured the stability of mRNA of selected genes Particularly interesting was the case of the most 5' genes of located in HOXB and in other HOX loci, in the absence and the HOXC and HOXD loci, HOXC13 and HOXD13 (Fig. 2c). in the presence of the anti-HOXB3 oligonucleotide. We did These genes are weakly expressed in untreated NT2/D1 cells not observe any significant change (data not shown). Most and their expression is downregulated by addition of RA (2, mRNAs were found to have a half-life of about 4 hr, as 9). In the presence of the anti-HOXB3 ODN this downregu- previously reported (9). This suggests that the observed lation was not observed, or at least not until very late (Fig. interactions are at the transcriptional level for genes of all 2c). This demonstrates that the downregulation ofthese far 5' HOX loci. genes is mediated, directly or indirectly, by the products of Fig. 3a summarizes the maximal extent of the downregu- 3' HOX genes. lation of analyzed genes, while Fig. 3 b and c show the The observed effect on genes of other HOX loci can be temporal profile of the expression of some HOX genes. The explained as a trans effect, whereby gene products of some observed effect was relatively rapid and became maximal HOXB genes act to regulate the expression of other HOX within a few hours (Fig. 3 b and c). At later times a certain genes. This is not surprising in light of what is known about degree of reduction of the induced downregulation was the nature ofHox gene products (1, 16-18). Nevertheless, we observed for some genes. This slight effect is difficult to cannot exclude the possibility that the anti-HOXB3 antisense explain but it may simply mean that the expression level of ODN might affect directly the expression ofotherHOXgenes these genes is the effect of several concurrent factors. The and in particular that ofthe paralogous HOXA3 and HOXD3. expression level of HOXB3 itself showed some recovery at Though this cannot be excluded, it seems unlikely because later times. the corresponding nucleotide sequences differ from that of Fig. 4 summarizes the most signiicant findings of this HOXB3 in 2 and 7 nucleotides out of 25 nucleotides, respec- work. Both anti-HOXB3 and anti-HOXBI antisense ODNs tively. Furthermore, the HOXC locus does not contain a affect the expression level ofHOX genes located upstream in group 3 gene, but upstream genes ofthis locus were affected, the locus and responding later to RA but leave unaffected even if at a lower level (Fig. 3a). those located downstream. Downloaded by guest on September 25, 2021 5338 Developmental Biology: Faiella et al. Proc. Nad. Acad. Sci. USA 91 (1994)

a (abd-A, Ubx, Antp) (Scr) (zen1, zen2) 5, Abd-B Dfd pb lab 31

25 3 V V

35 30 20 4 4 5 A v v v v v 10 anti HOXB3

5 5 3 A v v 20

30 10 5 V V V

I anti HOXB1 30 23 20 15 V V V V

-*X HOXB1 - * - HOXB1 + anti HOXB1 - X HOXB3 - HOXB2 - * - HOXB3 + anti HOXB3 - HOXB6 - * - HOXB2 + anti HOXB1 z HOXB3 z - w - HOXB6 + anti HOXB3 0 0 HOXB8 - * - HOXB3 + anti HOXB1

- a -HOXB8 + anti HOXB3 -_-- HOXB4 -_w - HOXB4 + anti HOXB1

72 96 120 144 24 36 TIME (hrs) TIME (hrs)

FiG. 3. Diagram of HOX gene expression. (a) The 38 homeobox genes (circles) in the four human HOX clusters. Drosophila homologues of the human HOX genes are indicated at the top. The transcriptional orientation of all 38 HOX genes is from left to right. HOX genes whose expression level was measured are indicated by bold circles. Below each bold circle is shown the maximal effect of antisense ODN treatment on steady-state mRNA abundance: numbers above an arrowhead pointing downward indicate fold decrease, whereas numbers below an arrowhead pointing upward indicate fold increase. Equality (=) signs indicate no change. (b and c) Temporal profile of expression of a few selected HOXB genes upon treatment with anti-HOXB3 (b) or anti-HOXBI (c) antisense ODN. Note the logarithmic scale ofrelative expression levels. Our results indicate that the RA-induced sequential acti- lation. This mechanism is somehow cumulative, since the vation of Hox gene clusters entails a cascade of molecular effect of the inhibition of an early gene on the activation of events, whereby the activation level of early-induced, 3' later ones is progressively stronger for progressively more-5' genes somehow affects the progressive induction of more- genes. Interestingly, inhibition of the activation of a given upstream genes. Induction of early genes appears to be a Hox gene has no effect on downstream genes, indicating that prerequisite for activation of later ones, both in cis (the the cross-regulation has a well-defined 3'-to-5' polarity. We upstream genes in the same cluster) and in trans (their and others have previously shown that Hox gene products paralogs on other clusters). The simplest explanation for this may interact with Hox regulatory elements and regulate their phenomenon implies the existence of a network of cross- activity in both an auto- and a cross-regulatory fashion regulatory events between Hox genes, in which the products (16-18), thus providing a molecularbasis forthe phenomenon of early genes participate directly to the activation of late described in this paper. Different mechanisms, such as an ones, possibly by cooperating in their transcriptional regu- increased accessibility of 5' genes caused by progressive Downloaded by guest on September 25, 2021 Developmental Biology: Faiella et A Proc. Natl. Acad. Sci. USA 91 (1994) 5339

5' 3' and Progetti Finalizzati Consiglio Nazionale delle Ricerche Biotec- nologia e Biostrumentazione, Ingegneria Genetica, and Applicazioni Cliniche della Ricerca Oncologica; the VII AIDS Progetto of the Ministero della Sanita; the Telethon-Italia Programme; and the BSH Associazione Italiana per la Ricerca sul Cancro. anti HOXB3 1. McGinnis, W. & Krumlauf, R. (1992) Cell 68, 283-302. 2. Boncinelli, E., Simeone, A., Acampora, D. & Mavilio, F. (1991) Trends Genet. 7, 329-334. 3. Lewis, E. B. (1978) Nature (London) 276, 565-570. 4. Tabin, C. J. (1991) Cell 66, 199-217. 5. Mavilio, F. (1993) Eur. J. Biochem. 212, 273-288. 6. Conlon, R. A. & Rossant, J. (1992) Development 116, 357-368. 7. Marshall, H., Nonchev, S., Sham, M. H., Muchamore, I., Lumsden, A. & Krumlauf, R. (1992) Nature (London) 360, anti HOXB1 737-741. 8. Simeone, A., Acampora, D., Arcioni, L., Andrews, P. W., Boncinelli, E. & Mavilio, F. (1990) Nature (London) 346, 763-766. 9. Simeone, A., Acampora, D., Nigro, V., Faiella, A., D'Es- posito, M., Stornaiuolo, A., Mavilio, F. & Boncinelli, E. (1991) FIG. 4. Diagram of HOX2 gene expression in both types of Mech. Dev. 33, 215-228. treatment described in this paper. The extent of inhibition is shown 10. Papalopulu, N., Lovell-Badge, R. & Krumlauf, R. (1991) Nu- to scale by vertical bars of appropriate length. The standard error of cleic Acids Res. 19, 5497-5506. these measures is also shown. Equality (=) signs indicate no change. 11. Duboule, D. (1992) BioEssays 14, 375-384. 12. Dekker, E.-J., Pannese, M., Houtzager, E., Boncinelli, E. & chromatin opening (19) induced by the transcription of 3' Durston, A. J. (1992) Mech. Dev. 40, 3-12. genes cannot be ruled out on the basis of the existing 13. Acampora, D., D'Esposito, M., Faiella, A., Pannese, M., evidence. However, the observed effect on the regulation of Migliaccio, E., Morelli, F., Stornaiuolo, A., Nigro, V., Sime- genes both in cis and in trans is more easily explained by a one, A. & Boncinelli, E. (1989) Nucleic Acids Res. 17, 10385- mechanism involving a direct action ofHox gene products on 10402. the transcription of upstream genes. 14. Agrawal, S. (1992) Trends Biotechnol. 10, 152-158. In conclusion, these data, obtained in cultured cells, sug- 15. Murray, J. A. H., ed. (1992) Antisense RNA and DNA (Liss, gest a cascade model of activation New York). sequential transcriptional 16. Zappavigna, V., Renucci, A., Izpisua-Belmonte, J. C., Urier, ofHox genes with a 3'-to-5' polarity. Whether a model ofthis G., Peschle, C. & Duboule, D. (1991) EMBO J. 10, 4177-4187. type might also represent an explanation of the observed 17. Arcioni, L., Simeone, A., Guazzi, S., Zappavigna, V., Bon- temporal colinearity in the activation of Hox genes in mouse cinelli, E. & Mavilio, F. (1992) EMBO J. 11, 265-277. and frog embryos (11, 12) remains to be assessed. 18. Popperl, H. & Featherstone, M. S. (1992) EMBO J. 11, 3673- 3680. This work was supported by grants from EC Biotech Programme 19. Gaunt, S. J. & Singh, P. B. (1990) Trends Genet. 6, 208-212. Downloaded by guest on September 25, 2021