(Received for publication, October 18, 1994; and in revised form, December 12, 1994)
From the
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.
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 ()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, ()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.
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
[
-
P]UTP (Amersham Corp.). 5-10 µg
of poly(A)
RNA were hybridized overnight with 0.5
10
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).
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.
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.
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 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`.
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
2
microglobulin (
2m)-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
2m 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).
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.
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 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.
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X78978[GenBank].