©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transcription Factor GATA-1 Regulates Human HOXB2 Gene Expression in Erythroid Cells (*)

(Received for publication, October 18, 1994; and in revised form, December 12, 1994)

Isabelle Vieille-Grosjean Philippe Huber (§)

From the From INSERM, unité 217, Laboratoire d'Hématologie, Département de Biologie Moléculaire et Structurale, Centre d'Etudes Nucléaires, 17 rue des Martyrs, 38054 Grenoble CEDEX 9, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The human HOXB2 gene is a member of the vertebrate Hox gene family that contains genes coding for specific developmental stage DNA-binding proteins. Remarkably, within the hematopoietic compartment, genes of the HOXB complex are expressed specifically in erythro-megakaryocytic cell lines and, for some of them, in hematopoietic progenitors. Here, we report the study of HOXB2 gene transcriptional regulation in hematopoietic cells, an initial step in understanding the lineage-specific expression of the whole HOXB complex in these cells. We have isolated the HOXB2 5`-flanking sequence and have characterized a promoter fragment extending 323 base pairs upstream from the transcriptional start site, which, in transfection experiments, was sufficient to direct the tissue-specific expression of HOXB2 in the erythroid cell line K562. In this fragment, we have identified a potential GATA-binding site that is essential to the promoter activity as demonstrated by point mutation experiments. Gel shift analysis revealed the formation of a specific complex in both erythroleukemic lines K562 and HEL that could be prevented by the addition of a specific antiserum raised against GATA-1 protein. These findings suggest a regulatory hierarchy in which GATA-1 is upstream of the HOXB2 gene in erythroid cells.


INTRODUCTION

The mammalian Hox gene family contains 38 homeobox gene members located in four independent complexes named HoxA, -B, -C, and -D(1, 2) that code for proteins with a highly conserved DNA-binding domain of 61 amino acids closely related to the fly Antennapedia(3) . These genes are expressed during embryonic development, during which they have a determinant role in the body plan organization(4, 5) . Hox genes could also be implicated in the regulation of hematopoietic cell growth and differentiation. In the mouse and in man, their expression is restricted to only some of the hematopoietic cell types in which aberrant expression may have a dramatic effect. Mutations and translocations involving homeobox genes have also been reported in cases of human leukemia (for a review, see (6) ). Several lines of evidence indicate that Hox gene products are candidates for the regulation of expression of some erythroid markers like the globin genes, as has recently been suggested for human HOXB6 and HOXB2(7, 8) . Therefore, Hox genes are thought to act as specific transcription factors that could control both the proliferation and the commitment of hematopoietic progenitor cells in the differentiation pathway.

Convergent data are available showing that human HOXB genes are expressed in erythro-megakaryocytic cells but not in granulo-monocytic cells. This striking characteristic is shared by eight of the nine genes in the complex(9, 10) . Furthermore, nothing is known about the regulation of homeobox gene expression in hematopoietic cells, including transcriptional regulation. The aim of our study was to determine how this specific expression is regulated.

We initially focused our attention on the human HOXB2 gene because of a previous study based on RT-PCR (^1)analysis of homeobox-containing transcripts, in which we established that the HOXB2 (HOX2H) gene was expressed not only in the erythroid line K562, the erythro-megakaryocytic line HEL, and the megakaryocytic line Meg01, as are the other members of the HOXB complex, but in two early myeloid cell lines, KG1a and KG1, as well(10) . This characteristic led us to believe that HOXB2 could be an early regulator of both erythroid and megakaryocytic lineages. HOXB2 gene is also expressed in normal human bone marrow (9) as well as in progenitor cells(11) . Other authors (12) have reported the existence of a transcript for HOXB2 of about 1.6 kb in hematopoietic cells. Very recently, using specific antibodies, Sengupta et al.(7) have demonstrated the presence of the HOXB2 protein in two erythroid cell lines, K562 and HEL. The same authors have suggested that the HOXB2 protein binds to specific sites within the A globin-regulating sequences(7) .

In this paper, we report our studies of the regulation of HOXB2 expression in human erythroid cell lines. Recently, Sham et al.(13) have reported the regulation of HoxB2 (Hox2.8) gene by Krox20 in the developing nervous system of the mouse(13) . This regulation implicates three sites for the Krox20 protein situated about 2 kb upstream from the first exon, in the intergenic region between HoxB3 and HoxB2. Krox20, or its human homologue EGR2(14) , is considered to be an early response gene that can be induced in various cell types, including hematopoietic cells, by mitogenic agents such as phorbol esters or after serum deprivation. Because the expression level of human HOXB2 mRNA was not affected by 12-O-tetradecanoylphorbol-13-acetate treatment of KG1, K562, and HEL lines, (^2)whereas, under the same conditions, 12-O-tetradecanoylphorbol-13-acetate modifies expression of different genes coding for erythroid and megakaryocytic markers(15) , we thought that HOXB2 gene could be regulated by different pathways during brain ontogenesis and erythropoiesis. Furthermore, the specificity of HOXB2 expression in early myeloid cell lines suggested the existence of specific regulating regions for HOXB2 gene expression. To study this, we isolated and characterized the 5`-flanking region of the gene. Because the nucleotidic sequence contained a consensus site for the erythroid factor GATA-1, which has been demonstrated to play a major role in the regulation of expression of various specific erythroid genes and in the terminal erythroid differentiation in vivo(16, 17, 18, 19) , we investigated the possible regulation of HOXB2 by GATA-1 factor. We report here the evidence that, in erythroid cell lines, the expression of HOXB2 gene is under major control of GATA-1 protein, suggesting that HOXB2 is a part of the cascade of regulating events that lead to the establishment of the erythroid phenotype.


MATERIALS AND METHODS

Cell Culture

Cell lines were obtained from the American Type Culture Collection. HEL (erythroleukemia), K562 (erythroid), HeLa (epithelial carcinoma), and KG1a cells were maintained in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (Boehringer Mannheim).

Isolation of the 5`-Flanking Region of HOXB2 Gene

A human genomic clone in a pcos2EMBL cosmid vector, BC206, containing 36 kb of HOXB complex, was generously provided by E. Boncinelli (20) and used as starting material. A 3.7-kb AsnI restriction fragment, including a large intergenic region between the HOXB3 last exon and the HOXB2 first exon, was subcloned and inserted into the SmaI site of the Bluescript I SK plasmid vector (pBSK, Stratagene) for restriction enzyme mapping and DNA sequencing. All sequencing was carried out by the chain termination procedure on both strands (21) using Sequenase (U. S. Biochemical Corp.).

Determination of Transcriptional Start Site

Poly(A) RNAs were used for RNase protection experiments. They were prepared from HEL, K562, and KG1a cells by the thiocyanate guanidinium method (22) and purified using the mRNA purification kit (Pharmacia Biotech Inc.). For RT-PCR studies, RNA from HEL and HL60 were prepared by Nonidet P-40 lysis and phenol extractions as previously described(10) .

RNase protection was carried out essentially as described (23) with an antisense RNA probe spanning the region from -114 (EcoRI) to +296 (NarI, in the first exon). Briefly, a 1.2-kb genomic fragment HindIII/NarI from the BC206 cosmid was subcloned into the plasmid pBSK and linearized with EcoRI. The antisense RNA probe (472 bases) was generated with T3 RNA polymerase (Boehringer Mannheim) and [alpha-P]UTP (Amersham Corp.). 5-10 µg of poly(A) RNA were hybridized overnight with 0.5 times 10^6 cpm of probe at 45 °C in standard buffer and digested with 13 µg of RNase A (Boehringer Mannheim) and 2700 units of RNase T1 (Life Technologies, Inc.). The RNase-resistant products were subsequently analyzed on a 6% acrylamide sequencing gel.

RT-PCR was performed according to the protocol that was previously described (10) using 750 ng of cytoplasmic RNA, and the amplification step was carried out for 25 cycles. Four sense oligonucleotides were synthesized (Cyclone Plus DNA synthesizer, Millipore) to be used as 5`-primers in the PCR: primer 1, 5`-CCCAAAATCGCTCCATTACATAAAT-3` from position +12; primer 2, 5`-TAAAAAAAAAGAGAGACCGAAATCTCCCCCT-3` from position -19; primer 3, 5`-CGCTTGTATTTATCAGCAATA-3` from position -62; and primer 4, 5`-TAGAGAGAGTCCCCATACGC-3` from position -79. The 3`-primer was the antisense oligonucleotide 5`-GCGGGGGAAGGAAGCAGACACTCGG-3` from position +201. The amplified fragments were revealed with a 35-mer antisense oligonucleotide probe, 5`-end-labeled with [-P]ATP (DuPont NEN), corresponding to the first 11 amino acids in the cDNA sequence (20) (5`-AACCCAATCTCCCTCTCAAATTCAAAATTCATGGC-3` from position 153).

Construction of CAT-expressing Vectors

The chloramphenicol acetyltransferase (CAT) plasmid used was pBLCAT3(24) . It contains the coding region of the bacterial CAT gene and the polyadenylation signal from SV40. The DraII genomic segment from pB2AsnI (Fig. 1), containing the cap site and 2.2 kb of 5`-flanking sequences, was inserted into the XbaI site of pBLCAT3. The construct (named -2200CAT) was sequenced in the 3`-side of the insert for confirmation. Two shorter constructs were derived as follows: the -865CAT was prepared by excision of the 1.3-kb HindIII fragment from the -2200CAT and recircularization, and the -323CAT was prepared by excision of the 1.9-kb HindIII/BstBI fragment from the -2200CAT, followed by Klenow treatment and recircularization (Fig. 4).


Figure 1: Structure of the 5`-flanking region of HOXB2 gene. The map shows the intron-exon organization of the human HOXB2 gene(20) . A 3.7-kb AsnI fragment was subcloned from the BC206 cosmid clone into pBlueScript (pB2AsnI) for restriction mapping and sequencing (Fig. 3). All restriction sites for the indicated enzyme are shown on the map. A, AsnI; B, BamHI; D, DraII; E, EcoRI; Pv, PvuII; Ps, PstI.




Figure 4: Cell-specific expression of the HOXB2 gene 5`-flanking sequence. On the left are diagrams of HOXB2 promoter-CAT fusion constructs. Restriction sites indicated are: D, DraII; H, HindIII; B, BstBI. The DraII fragment containing 2200 bp upstream and 58 bp downstream from the transcription start site was inserted before the CAT gene in the XbaI site of pBLCAT3. The -865CAT and -323CAT constructs contain the HindIII-DraII and the BstBI-DraII fragments, respectively, in pBLCAT3. Each plasmid was transfected into K562 and HeLa cells, and respective CAT activities were measured 48 h after transfection. In each assay, the pRSVL plasmid was cotransfected, and CAT assays were normalized according to the luciferase activity. The promoterless plasmid pBLCAT3 was used as a negative control and measured the experimental background. On the right, the relative CAT activity of K562 and HeLa cells transfected with each construct, normalized against pBLCAT3 activity that was taken as 1, is plotted. Each data is the average of three independent experiments performed in duplicate. The standard variations are indicated in the figure.




Figure 3: Nucleotide sequence of the 5`-flanking region of the HOXB2 gene. The sequence contains 1143 nucleotides upstream from the ATG codon and the beginning of the first exon. All numbering is relative to the transcriptional start site (+1). The HindIII(-865), BstBI(-323), and DraII (58) sites used for construction of the expression vectors are underlined. Putative regulatory elements are indicated in boldfacetype; the TATA box is underlined in italics, the GATA element is doubleunderlined, Ets binding elements are underlined, and the ATTA sites are in italics.



The mutated HOXB2 fragments were obtained by site-directed mutagenesis in the -48 GATA site, using the polymerase chain reaction as described(25) , except that we used Pfu DNA Polymerase (Stratagene) as the enzyme. The plasmid -323CAT was used as matrix. Briefly, two simultaneous PCR reactions were performed. The first one used two primers homologous to the pBLCAT3 vector sequence, 5`-AAGTTGGGTAACGCCAGGGT-3` from position 341 (24) and 5`-GAGCTAAGGAAGCTAA-3` from position 471, containing a mismatched 3`-end. The second one used the primer mutated on the -48 GATA site, 5`-ATACGCTTGTATTTAGAAGCAATATACAATTA-3` from position -65 in HOXB2 gene (the T and C in the GATA site were mutated to G and A), and a more external primer in the CAT gene, 5`-GGCATTTCAGTCAGTTGCTC-3` from position 556 in pBLCAT3 vector. Amplified fragments from each PCR reaction were purified, mixed, and subjected to another round of PCR with the two most external primers (341 and 556). The amplified fragments were digested with TaqI enzyme, end flushed with Klenow fragment, digested with BamHI, purified, and ligated into the pBLCAT3 vector that was itself first digested with HindIII, end flushed, and then digested with BamHI. The sequence of the entire insert was verified, and the insert borders were identical to those in the wild type -323CAT plasmid.

DNA Transfections

All plasmids used for transfection were prepared with the Qiagen columns protocol.

K562 were transfected by the electroporation method (26) using a gene pulser (Bio-Rad), set at 960 microfarads, 400 V, in a total volume of 800 µl. Each assay was done with 40 µg of one of the CAT constructs. 5 µg of pRSVL plasmid, containing the firefly luciferase gene under the control of the Rous sarcoma virus promoter (27) were cotransfected with the HOXB2/CAT constructs for internal control of transfection efficiency. The pRSVCAT plasmid containing the CAT gene driven by the Rous sarcoma virus promoter was used as positive control.

HeLa cells were transfected by the calcium phosphate method(28) , each assay containing 10 µg of CAT constructs and 15 µg of pRSVL.

Luciferase and CAT Assays

Cells were harvested 48 h after transfection, and cell extracts were obtained by three cycles of freeze and thaw lysis. Luciferase activity of the extracts was measured using the luciferase assay system (Promega Biotec). CAT assays were performed essentially as described by Gorman et al.(29) . The amount of cellular extracts used in the CAT assay was normalized according to luciferase activities.

Nuclear Extracts

The nuclear extracts were prepared from HEL, K562, and HeLa cells according to the method of Dignam et al.(30) from exponentially growing cells.

Gel Mobility Shift Assays

Gel mobility shift assays were performed by a combination of the procedures of Halligan and Desiderio (31) and of Singh et al.(32) . For the binding reaction, 0.5-0.9 ng of radiolabeled DNA fragment (2 times 10^4 cpm) were mixed with 2.5-5 µg of nuclear extracts in a final volume of 10 µl containing 10 mM Tris-HCl (pH 7.5), 25 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 5% glycerol, and 2 µg of poly(dI-dC)bulletpoly(dI-dC) (Pharmacia) used as nonspecific competitor. Specific competitors and antisera were added to the reaction mixture before the addition of the end-labeled probe as described in individual experiments and incubated 10 min at +4 °C. Finally, samples were incubated for 15 min at room temperature and analyzed on 5% polyacrylamide, 2.5% glycerol gels that were run in 0.5 times TBE buffer (1 times TBE buffer = 0.089 M Tris-HCl, 0.089 M boric acid, 0.002 M EDTA) for 1 h and 30 min at 200 V and then dried prior to overnight autoradiography.

The double-stranded DNA probes used in gel mobility shift assays were the following: 5`-CTGATGGGCCTTATCTCTTTACCCACCT-3` from position -83 in the erythroid promoter of human porphobilinogen deaminase gene (PBGD) used as a GATA-1 standard binding site(33) ; the HNF-1-binding site from position -92 in the promoter of human beta fibrinogen gene, 5`-AAAATTAAATATTAACTAAGGA-3`(34) ; the wild type and the mutated GATA-binding sites from position -67 in the human HOXB2 gene promoter, respectively, 5`-CCATACGCTTGTATTTATCAGCAATATAC-3` and 5`-CCATACGCTTGTATTTAGAAGCAATATAC-3`.

Antiserum

Specific rabbit antiserum directed against the human protein GATA-1 was generously provided by F. Martin. Anti-GATA-1 antibody was prepared by injecting a synthetic peptide corresponding to a hydrophilic region of GATA-1 having no homology with any sequence of other GATA proteins. The specificity of the antibody has already been established(35) .


RESULTS

Isolation of the 5`-Flanking Region of the Human HOXB2 Gene

To identify the promoter and the regulatory sequences of HOXB2, we subcloned the HOXB2 upstream genomic sequences from the human genomic clone BC206(20) . This clone contains 36 kb of the HOXB complex from chromosome 17, encompassing exons 2, 3, and 4 of HOXB3 gene, the entire HOXB2 gene, and the two exons of HOXB1, which is the most 3`-gene in this complex. A 3.7-kb AsnI restriction fragment containing the 5`-flanking region of HOXB2 was subcloned into pBSK and analyzed by restriction mapping (Fig. 1); 1 kb of proximal sequence was determined (Fig. 3).

Determination of the Transcriptional Start Site

To define the transcriptional start site of HOXB2 gene, three assays were used. The RNase protection assay was performed using an antisense RNA EcoRI/NarI probe. A strong protected band of 300 nucleotides in length was produced from mRNA from K562, HEL, and KG1a cells but not from control tRNA (Fig. 2A). A band of smaller size (216 nucleotides) was also weakly detected with the three cell lines.


Figure 2: Identification of the transcriptional start site of HOXB2 gene. A, RNase protection analysis. 5-10 µg of poly(A) RNA from HEL, K562, KG1a (lanes1 and 2, 5 µg; lane3, 10 µg), or 10 µg of tRNA (lane4) were hybridized with the antisense RNA probe. After treatment with RNase, the protected products were run on a 6% sequencing gel. Sequencing reaction products of a non-related DNA were used as size markers. As a control of the whole experiment, the beta2 microglobulin (beta2m)-protected products were run in parallel (amounts of RNA were as follows: lanes5 and 6, 0.25 µg; lane7, 0.5 µg; lane8, 10 µg). The major band corresponding to the HOXB2 protected fragment is indicated with a closedarrow, and the band corresponding to beta2m is indicated with an openarrow. B, RT-PCR analysis. Relative positions of primers (arrows) and probe (hatchedbox) used in the analysis are indicated in the diagram. On the leftpanel, the control of 5`-primers efficiency tested with DNA from HeLa cells is shown. Sizes of amplified fragments were, respectively, 280, 263, 220, and 190 bp. On the rightpanel, the RT-PCR experiment performed with 750 ng of cytoplasmic RNA from HEL and HL60 as negative control is reported. Appropriate 5`-primer is indicated for each amplification (primer 1 (+12/+36), primer 2 (-19/+11), primer 3 (-62/-42), and primer 4 (-79/-60). The +201/+177 antisense oligonucleotide was the 3`-primer. After 25 cycles of amplification following the reverse transcription step, the PCR products were electrophoresed, blotted, and hybridized with the P end-labeled 35-mer antisense (+153/+119) oligonucleotide. Plus (+) indicates samples treated with reverse transcriptase, and minus(-) indicates non-treated samples (see reference 10).



Primer extension experiments with a 35-mer antisense primer (from position +153 to +119) allowed us to observe an extension product of approximately 150 nucleotides (data not shown), corresponding to the major band in the RNase protection assay.

For a definitive approach, we performed a RT-PCR analysis (Fig. 2B). The antisense primer used in the reverse transcription step was chosen 55 bases downstream from the ATG codon and was also used in the amplification step. The four 5`-primers were chosen in the region that overlaps the previously identified start site (from -60 to +36). Each 5`-primer together with the 3`-primer was able to produce the correct PCR fragment from genomic DNA. The RT-PCR experiment was performed in parallel with mRNA from HEL cells and from HL-60 cells as a negative control(10) . After electrophoresis, the PCR products were blotted and hybridized with a labeled probe consisting of an oligonucleotide located within the smallest fragment. A positive signal was observed with HEL mRNA, only when primer number 1 (from position +12 to +36) was used and not with the upstream oligonucleotides. This result was consistent with RNase protection analysis and allowed us to confirm the localization of the transcriptional start site. This site is common for HEL, K562, and KG1a cells and is situated 122 bp upstream from the ATG codon (Fig. 3).

Structural Features of the 5`-Flanking Region

The sequence of part of the first exon and about 1.1 kb upstream from the ATG codon is shown in Fig. 3. Two independently isolated clones were sequenced, and no difference was observed. A canonical TATA box was found 35 bp upstream from the transcription start point but no CCAAT box(36) . A number of potential regulatory elements are present, including at position -48 in an inverted orientation, a putative binding site for the GATA family of zinc finger transcription factors which consensus is 5`-(A/T)GATA(A/G)-3`(37) , six elements at positions -303, -312, -381, -467, -498, and -776 containing the sequences 5`-AGGAA(A/G)-3` or 5`-GAGGAA-3`, which are recognized by factors belonging to the Ets class of oncoproteins(38) , and nine elements at positions -21, -37, -82, -105, -178, -189, -400, -411, and -543 containing an ATTA core sequence that is similar to the binding site (5`-TCAATTAAAT-3`) for Antennapedia class of homeoproteins(39, 40) . Among them, four are organized in tandem sites in an inverted orientation and are separated by exactly 7 bp (-178 and -189 sites, -400 and -411 sites).

A 381-bp Fragment from the 5`-Flanking Region Contains the Promoter Region and Is Sufficient for Tissue-specific Expression in Erythroid Cells

To test whether the 5`-flanking region of the HOXB2 gene had promoter activity, we inserted the HOXB2 5`-region upstream from a promoterless CAT gene in the plasmid pBLCAT3. Three constructs of different sizes (-2200CAT, -865CAT, and -323CAT) were analyzed in transient CAT assay, both in erythroid cells K562, which express HOXB2, and in HeLa cells, which do not normally synthesize HOXB2(10) . The three constructs allowed the expression of the reporter gene in K562 cells, whereas very weak expression was detected in HeLa cells (Fig. 4). Although the -323CAT was slightly less active than the other two in K562, it contained sufficient elements to promote the specific activity of the gene in erythroid cells. This characteristic feature allowed us to think that the GATA motif at -48 could be a functional binding site for erythroid factors belonging to the GATA family.

The GATA Element at -48 Is Required for Optimal Function of the HOXB2 Gene

The GATA motif at position -48 is located 15 bp upstream of the TATA box and exhibits the strict consensus 5`-(A/T)GATA(A/G)-3` in an inverted orientation (Fig. 3). To characterize the contribution of this motif to the promoter activity, we mutated the wild type sequence TTATCA to TTAGAA in the -323CAT construct. This mutation is known to disrupt the binding activity of GATA proteins. The mutation resulted in an 85% decrease in activity of the -323CAT construct (Fig. 5).


Figure 5: Effect of point mutation on the GATA putative binding site in K562 cells. A mutation in the -48 GATA motif was introduced in the -323CAT construct using a PCR strategy as described under ``Materials and Methods.'' The wild type (WT) -323CAT and the mutated (Mut) -323CAT constructs were introduced into K562 cells for transient transfection assays. The CAT values obtained with the different plasmids were expressed relative to the wild type -323CAT, which was taken as the 100% value. pBLCAT3 was used as a negative control. Each data is an average of three independent replicates. Errorbars represent standard deviations.



Thus, this clearly demonstrates that the -48 GATA motif is functional and essential for the promoter activity of HOXB2 in K562 cells.

The Function of the -48 GATA Element Correlates with Its Ability to Bind GATA-1 Protein

To determine whether the -48 GATA element bound nuclear proteins, we synthesized a 29-bp double-stranded oligonucleotide probe encompassing this sequence. Incubation of the P end-labeled probe with HEL and K562 nuclear extracts resulted in the formation of a single complex (Fig. 6). This complex was sequence specific, as it was prevented by addition of a 200-fold molar excess of the unlabeled oligonucleotide (lanes3 and 8) but not by a 200-fold excess of a heterologous oligonucleotide (lanes6 and 11). Furthermore, the competition with 200-fold excess of the HOXB2 oligonucleotide mutated on the -48 GATA site (same mutation than in Fig. 5, the TTATCA motif mutated in TTAGAA) did not prevent the complex formation (lanes4 and 9), suggesting that the core GATA sequence is required for the binding.


Figure 6: Gel mobility shift assays of the HOXB2 promoter probe containing the -48 GATA motif. 5 µg of HeLa, HEL, or K562 nuclear extracts were incubated with the end-labeled HOXB2/-48 GATA probe (lanes 1-11) and with the PBGD/-75 GATA probe (lanes 12-15) as described under ``Materials and Methods.'' The binding reactions were performed in the absence or presence of the indicated unlabeled double-stranded competitor oligonucleotide (lanes3 and 8, homologous HOXB2/-48GATA; lanes4 and 9, HOXB2/mutated -48GATA; lanes5, 10, and 13, PBGD/-75GATA; lanes6, 11, and 14, non-homologous HNF1. The mutated oligonucleotide contained two nucleotides changes in the core GATA motif, the same changes that were used in the mutated construct listed in Fig. 5. The PBGD oligonucleotide corresponded to the -75 GATA element in the human porphobilinogen deaminase gene(33) . The HNF1 oligonucleotide corresponded to the HNF1 element in human beta fibrinogen gene(34) . In lane15, the K562 nuclear extract was incubated with 1 µl of antiserum against GATA-1 protein (35) before the addition of the PBGD probe.



In HEL and K562 cells, it has been shown that two GATA proteins were expressed: GATA-1 and GATA-2(17, 41, 42) . Both were therefore candidates for this interaction. To discriminate between these two proteins, we used HeLa cells that contain GATA-2 but not GATA-1 (41, 42, 43) . With these extracts, no complex was observed (Fig. 6, lane1), suggesting that in erythroid cells, the GATA-binding protein involved in the interaction with HOXB2 promoter probe is GATA-1. In addition, we performed a competition experiment with an oligonucleotide encompassing the -75 GATA element of the PBGD promoter gene that was used as a standard GATA-1-binding site(33) . The addition of a 200-fold excess of this oligonucleotide prevented complex formation with the HOXB2 probe (Fig. 6, lanes5 and 10). The -75 GATA site of PBGD gene was also used as a probe to compare the electrophoretic mobility of the two complexes. The identity of the GATA-binding protein to the PBGD probe was verified using specific antiserum raised to a region of human GATA-1. This antibody has been shown to bind specifically GATA-1 protein but not GATA-2 protein(35) . When antiserum was added to the gel shift incubation mixture containing K562 cell nuclear extracts, no interaction was observed (Fig. 6, lane15). Without competitor (lane12), the complex formed with GATA-1 protein comigrated with HOXB2 site complex (Fig. 6).

To definitively and directly address the identity of the GATA protein involved in the interaction with the GATA site of HOXB2 promoter, we performed mobility shift assays with the antiserum against human GATA-1 protein. Antiserum was incubated with K562 or HEL extracts before the addition of the labeled probe. In the two cell lines, formation of the specific DNA-protein complex was not affected by the preimmune serum, whereas competition with the antibody against GATA-1 completely inhibited the formation of the complex with the DNA probe (Fig. 7). No other band was visible on the autoradiography except a weak band corresponding to the supershifted ternary complex.


Figure 7: Immunological identification of protein that binds to the -48 GATA motif in HOXB2 promoter. 5 µg (lanes1-3) or 2.5 µg (lanes 4-9) of K562 or HEL nuclear extracts were incubated with the end-labeled oligonucleotide corresponding to the -48 GATA element in HOXB2 promoter. Prior to addition of the probe, the mixtures were incubated 10 min on ice without antiserum (lanes1, 4, and 7) or with 1 µl of undiluted preimmune serum (lanes2, 5, and 8) or with 1 µl of undiluted antiserum raised to a human GATA-1 specific peptide (35) (lanes3, 6, and 9).



These data show that GATA-1 is the GATA protein that binds to the -48 GATA site of the HOXB2 gene in vitro and strongly suggest, together with transfection experiments, that GATA-1 is the major regulator of the erythroid-specific expression of HOXB2 gene.


DISCUSSION

In this paper, we present the characterization of the 5`-flanking region of the HOXB2 gene and some insights into the mechanisms that govern its regulation. The expression pattern of this gene, as well as other HOX genes, in hematopoietic cell lines has been established by different groups(9, 10, 12, 44) . In erythroid cell lines, a major transcript for HOXB2 of about 1.6 kb was detected(12) . We identified a major cap site located 121 bp upstream from the suggested initiation codon. This site is common to K562 (erythroid), HEL (erythro-megakaryocytic), and KG1a (early myeloid) cell lines. RNase protection experiments showed that these cell lines contain similar amounts of HOXB2 mRNA.

Three deletion CAT constructs of 2200, 865, and 323 bp produced comparable and substantial CAT activities when transfected in K562 cells. We could observe a slight but consistent decrease between the -865 and -323CAT constructs that may have functional significance, but we did not attempt to study it more precisely. Thus, within the 5`-flanking region examined, most of the HOXB2 gene regulatory sequences are contained in the 323-bp proximal domain. It also contains erythroid-specific information as it directs only basal expression in HeLa cells. Whether this sequence can mediate tissue-specific expression in vivo must be verified in a transgenic mice model.

The examination of the 5`-flanking sequence revealed some putative regulatory elements. A TATA box is present at -35 bp, which is not usual for HOX genes. We could identify nine ATTA or TAAT sites concentrated in 530 bp. This motif is the core consensus sequence for homeoprotein binding. Similar clusters of homeobox protein DNA-binding sites are found in cis-regulatory regions of several Drosophila homeotic genes (45) and in human HOXD9 (HOX4C)(46) . Interestingly, four of them are organized in tandem sites. Actually, Galang and Hauser (47) showed that human HOXA5 (HOX1C) and HOXB5 (HOX2A) homodimers exhibit cooperative DNA binding on tandem ATTA sites. These elements could be involved in cross-regulation by other homeoproteins or in autoregulation mechanisms by the HOXB2 protein, as has been suggested for HOXD9(46) , HOXC5 (HOX3D)(48) , or mouse HoxD4 (Hox4.2)(49) .

Six putative binding sites for members of the Ets family of transcription factors are present in the -865CAT construct(50) . Some of these factors play a role in hematopoietic gene expression. Implication of Ets proteins in the regulation of the megakaryocyte-specific gene for glycoprotein IIb has been reported (51) . In HOXB2 gene, two of these Ets recognition sequences are still present in the -323CAT plasmid and thus are more candidates as cis-acting elements. The small decrease in CAT activities between the -865CAT and the -323CAT constructs may be due to the loss of some Ets binding elements or ATTA sites. We found neither CACCC nor TGAGTCA (AP1-binding site) motifs, which are often present in erythroid promoters(33, 52, 53) .

A binding site for the GATA family of transcription factors (54) is located at position -48. Three members of this family are expressed in hematopoietic cells. GATA-1 is present in erythroid, megakaryocyte, mast cell, and eosinophil lineages(42, 55, 56, 57) , whereas GATA-2 is expressed in mast cell, basophil, eosinophil, neutrophil, megakaryocyte, and erythroid lineages. Finally, GATA-3 expression is restricted to T-cell, mast cell, and eosinophil lineages(42, 54, 57) . GATA elements play a central role in erythroid and megakaryocytic gene regulation. Functional GATA elements have been described in the regulatory regions of the vast majority of promoters of erythroid-expressed genes including the genes for globins(58) , porphobilinogen deaminase(51, 52) , and erythropoietin receptor(59) , as well as several megakaryocytic expressed genes like the genes for platelet glycoprotein IIb(35) , platelet factor 4(60) , and P-selectin (61) . In this paper, we demonstrated that the GATA element upstream from HOXB2 gene is functional since its mutation reduced promoter activity by 85% and abolished its protein binding properties in gel mobility shift assays. We showed that GATA-1 binds in vitro to this sequence as indicated by three experiments: 1) the use of a negative control cell line (HeLa) that expresses GATA-2 but not GATA-1 protein, 2) the DNA-protein complex electrophoretic mobility and the competition with a DNA-binding site for GATA-1, and 3) the use of a specific antibody against GATA-1. Further investigation is necessary to determine if the other HOXB genes that are essentially expressed in the erythro-megakaryocytic compartment are also GATA-1 regulated. From the sequences available in the literature, we could identify GATA consensus sequences in mouse HoxB7 (Hox2.3) (62) and HoxB9 (Hox2.5) (63) promoters. Therefore, it is possible that GATA-1 mediates erythro-megakaryocytic expression of HOXB genes through cis-elements in each promoter.

In conclusion, the study presented here is the first characterization of HOX gene promoter in hematopoiesis. We identified the transcription factor GATA-1 as one of the major regulators of HOXB2 gene expression in erythroid cell lines. GATA-1, strongly expressed in erythroid and megakaryocytic cell lineages, is likely to be a major component of HOXB2-specific expression in the hematopoietic compartment.


FOOTNOTES

*
This work was supported by the Commissariat à l'Energie Atomique, the Institut National de la Santé et de la Recherche Médicale, and the Association pour la Recherche sur le Cancer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X78978[GenBank].

§
To whom correspondence should be addressed. Tel.: 33-76-88-41-18; Fax: 33-76-88-51-23.

(^1)
The abbreviations used are: RT-PCR, reverse transcriptase-polymerase chain reaction; kb, kilobase(s); bp, base pair(s); PBGD, porphobilinogen deaminase; CAT, chloramphenicol acetyltransferase.

(^2)
I. Vieille-Grosjean and P. Huber, unpublished data.


ACKNOWLEDGEMENTS

We thank E. Boncinelli for the generous gift of BC206 genomic clone and F. Martin for providing the GATA-1 antiserum. We are grateful to F. Aubouy for artwork, to G. Uzan for useful discussions, and to E. Dejana and D. DiMichele for careful reviewing of the manuscript.


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