Identification of a novel vertebrate homeobox gene expressed in haematopoietic cells

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1 1992 Oxford University Press Nucleic Acids Research Vol. 20 No Identification of a novel vertebrate homeobox gene expressed in haematopoietic cells Mark R.Crompton + Terence J.Bartlett Angus D.MacGregor Guidalberto Manfioletti Emanuele Buratti Vincenzo Giancotti and Graham H.Goodwin* Chester Beatty Laboratories Institute of Cancer Research 237 Fulham Road London SW3 6JB UK Received July ; Revised and Accepted September EMBL accession nos X64711 and X67235 ABSTRACT This paper describes the characterisation of a novel chicken homeobox gene Prh whose encoded homeodomain sequence differs significantly from those of other factors which have been described. As expected a portion of the encoded protein containing the homeodomain is capable of sequence-specific DNA-binding. Outside the homeodomain Prh possesses an N-terminal region extremely rich in proline residues and a C-terminal acidic portion either of which may function as transcription regulatory domains. Since among the chicken tissues tested its transcription is restricted to haematopoietic cells lung and liver it may function in tissue-specific patterns of gene regulation. Human and murine Prh homologues have also been identified; so it is likely that such genes are a general feature of vertebrate genomes. INTRODUCTION Variations of a ~ 60 amino acid motif called the homeodomain were originally predicted to reside within the protein products of genes which regulate the specification of the Drosophila body plan and segment identity during early development by acting as DNA-binding transcription factors. The homeodomain itself folds to form three alpha-helices encompassing a helix-turn-helix motif and is responsible for sequence-specific binding to B-form DNA (reviewed in 12). Vertebrate genomes have also been demonstrated to encode many such proteins and the spatial and temporal patterns of expression of some of their genes implicate them in the regulation of body pattern development (reviewed in 34). Direct roles for homeodomain proteins in vertebrate development have been demonstrated by recent studies in which their expression has been modified by targeted disruption of murine homeobox genes (5 8 reviewed in 9). Homeodomain factors also regulate cell lineage specific gene expression patterns in vertebrates; liver- (10 12) thyroid- (13) and pancreas- (14) specific genes are regulated by DNA binding proteins containing homeo-like domains. Some of the POU-domain proteins which contain divergent homeodomains are also tissue-specific transcription factors (reviewed in 15). Multiple members of the homeobox gene family are expressed in haematopoietic cell lines from various vertebrate species (16 29) implicating these factors in the orchestration of differentiation within these lineages. However not all of the studies referenced above went on to investigate primary cultures or normal cells. An interest in systems in which the biochemical and cellular functions of homeodomain proteins could be studied in a relatively normal context led us to investigate the chicken haematopoietic system. Infection by avian transforming retroviruses causes the outgrowth of populations with defined phenotypes; in particular infection of chick bone marrow cultures with Avian Myeloblastosis Virus (AMV) in vitro results in the proliferation of transformed monoblast-like cells (30). We have previously demonstrated that a pair of redundant oligonucleotides can be used to amplify nucleic acid sequences similar to the prototypical Antennapedia homeobox (31). This paper describes the characterisation initially in AMV-transformed cells of a novel type of homeobox gene which is expressed in transformed and normal haematopoietic cells lung and liver and which encodes a sequence-specific DNA-binding protein. MATERIALS AND METHODS Culture of cells and cell lines To isolate primary transformed monoblasts the long leg bones of 19-day white leghorn chick embryos were dissected the ends were removed and the marrow was washed out with medium (HEPES-buffered RPMI/5% chick serum/10% foetal calf serum/10% tryptose phosphate broth) using a syringe and needle. Cultures were infected with serum from AMV-infected chickens (provided by Dr L.N. Payne Houghton Poultry Research Centre) and cultured in tissue culture flasks at 41 C in 5% CO 2 for 1 week. Adherent cells were discarded and the non-adherent cells which were by now of a uniform morphology were propagated under the same conditions for up to 6 weeks (non-adherent cells failed to proliferate in the absence of virus). Chick bone marrow and peripheral white cells were separated from red cells as follows: cell suspensions in DMEM were underlaid with 1/5 volume of Lymphoprep (Nyegaard and Co. A/S Oslo) and centrifuged for 15 minutes at 2.5 xlo 3 * To whom correspondence should be addressed Present addresses: + The Institute of Cancer Research Royal Cancer Hospital Haddow Laboratories 15 Cotswold Road Belmont Sutton Surrey SM2 5NG UK and 5 Universita di Trieste Dipartimento di Biochimica Biofisica e Chimica delle Macromolecole Trieste Italy

2 5662 Nucleic Acids Research Vol. 20 No. 21 revolutions per minute. Red cells formed a pellet whereas white cells collected at the interface. Cells were washed in phosphate buffered saline to remove excess Lymphoprep. BM2 cells (32) and HPRS line 2 cells (33) were grown at 37 C/5% CO 2 in HEPES-buffered RPMI/5% chick serum/10% foetal calf serum/10% tryptose phosphate broth and HD3 cells (34) were grown at 37 C/5% CO 2 in DMEM/8% foetal calf serum/2% chick serum. BM2 cells were induced to differentiate to a macrophage-like morphology by addition to the medium of 12-O-tetra-decanoylphorbol-13-acetate (TPA) at 250 ng/ml for 48h. RNA purification and analysis RNA was purified from cells and tissues of 3 week-old chickens by acid guanidinium thiocyanate/phenol/chloroform extraction (35). mrna was isolated on oligo-dt cellulose and analysed on Northern blots (Hybond-N Amersham International) after electrophoresis through formaldehyde gels by standard protocols (36). 32 P-labelled DNA probes were prepared by the method of Feinberg and Vogelstein (37). Probed blots were washed at 65 C in 0.2XSSC. Polymerase chain reaction and analysis of products Total cellular RNA substrate (50 ng) was pre-digested with RNAse-free DNAse and reverse transcribed using AMV-reverse transcriptase and oligo-dt primer (36). This sample and a control which had not been reverse-transcribed were subjected to polymerase chain reaction (PCR) amplification (38). The oligonucleotides were 5'- CAA/GACNT/CTNGAA/GT/CTNGAA/ GAAA/GGAA/GTT-3' and 3'-TTT/CTAA/G/TACCAAA/ GGTT/CTTA/GT/GCNT/GCNTACTT-5' directed at the amino acid sequences QTLELEKEF and KIWFQNRRMK (single letter amino acid code) respectively. These sequences represent regions common to a large number of Antennapedia-like homeodomains (2). The temperature cycle parameters were: 94 C 1 min 55 C 1 min 72 C 1 min 30 cycles. Specific products were subcloned by blunt-end ligation into M24mpl8 and sequenced by the 'dideoxy' method (39) with Sequenase (U.S. Biochemicals) and M13 primers. Isolation and characterisation of cdnas PCR was used to amplify inserts from Ml3 subclones (see above) and the products were labelled with 32 P by the method of Feinberg and Vogelstein (37). 5X10 5 clones of a chicken BM2 phage cdna library (40) were screened using Hybond-N membranes (Amersham International) and methods recommended by the manufacturer. Probed blots were washed at 65 C in 0.2XSSC. cdnas isolated in this way were labelled and used to rescreen the library by the same method. The longest cdna was cloned into plasmid vectors and nested deletions from each end were generated using exonuclease HI digestion (41). Selected products were sequenced with Sequenase (U.S. biochemicals) and oligonucleotide primers. Regions of high G/C content were sequenced using 7-deaza-dGTP in all solutions instead of dgtp. The cdna of the human homologue of Prh was also isolated using oligonucleotides based on the chicken Prh sequence a short DNA fragment was amplified by PCR from cdna from the human promyelocytic leukaemia cell line HL60. This was used as a probe to screen a cdna library from the human acute myelogenous leukaemia cell line KG la. The largest cdna was 1.7 kb in length and corresponded to the size of the mrna. This was fully sequenced in both strands. Expression of engineered fusion proteins in E.coli A Notl-EcoRI fragment encoding the chicken Prh homeodomain and C-terminal amino acid sequences was subcloned using a BamHI-NotI adaptor (oligonucleotide 5'GATCCATCCAGC annealed with 5'GGCCGCTGGATG) into BamHI-EcoRI digested pgex-2t (42). Glutathione S-transferase (GST) and GST-Prh fusion protein were purified from lysates of transformed E.coli TGI using glutathione sepharose 4B (Pharmacia) and standard protocols recommended by Pharmacia. Proteins were eluted in 5mM glutathione/50mm Tris-HCl ph8 and stored at -20 C after addition of glycerol to 25%. Binding site selection This procedure was modified from that of Weston (43). A single stranded oligonucleotide (R76) was synthesised to comprise 26 residues of random sequence flanked by unique 25 base sequences containing an EcoRI recognition sequence at one end and a BamHI sequence at the other. Oligonucleotides corresponding to the flanking sequences were synthesised to allow the amplification of double stranded (ds) R76 by PCR. Labelled dsr76 was synthesised by annealing 400ng R76 with the appropriate PCR primer and filling in the complementary strand with die Klenow fragment of DNA polymerase I 32 P-labelled dctp and the other three dntps. l/50th of this labelled dsr76 was incubated with 50ng GST-Prh fusion protein (see above) for 30 mins on ice in 4% ficoll/lmm MgCl 2 /20mM Hepes-NaOH ph7.9/lmm dithiothreitol/50mm NaCl (total volume 20/tl) with varying masses of HindHI digest of lambda DNA (up to lojig) competitor DNA. After resolution at 4 C in 4% polyacrylamide gels buffered with 0.25 X TBE drying and autoradiography protein-dna complexes in dried polyacrylamide were excised and placed in reaction tubes. The bound DNA sequences were then amplified directly by PCR and purified from 3% agarose gels. Approximately 40ng of this selected dsr76 was then labelled as above (using both of the PCR primers) for use in subsequent rounds of sequence selection in the electrophoretic mobility shift assay. In successive rounds of selection ever lower masses of fusion protein could be employed to produce a detectable protein DNA complex in this way the stringency of the selection was increased. After five rounds of selection the amplified sequences were diluted and subjected to a single PCR cycle (to ensure that all molecules were homoduplexes) digested with BamHI and EcoRI and subcloned into pbluescript SKII + (Stratagene). The inserts were then sequenced as before. DNAse I footprinting Three selected sequences ligated in tandem in pbluescript (see above) were amplified as a unit by PCR using M13 and T3 primers digested with Clal and NotI (which cleave in the vector poly linker) and labelled at one end by filling in the Clal overhang with the Klenow fragment of DNA polymerase I and 32 P- labelled dctp. 5 fmol of this DNA were incubated with 50ng GST-Prh fusion protein for lh on ice in 20^120mM Hepes-NaOH (ph 7.6)/50mM NaCl/2mM CaCl 2 /5mM MgCl 2 /lmm DTT/10% glycerol. Samples were digested with 20ng DNAsel for 30s at room temperature and the reactions were stopped with 20/J 2% SDS/lOmM EDTA. After organic extraction and ethanol precipitation the products were resolved on a standard DNA sequencing gel and detected by autoradiography. 'Maxam and Gilbert' cleavage reaction products of the same substrate were co-electrophoresed as size markers.

3 Nucleic Acids Research Vol. 20 No RESULTS Identification of homeobox genes expressed in AMVtransformed monoblasts RNA was purified from a population of primary cultured AMVtransformed chick monoblasts (see Materials and Methods) treated with RNAse-free DNAse and used as a substrate for cdna synthesis. The DNAse treatment was found to be necessary due to contaminating genomic DNA which persisted in the preparation even after mrna purification using oligodt cellulose. The Antennapedia-like homeobox sequences represented in the cdna were amplified by PCR using a pair of redundant oligonucleotides (Materials and Methods) subcloned and sequenced. Of nineteen clones three were singlyrepresented sequences; one was derived from Chox M (31) and the other two were derived from previously unidentified homeobox genes (data not shown). The remaining sixteen were identical (allowing for PCR-induced or allele-specific single base pair differences) and again contained a novel homeobox sequence. This sequence was subsequently named Prh (for reasons explained below). Isolation and characterisation of Prh cdnas A radiolabelled probe derived from a Prh PCR subclone (Materials and Methods) was used to screen a cdna library (40) made from BM2 cells; a non-producer AMV-transformed chicken cell line (32). Hybridising clones were present at a frequency of approximately 1 in 5 X10 4 ; 16 were analysed. The longest cdna insert (clone 16) was fully sequenced on both strands; it was 1845 base pairs (bp) long (excluding the cloning linkers and oligo-dt sequence at the 3' end). Northern blotting analysis allowed the length of the Prh mrna to be estimated at approximately 2.7 kilobases (kb) (see below) so in an attempt to isolate longer or overlapping cdna clones a 33 bp sequence from the extreme 5' end of clone 16 was used to rescreen the cdna library. This resulted in the isolation of a shorter cdna lacking 3' sequences relative to clone 16 (data not shown). At their 5' ends however the two clones were identical the likely explanation being that they both represented the extreme 5' end of the cognate mrna. Other cdna clones had been isolated which overlapped clone 16 at its 3' end and lacked polyadenylated tails; thus clone 16 most likely lacked sequences relative to the mrna only at its 3' end. The sequence of the clone 16 insert contained a long open reading frame extending from the 5' end to nucleotide 917 with a single ATG codon beginning at nucleotide 87 (Figure 1). This codon was in a relatively good context for it to be the site of translation initiation (44). When the cdna insert was transcribed in vitro and translated in reticulocyte lysates it directed the synthesis of a polypeptide whose size was estimated to be greater than 30 kilodaltons by SDS-polyacrylamide gel electrophoresis (data not shown); this was consistent with nucleotides functioning as a full coding region. Based upon this assignment the translation product was 277 amino acids in length of which residues comprised a homeodomain (Figure 1). Confirmation that the open reading frame of Fig. 1 represents the full length protein was obtained by isolating a human Prh cdna the length of which (1.7 Kb) corresponds to the size of the mrna seen on Northern blots (see below). The open reading frame of 270 amino acids is highly homologous to the chicken Prh protein starting with an initiation codon within a good 'Kozak' consensus (44) eight bases from the 5' end of the clone. When this amino acid sequence was aligned with that of chicken Prh 75% of residues were identical (97% identity in the homeodomain) (Fig.l). Thus the human and chicken proteins are highly conserved over the whole of the protein with the exception of MQYQAPGAAPAAALGVGVPLYAPTPLLQPAHPTPFYIEDILGRGPAAAPAPHSLPAPPPPTLPSPNSSF PH-- GA T- -- TSLVAPYRTPVYEPTPIHPAFSHH LAATYGTGAYAGPLYSFPRAVGDYTHALIRQDPLGKPLLWS S SAAA A--P-GFG P T-N L-H PFIQRPLHKRKGGQVRFSNEQTIELEKKFETQKYLSPPERKRLAKLLQLSERQVKTWFQNRRAKWRRLK L D M QENPQATKKEEAEGTGDHGDPR SEGSPSPAGGGEAEPQDSPSAASQEDPESDVSDDSDQEVDIEGDKG SN L-SLDSSC-Q-QDLP-EQNK-ASLDSS-C P L--EI-E S FYSATR YFN-G Figure 1. Amino acid sequences of the chicken and human Prh proteins. The top line is the chicken protein. Dashes represent identities. The nucleic acid sequences are in the EMBL data library under accession numbers X64711 and X I I I I I I I Prh KRKGGQVRFSNEQTIELEKKFETQKYLSPPERKRLAKLLQLSERQVKTWFQNRRAKWRRLK MORI OF KPRTS RS-VL RR-LR ASA AA A-RMTDA T QT 56% HLX/HB24 SWSRAV L-RKG R I VTK-D Q AM-G-TDA V M HS- 56% Hoxll/tcl-3 -K-KPRTS-TRL-IC R-HR ASA A» ^-KMTDA- T QT Chox7 54% -SRRRRTA-TS LL E-HCK LT SQI-HA-K V 1 K-I- Ceh-9 54% KARTT GK-VF R AK SSD-SE R-DVT-T 1 T KKIE Antp 53% E R-RQTYTRY L E-HFNR TRRR-IEI-HA-C-T I-I M KKEN 46% Figure 2. Alignment of the Prh homeodomain amino acid sequence (single letter code) with those of other factors (for descriptions and references see text). Dashes represent residues identical to that at the equivalent position in Prh percentage figures refer to amino acid identities with Prh.

4 5664 Nucleic Acids Research Vol. 20 No. 21 a 25 amino acid section c-terminal to the homeodomain. A partial cdna was also isolated from a murine 416B myeloid cell line cdna library. The deduced open reading frame of this partial clone (by comparison with the sequences of chicken and human clones) included a homeodomain identical to the human Prh homeodomain and the c-terminal domain more closely resembled that of the human rather than the chicken Prh c-terminus (data not shown). The Prh protein contains a homeodomain of which 81% of the residues correspond to amino acids in a consensus sequence for all higher animal homeodomains (including all four invariant residues) (1). The homeodomain is obviously related to those of the antennapedia-type family of homeobox proteins having similar first and third helices (Fig.2). Whilst searches of the dafc banks reveal that Prh is related to a large number of such proteins the closest similarity was found with the homeodomains of the murine factors Hlx (20) and MUR10F (45) and human HB2^ (24) (Fig.2). In these cases 56% of the residues were identical Other homeodomains showing similarity were those of humar Hox 1 l/tcl-3 (4647) chicken Chox 7 (48) and C.elegans Ceh-S (49) (Fig.2). Ascidian AHox 1 (50) chicken CHox E (51) ra TTFl (13) and Drosophila H2.0 (52) showed lesser degrees of similarity (49 52%). Of these homeodomains those of Hlx/HB24 AHoxl and H2.0 comprised a subfamily with similarities of 67 79%; Prh therefore represented a novel type of homeodomain sequence which could not be assigned to a particular subfamily. It is noteworthy that Prh does not have the conserved arginine at position 5. No other regions of the predicted Prh amino acid sequence showed significant similarity with others in the databases apart from some short stretches of identity (three or four amino acids) in the C-terminal regions of Chicken Prh and HLx/HB24. Since these were in the non-conserved part of Prh it is difficult to assess their significance. Of the 142 amino acids N-terminal of the homeodomain 29 (20%) were proline residues (Figure 3). This observation coupled with the presence of two consecutive proline residues within the homeodomain (Figure 1) lay behind this factor being named Prh (for grolinerich homeobox). The two prolines in the homeodomain can probably be accommodated in the N-terminus of the second helix. The region C-terminal of the homeodomain carried almost four times as many acidic as basic residues whereas the homeodomain and N-terminal portion carried positive net charges (Figure 3). Expression of Prh The Prh cdna insert was to be used as a hybridisation probe on Northern blots to study its pattern of expression. However there was the possibility that a clone representing only one of a family of closely related genes had been isolated and that crosshybridisation between these sequences might occur. The chicken probe was therefore hybridised with Southern blots of restriction enzyme-digested chicken genomic DNA and washed at high stringency. Digestion with either EcoRI or HindlE produced single hybridising bands between 6.6 and 9.4 kbp in length (data not shown). It was therefore concluded that only one chicken gene hybridised with the probe under stringent conditions and therefore that any hybridising mrna species detected on appropriately washed Northern blots would represent transcription from the same gene. Polyadenylated RNA was purified from primary AMVtransformed chicken monoblasts and from several chicken haematopoietic cell lines. Northern blotting revealed that steady state levels of a Prh mrna approximately 2.7 kb in length were Homeodomain Pro Arg/Lys/His Asp/3lu 1 1 "" ll'l ' '" " Figure 3. Schematic representation of the distribution of selected amino acids throughout the chicken Prh sequence. The homeodomain is shaded vertical dashes correspond to single occurrences of the appropriate residues. Amino acids are represented by the three letter code N = amino-terminus C = carboxy-terminus. Figure 4. Northern analysis of polyadenylated RNA ( 3/jg) purified from various tissues of 3 week old chickens from AMV-transformed monoblasts and from BM2 cells. The filter was probed sequentially with cdnas for Prh and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Lu = lung In = intestine He = heart Ki = kidney Li = liver Mu = thigh muscle Br = brain Sp = spleen Th = thymus Bu = bursa of Fabricius BMW = bonemarrow 'white' cells BMR = bone marrow 'red' cells PBW = peripheral blood 'white' cells PBR = peripheral blood 'red' cells AMV-B1 = AMV transformed monoblasts. equivalent in primary transformed monoblasts and in BM2 cells (Figure 4). The latter are an AMV-transformed monoblastic cell line which can be induced to differentiate to a macrophage-like morphology by treatment with phorbol esters (53). Unlike ChoxM another homeobox gene which is expressed in BM2 cells (31) Prh mrna levels did not change as a result of differentiation (data not shown). Prh expression was high in the chicken erythroblastoid cell line HD3 (34) but undetectable in the chicken T-lymphoblastoid cell line HPRS line 2 (33) (data not shown). To investigate Prh expression in normal chicken cells and tissues mrna was purified from the tissues of 3 week old chicks for analysis by Northern blotting (Figure 4). Prh transcription was detected in all of the haematopoietic tissues tested with the exception of peripheral blood erythrocytes (although GAPDH mrna could not be detected in the latter sample that for chicken SCL (54) could thus confirming the integrity of the preparation (data not shown)). Amongst these Prh was particularly highly expressed in white cells from the bone marrow and peripheral blood and in cells of the bursa of Fabricius. Low transcript levels were detected in the spleen thymus and dense cells in the bone marrow (which included a high proportion of red erythroblasts). Among the nonhaematopoietic tissues tested Prh mrna showed a very limited range of expression; high levels in the lung and low levels in the liver were detected. Multiple experiments failed to detect expression in the intestines heart kidney thigh muscle and brain (Figure 4 data not shown). When the human Prh cdna was hybridised with Northern blots of total RNA from human cells expression of a specific 1.7 kb mrna was detected in myeloid cell lines (HL60 K562 and KG1) a hepatoma cell line (PLC/PRF/5) and liver whereas it was undetectable in lymphoid

5 Nucleic Acids Research Vol. 20 No Table 1. Sequences selected by Prh G Consensus: G+A I 5'- AGGCAATTAAACTTTAGCAAGCCACC 5'- AAAGCTATTAAGCTCCGAAGATCCTG 5'- TACGCTTATTAAGTAGCCGTTCTCGT 5 ' - GCATTAAAAACGGGCGGAAGGAGCCCTG 5'- AAGTAATTAAATAAGCTATTTAAATT 5'-AfiArTTAATTAACCCTTAACCTATTA CTGGGAAGCAAJT1AAAAAATGGCTCG 5'- ACATfiTAGGCAATTAAAGTTATGATC 5 ' -CAGCGACAATACATCCCGlATTMaG 5' - ATCACGCAAX1AX2AA GATTGCGTC TCTAfinfiTACTTATTAAAGATCAAAT AAAAGCAATTAAGCAGATACTACTAC CAATTAAAACCTCGAATCCTCCGCGC TTATCCAGGTCGCAAGGCAATTAAAG 5 _ 5'- ATTTAGCTATTAAGAATGACTTAATA 5'- TGCAGGGTGCA1AXTAAC.TTCTCAAC 5'- ACTCCACTACCATTAAATCTCCGAAG AAAGACCAATTAAACTCCNGGCCCCA 5'- TGCTGCAATTAAATTGCCAGTGGATG 5'- TACCCCTTATTAACAGGTTAATCTGT 5'- ATTGTGGAGGCTATTAGCGTTACGAT 5'- CGTATCAGGC1AAI1CCCGTCCTCAT 5' - NGCCTTGCTTAC2AXXCGA.TTGCCCC 5'- GCGTGCCCTAA AHACAGTTAGAAA 5'- ATCTTGCGCCACATTCACAAGGCAAT 5'- AGTATCGGTTGTATACAA.CCCTA.CTT -PRH +PRH I I 5'- CAATTAAA TT G cell lines (Molt4 and Jurkatt) peripheral blood lymphocytes fibroblasts (IMR-90) placenta and epithelioid carcinoma cells (HeLa) (data not shown). The C-terminus of Prh can bind DNA with sequence specificity In order to ascertain whether chicken Prh was capable of binding DNA (as predicted from the presence of a homeodomain in the conceptual translation product) a bacterial expression system was used. A C-terminal fragment of Prh encompassing the homeodomain and sequences C-terminal of it was purified as a fusion protein with glutathione-s-transferase (GST) at its Nterminus (see Materials and Methods). The whole Prh protein could not be purified due to the instability imparted by its prolinerich N-terminal region. The fusion protein but not GST purified in parallel from bacterial lysates was capable of binding ds oligodeoxynucleotides in a population containing 26 bp random sequences between constant flanking sequences when assessed by an electrophoretic mobility shift assay (see Materials and Methods) (data not shown). By increasing the ratio of competitor DNA to fusion protein and selecting the ds oligodeoxynucleotides participating in protein-dna complexes over multiple rounds of electrophoretic mobility shift assays (see Materials and Methods) a set of sequences likely to be bound by the fusion protein with high affinity were identified. When twenty-six individual oligonucleotides selected by this procedure were sequenced twenty of them were found to contain the motif 5'ATTAA (Table 1). This suggested that this sequence might form the core of a specific Prh recognition consensus. Although a reliable statistical analysis was not obtainable from the small sample size there were preferences for particular residues at specific positions flanking the ATTAA motif. Thus a consensus sequence YHATTAAV can be discerned where Y is usually C or T (11/20 have C 6/20 have T 2/20 have G 1/20 have A) H is A or Figure S.DNAse I footprinting assay using a cloned binding motif and GST-Prh fusion protein. For protocol see Materials and Methods. Lane a: 'G' Maxam and Gilbert cleavage products lane b: 'G+A' Maxam and Gilbert cleavage products lane c: DNAse I cleavage products lane d: DNAse I cleavage products after preincubation of the DNA with - 50ng GST-Prh fusion protein. The sequence of the region protected by protein (5'-GAAGCAATTA-3') is shown on the right. The dashed lines indicate where DNAse does not cut extensively in the control samples and therefore reflects uncertainty of the borders of the footprint. T (10/20 have A 8/20 have T 2/20 have C no G) and V is usually A or G (12/20 have A 5/20 have G 3/20 have C no T). In order to confirm that the Prh fusion protein could specifically bind an ATTAA-containing sequence DNAse footprinting was performed on dsdna containing such a sequence incubated with the fusion protein. The data shown in Figure 5 confirm that Prh fusion protein can specifically protect dsdna containing an ATTAA motif from digestion with DNAse I. DISCUSSION As part of a study aimed at the characterisation of candidate transcription factors expressed in haematopoietic cells the novel homeobox-containing gene Prh has been identified. The PCR oligonucleotides employed to clone its homeobox sequence were designed to amplify any of the large number of sequences which

6 5666 Nucleic Acids Research Vol. 20 No. 21 comprise the highly interrelated family of which the Drosophila gene Antennapedia is the prototypical member. In spite of this the amino acid sequence of the Prh homeodomain is only 46% identical with that of Antennapedia. In fact there are two amino acid differences in each of the regions upon which the oligonucleotide design was based. Further Prh lacks a short sequence motif (containing Tyr-Pro-Trp-Met) which is characteristic of the Antennapedia class of proteins (reviewed in 1). Prh cannot therefore be classified as being an Antennapedialike sequence. Among those amino acid sequences predicted from genes sequenced to date the greatest similarity is with the homeodomains of murine MUR10F (45) murine Hlx (20) and human HB24 (24) (the latter two sequences may represent equivalent genes from the respective species). Since the Hlx/HB24 homeodomain sequence is significantly more similar to those of H2.0 (52) and AHoxl (50) than to Prh the latter must be regarded as a novel type of homeodomain. The characterization of Prh further extends our appreciation of the diversity that molecular evolution has generated around the homeodomain structural motif. The fact that expression of a highly related sequence has also been detected in human and murine cells implies that Prh equivalents are likely to be a general feature of vertebrate genomes. It will be of interest to ascertain whether such genes are present in representatives of lower phyla (eg Drosophila). Although many investigators have described the expression of homeobox genes in haematopoietic cell lines only some of these studies have included normal cells. Prh expression was originally detected in transformed chicken monoblasts and its cdna was isolated from a cell line-derived library; however our studies found this gene to be expressed in normal white blood cells and tissues which contain high densities of haematopoietic cells. Prh has a very limited expression profile in chicken tissues but is most likely not confined to haematopoietic cells. The expression detected in the lung and liver (the only 'non-haematopoietic' tissues giving a signal on a Northern blot) could have represented the presence of infiltrating haematopoietic cells such as macrophages. However the latter possibility was discounted by re-probing the filter shown in Figure 4 with a cdna for chicken lysozyme. This gene is expressed constitutively in macrophages (55 and references therein) and its mrna was only detected in bone marrow cells the bursa of Fabricius and the spleen; neither the lung nor the liver contained detectable levels of the mrna (data not shown). The Prh expression detected in the latter tissues was therefore probably not solely a result of the presence of macrophages. Thus Prh has a restricted expression pattern including but probably not confined to haematopoietic cells. Structural and mutational studies of the homeodomains of a number of homeodomain proteins have demonstrated that DN A sequence-specific binding of the homeodomain is achieved by multiple interactions (reviewed in 56). Amino acid side chains in the third ('recognition') helix interact with bases in the major groove and basic amino acids in the extended polypeptide chain N-terminal to the first helix interact with A or T bases in the minor groove. When comparisons have been made between the homeodomain and the sequences to which they bind some general principles have emerged. Thus a methyl group of the isoleucine commonly found at residue 47 of the homeodomain interacts with the methyl group of T in the major groove. Asparagine is frequently found at residue 51 and forms hydrogen bonds with A in the major groove whilst the arginines conserved at residues 3 and 5 in many homeodomain factors bind to AT base pairs in the minor groove. These interactions largely account for the 5'-ATTA-3' core motif bound by a large number of homeodomain factors. Additional discrimination is imparted by an amino acid at residue 50 in the third helix. Thus for example a glutamine at this position in Fushi tarazu probably interacts with C (or T) and A bases immediately 5' of the ATTA core motif (57). Similarly Prh (which has glutamine and asparagine at residues 50 and 51 respectively) binds specifically over a region containing the sequence CAATTAA (Figure 5). Presumably the methyl group of threonine 47 can interact with T in the same way as the more commonly found isoleucine. However Prh does not have the conserved arginines at positions 3 and 5 and therefore minor groove binding to AT bases at the 3' end of the consensus sequence may involve the arginine at position 7 as is the case for Mat alpha-2 (58). It is conceivable that the glutamine at position 5 in Prh is also involved in minor groove interactions. In the light of recent data showing the importance of the homeodomain N-terminal region in determining functional specificity in vivo (59) the divergent N-terminal of the Prh homeodomain is of particular interest. Thus although Prh may share some binding specificities with proteins such as Antennapedia and Fushi tarazu it could also manifest novel binding activities determined by the divergent regions of its homeodomain. Work is presently in progress to characterise more accurately the optimum DNA sequences for Prh binding in vitro and to identify the particular amino acids which mediate affinity and specificity of binding. Many homeodomain proteins have been shown to modulate transcription either positively or negatively by binding to gene regulatory sequences in cis (reviewed in 2) although other regulatory mechanisms have been described for a homeodomain protein incapable of binding DNA (60). Based on the assumption that it is likely to be a DNA-binding transcriptional regulator it is possible that Prh functions to positively regulate gene expression since it contains proline-rich and acidic regions both of which are features of some transcriptional activator proteins. However there are precedents for individual homeodomain factors manifesting either activation or repression activities depending on the sequences they interact with or their cellular context (eg 61 and references therein). Therefore it will be important to identify the gene regulatory sequences with which the Prh protein interacts in vivo in order to set future functional experiments in an appropriate context. ACKNOWLEDGEMENTS We thank Drs. U.Kruse and A.E.Sippel for the kind gift of the BM2 cdna library and for supplying the BM2 cells and chicken lysozyme cdna and Dr. L.N.Payne for providing AMV stocks. We are indebted to colleagues for helpful comments in particular Robert H.Nicolas and Kathleen Weston who provided the binding site selection oligonucleotides. We wish to thank Alessandra Rustighi for help in sequencing the human Prh and R.Pellicci for providing the KGla cdna library. This research was funded by grants from the Leukaemia Research Fund the Cancer Research Campaign and the Associazione Italiano per la Ricerca sul Cancro (AIRC) Milano Italy; Consiglio Nazionale per le Ricerche (CNR) Roma Italy; and Ministero dell'universita e della Ricerca Scientifica e Tecnologica Roma Italy.

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