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A Novel Retinoic Acid-Responsive Element Regulates Retinoic Acid-Induced BLR1 Expression

Department of Biomedical Sciences, Cornell University, Ithaca, New York 14853

Received 14 July 2003/ Returned for modification 27 August 2003/ Accepted 10 December 2003

	ABSTRACT

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The mechanism of action of retinoic acid (RA) is of broad relevance to cell and developmental biology, nutrition, and cancer chemotherapy. RA is known to induce expression of the Burkitt's lymphoma receptor 1 (BLR1) gene which propels RA-induced cell cycle arrest and differentiation of HL-60 human myeloblastic leukemia cells, motivating the present analysis of transcriptional regulation of blr1 expression by RA. The RA-treated HL-60 cells used here expressed all RA receptor (RAR) and retinoid X receptor (RXR) subtypes (as detected by Northern analysis) except RXR. Treatment with RAR- and RXR-selective ligands showed that RAR synergized with RXR to transcriptionally activate blr1 expression. A 5'-flanking region capable of supporting RA-induced blr1 activation in HL-60 cells was found to contain a 205-bp sequence in the distal portion that was necessary for transcriptional activation by RA. Within this sequence DNase I footprinting revealed that RA induced binding of a nuclear protein complex to an element containing two GT boxes. Electromobility shift assays (EMSAs) and supershift assays showed that this element bound recombinant RAR and RXR. Without RA there was neither complex binding nor transcriptional activation. Both GT boxes were needed for binding the complex, and mutation of either GT box caused the loss of transcriptional activation by RA. The ability of this cis-acting RAR-RXR binding element to activate transcription in response to RA also depended on downstream sequences where an octamer transcription factor 1 (Oct1) site and a nuclear factor of activated T cells (NFATc) site between this element and the transcriptional start, as well as a cyclic AMP response element binding factor (CREB) site between the transcriptional start and first exon of the blr1 gene, were necessary. Each of these sites bound its corresponding transcription factor. A transcription factor-transcription factor binding array analysis of nuclear lysate from RA-treated cells indicated several prominent RAR binding partners; among these, Oct1, NFATc3, and CREB2 were identified by competition EMSA and supershift and chromatin immunoprecipitation assays as components of the complex. RA upregulated expression of these three factors. In sum the results of the present study indicate that RA-induced expression of blr1 expression depends on a novel RA response element. This cis-acting element approximately 1 kb upstream of the transcriptional start consists of two GT boxes that bind RAR and RXR in a nuclear protein complex that also contains Oct1, NFATc3, and CREB2 bound to their cognate downstream consensus binding sites.

	INTRODUCTION

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Certain features of the biology of Burkitt's lymphoma receptor 1 (BLR1) have been established which suggest a regulatory role in cell proliferation and differentiation and development. The blr1 gene was originally identified in a screening for differentially expressed genes conferring metastatic capability to human B-cell lymphomas. It encodes a putative serpentine heterotrimeric G protein-coupled chemokine receptor (also known as CXCR5) (13, 14, 36). The blr1 cDNA sequence contains an open reading frame of 1,116 bp encoding a 372-amino-acid protein. Analysis of the genomic structure of the blr1 gene indicated that the predicted protein is derived from two exons, with the first encoding 17 amino acids about 10 kb upstream of the second exon (13). The sequence is highly related to receptors for interleukin-8 (IL-8) and other neutrophil chemoattractants (13). Other than in the human Burkitt's lymphoma cells from which it was originally isolated, BLR1 expression in human hematopoietic system cells is restricted to mature resting B cells (13) and a subset of T-helper memory cells (16), thus identifying BLR1 as a member of the lymphocyte-specific chemokine receptor family (15, 16). Murine expression of the receptor, however, has also been found in defined areas of the cerebellum (17), monocytes, peripheral blood leukocytes (4), and neuronal tissues (31). Knockout of the murine blr1 function by gene targeting disrupted the formation of inguinal lymph nodes and severely affected the development of Peyer's patches. The formation of follicles in the spleen of blr1-negative mice was aberrant, and B cells failed to migrate into the B-cell follicles (15). Two CXC chemokines, B-lymphocyte chemoattractant and B-cell-attracting chemokine 1 (BCA-1), were found to function as physiological BLR1 ligands. B-lymphocyte chemoattractant directed the migration of murine B lymphocytes to follicles in secondary lymphoid organs (24), and BCA-1 selectively attracted murine and human B lymphocytes via BLR1/CXCR5 (38). However, BCA-1 was chemotactic only for human B lymphocytes and not for T lymphocytes, monocytes, or neutrophils.

Regulation of blr1 expression has been studied in a limited number of contexts. Alignment of the nucleotide sequences showed that neither the human blr1 promoter nor its murine counterpart has TATA or CCAAT boxes, a feature that is not uncommon for genes encoding serpentine receptors. The human blr1 promoter directs initiation of transcription from a single start site (54). In murine B lymphocytes, regulatory elements lying between positions -78 and +215 are sufficient to confer basal activity of the murine blr1 promoter. Three essential regulatory elements in the blr1 promoter have been found to be necessary for cell type-specific and differentiation lineage-specific blr1 expression (54). These include binding sites for the transcription factors (TFs) NF-B, Oct2, and Bob1, which cooperatively regulate the blr1 gene in B cells. As a B-lymphocyte-restricted octamer factor, Oct2 is the key determinant for blr1 promoter activity. Bob1 acts as a B-cell coactivator of octamer binding factors (54). Both are also expressed in myeloid cells and regulate myeloid lineage development (2, 48).

BLR1 mRNA expression can be induced by retinoic acid (RA) (7). RA is a developmental morphogen, a necessary dietary factor for proper development in juveniles, and a cancer chemotherapeutic agent used in differentiation induction therapy. Reflecting these physiological functions, RA regulates cell proliferation, differentiation, and apoptosis (1, 12, 19, 30, 40, 47). RA and its retinoid metabolites act as ligands for RA receptors (RARs) RAR, RARß, and RAR and retinoid X receptors (RXRs) RXR, RXRß, and RXR, which are ligand-activated TFs that are members of the steroid thyroid hormone superfamily of nuclear receptors (32, 37, 42, 44, 62). RAR and RXR regulate transcription by binding to particular cis-responsive elements, RA response elements (RAREs), and regulate their transactivation function (8, 32, 42, 43, 62). In HL-60 human myelomonocytic precursor cells, RA induced BLR1 expression through RAR and RXR activation, which caused activation of the extracellular signal-regulated kinase 2 (ERK2) mitogen-activated protein kinase and propelled cell cycle arrest and myeloid cell differentiation. Ectopic expression of BLR1 in these myeloblastic leukemia cells enhanced ERK2 activation and accelerated RA-induced myeloid differentiation (6, 7). BLR1 also enhanced ERK2 activation and RA-induced differentiation along the monocytic lineage in U937 monoblastic leukemia cells (5). There is thus a role for BLR1 in regulating cell proliferation and differentiation. The transcriptional mechanism by which RA induced BLR1 expression in HL-60 cells to propel their differentiation and G₀ arrest is thus of interest, but the cis-acting elements and TFs conferring RA responsiveness are not known.

The present study identifies an approximately 1.3-kb 5'-flanking region capable of supporting RA-induced blr1 expression. A cis-acting element that contains a novel RA-responsive element with two GT boxes is located in the distal portion of this region. The element binds a TF complex in response to RA. The complex contains RAR, RXR, octamer TF 1 (Oct1), and nuclear factor of activated T cells 3 (NFATc3) throughout blr1 activation (as well as cyclic AMP [cAMP] response element binding factor 2 [CREB2] transiently at an early stage of blr1 activation). The cis-transactivating capability of this element in response to RA depends on downstream Oct1, NFATc, and CREB sites. Activation of the blr1 gene by RA thus seems to require formation of a multimolecular TF complex to a novel RA-responsive element in the blr1 promoter.

	MATERIALS AND METHOD

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Reagents Plasmids pGL3-basic, pRL-TK, and pGEM-T Easy and a luciferase reporter assay system as well as a footprinting kit were purchased from Promega. Rabbit polyclonal antibodies for each of RAR, RXR, Oct1, Oct2, NTATc1, NFATc2, NFATc3, NFATc4, NFATc5, CREB1, and CREB2 and normal anti-rabbit immunoglobulin G (IgG) were purchased from Santa Cruz Biotechnology Inc. [-³²P]dCTP, [-³²P]dATP, [-³²P]dTTP, and [-³²P]ATP were obtained from Perkin Elmer Life Sciences. A TITANIUM One-Step reverse transcriptase PCR (RT-PCR) kit was purchased from BD Biosciences. Probes for Northern analysis and oligonucleotides based on blr1 promoter sequences for electromobility shift assays (EMSAs) were ordered from Operon Qiagen. A random primer labeling system and mutagenesis reagents were obtained from Stratagene. An end extension labeling kit and a biotin labeling kit were obtained from Perkin Elmer Life Sciences. All-trans-RA and actinomycin D were purchased from Sigma-Aldrich. An EMSA kit, oligonucleotides for Sp1, early growth response factor 1 (EGR1), DR5, DR2, DR1, NFATc, CREB, NF1, Pbx1, and DNA-protein array and TF-TF array kits were purchased from Panomics (Redwood, Calif.). A rabbit reticulocyte lysate system was purchased from Novagen (Madison, Wis.).

A chromatin immunoprecipitation (ChIP) assay kit was purchased from Upstate Cell Signaling Solutions (Lake Placid, N.Y.).

Cell culture HL-60 cells were grown in RPMI 1640 supplemented with 5% fetal calf serum in a 5% CO₂ humidified atmosphere at 37°C. The cultures were initiated every 2 or 3 days at a density of 0.2 x 10⁶ or 0.1 x 10⁶ cells/ml, respectively. Cell viability (as determined by trypan blue exclusion) routinely exceeded 95%. For experimental cultures, cells were initiated at a density of 0.2 x 10⁶ cells/ml. All-trans-RA, actinomycin D, and RAR-selective ligands were added at indicated concentrations from 1 or 5 mM stock dissolved in 100% ethanol and stored protected from light at -20°C (RA and actinomycin D) and -80°C (ligands).

Plasmid constructions The human blr1 promoter sequence spanning 1,360 bp upstream of the translational start codon (or 1,096 bp upstream of the initiation site for transcription) was obtained by PCR from a genomic DNA template that was prepared from HL-60 cells. The primers were designed on the basis of the reported sequences (54) with GenBank/EBI database accession numbers X83755 and X83756. The 1,360-bp PCR product was inserted at the KpnI-HindIII sites in pGL3-basic, a promoterless luciferase reporter vector, resulting in a blr1 promoter-controlled luciferase reporter construct, BLR1-Luc. 5' end unidirectional deletion from -1,096 to +78, 3' end deletion from +1 to +266, and deletion between -891 and +1 of the BLR1 promoter were performed by PCR using the BLR1-Luc plasmid as a template to generate 10 BLR1-Luc constructs with different length blr1 promoter fragments. Two pairs of primers for amplification of RAR and RXR cDNAs, each 1,386 bp in length, were synthesized (incorporated restriction sites NotI-SalI and NotI-PstI are denoted by underlining): RAR forward, 5'-AATGCGGCCGCCATGGCCAGCAACAGCAGCTCCTGCCCG-3'; RAR reverse, 5'-AATGTCGACTCACGGGGAGTGGGTGGCCGGGCTGC-3'; RXR forward, 5'-AATGCGGCCGCCATGGACACCAAACATTTCCTGCCGC-3'; RXR reverse, 5'-AATCTGCAGCTAAGTCATTTGGTGCGGCGCCTCC-3'. Total RNA from RA-treated HL-60 cells was prepared and used as a starting template to amplify the cDNAs by RT-PCR using a TITANIUM One-Step RT-PCR kit according to the manufacturer's protocol. The expected PCR products were digested with NotI-SalI or NotI-PstI and ligated with a NotI-SalI- or NotI-PstI-digested plasmid (pGEM-T Easy) to produce the expression vectors (pGEM-RAR and pGEM-RXR). The Kozak sequence was incorporated immediately upstream of the open reading frames of the receptors to achieve high-level in vitro expression. All of the PCR products for construct building were confirmed by sequencing.

Transient transfection and luciferase assay HL-60 cells were harvested by centrifugation and washed with RPMI 1640 medium supplemented with 5% fetal calf serum, and 10 million cells were suspended in 0.4 ml of this medium. Transient cotransfection into cells of the BLR1-Luc luciferase (firefly) reporter plasmid and luciferase (Renilla [sea pansy]) expression vector, pRL-TK (for normalization of transfection efficiency), was performed by DEAE-dextran electroporation. A total of 50 µg of BLR1-Luc plasmid (1 µg/µl) and 15 µg of pRL-TK (1 µg/µl) with 2 µl of DEAE-dextran (1 µg/µl) were added to the 10⁷ cells, and the mixture was incubated for 10 min at room temperature followed by 5 min on ice and electroporated with a Gene Pulser apparatus (Bio-Rad) at 320 V and 960 µF. Right after electroporation, the cells were placed on ice for 15 min prior to dilution with 5 ml of prewarmed RPMI 1640 medium containing 5% fetal calf serum without antibiotics. The cells were harvested by centrifugation, rinsed once with room temperature phosphate-buffered saline (PBS), and suspended in passive lysis buffer. The protein concentration of the samples was measured, and each sample was diluted to 3 µg/µl with lysis buffer. Luciferase (30 µg) activity was measured with a luminometer (Lumat LB950; Berthold Japan K.K., Tokyo, Japan) using a Promega dual-luciferase reporter assay system.

RT-PCR and Northern analysis Total RNA was extracted from HL-60 cells by using an RNA-Bee kit (Tel-test, Friendswood, Tex.) according to the manufacturer's protocol. Specific primers for detecting mRNA transcripts of RAR and RXR as well as BLR1 with a TITANIUM One-Step RT-PCR kit are given in Table 1. In each case, reactions with 500 ng of total RNA were run on a PTC-100 thermal cycler (MJ Research, Inc.) with the following program cycle: 50°C for 1 h (1 cycle); 94°C for 5 min (1 cycle); 94°C for 30 s, 65°C for 30 s, and 68°C for 1 min (35 cycles); and 68°C for 2 min (1 cycle). Negative controls with Taq but not RT were included in each set of samples to confirm a lack of genomic DNA contamination in the RNA samples. The product was resolved by agarose electrophoresis, and bands representing the appropriate size product were recovered and used as probes in subsequent Northern analysis of RAR and RXR expression. A probe for the RXR transcript, which yielded no RT-PCR product from HL-60 cells, was amplified from a plasmid, pSG5/RXR (36, 44), by PCR with the following program cycle: 95°C for 2 min (1 cycle); 95°C for 30 s, 74°C for 30 s, and 72°C for 1 min (35 cycles); and 72°C for 10 min (1 cycle). Due to the unavailability of the complete RAR2 mRNA sequence, the probe for RAR2 extended from its 5' untranslated region into the first exon and was amplified from 500 ng of genomic DNA by PCR using the thermal cycler program described above.

Preparation of nuclear extracts and in vitro translation receptors HL-60 cells were grown in cultures for 12, 24, and 48 h in the presence or absence of 1 µM all-trans-RA. The nuclear extracts were prepared using a Sigma-Aldrich nuclear extract kit. Briefly, cells were rinsed with PBS, resuspended in hypotonic lysis buffer, and lysed in a tight-fitting Dounce homogenizer with 45 strokes. The cells were incubated on ice for 10 min and observed by phase-contrast microscopy to verify that 95% lysis had occurred. A nuclear pellet was obtained by centrifugation in a refrigerated microcentrifuge (30 min at 13,000 x g). Protein concentration of the samples was determined by a Bradford assay. Nuclear extracts were aliquoted and stored at -80°C until use. RAR and RXR proteins were prepared using a rabbit reticulocyte lysate translation system kit (Novagen). pGEM-RAR and pGEM/RXR plasmids were used as templates for RNA synthesis with T7 RNA polymerase, and the resultant mRNAs were used as templates for in vitro translation according to the manufacturer's instructions. Translation of the appropriate protein was verified by Western blotting after SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

DNase I footprinting DNase I footprinting was performed with reagents from Promega's Core footprinting system and a modification of the manufacturer's protocol. Briefly, the DNA fragment for footprinting experiments was amplified by PCR (using BLR1-Luc plasmid as a template) with a primer incorporating EcoRI at the 5' end and a primer incorporating PstI at the 3' end. To get better resolution of the 5' end of the footprinting probe in the sequencing gel, the PCR-amplified probe covered 217 bp from the blr1 gene 5'-flanking region (-1096 to -891) and 25 bp at the 5'end from the vector backbone plus 8 nucleotides (nt) from the incorporated restriction sites (EcoRI at the 5' end of the fragment and PstI at the 3' end). The footprinting forward primer was 5'-CGTGCGAATTCCAGGTGCCAGAACATTTCTC-3' and the reverse primer was 5'-CGTGCCTGCAGGTCCTGTCTGAGGGCCGCTTCC-3' (the incorporated EcoRI and PstI restriction sites, respectively, are denoted by underlining). The PCR product was then digested with EcoRI and PstI to produce a 250-bp DNA fragment with a 3' recessed end and 3' protruding end 5' and 3' of the double-stranded DNA (dsDNA) fragments, respectively, to ensure that there was only one end with a 3' recessed terminus to be radioactively labeled. The digested fragment was then exclusively end labeled with [-³²P]dATP and [-³²P]dTTP by filling in the 3' recessed end at the EcoRI restriction site with the Klenow fragment of DNA polymerase I and purified by a spin column. The DNA-protein binding reaction mixture (50 µl) contained 200,000 cpm of labeled DNA probe, 50 µg of nuclear extract (prepared as described above), 25 mM Tris-HCl (pH 8.0), 50 mM KCl, 6.125 mM MgCl₂, 0.5 mM EDTA, 10% glycerol, and 1 mM dithiothreitol. After incubation for 20 min on ice, 50 µl of 10 mM MgCl₂ and 5 mM CaCl₂ were added followed by the addition of 0.1 U of DNase I and incubation was continued at room temperature for 3 min. DNase I digestion was quenched by the addition of 90 µl of stop solution (200 mM NaCl, 30 mM EDTA, 1% SDS), and the DNA template was purified by phenol-chloroform (1:1) extraction and ethanol precipitation. Samples were run on denaturing 6% polyacrylamide-urea sequencing gels. Gels were dried on Whatman 3MM paper for 3 h with a gel dryer and visualized by autoradiography. A 10-bp DNA ladder labeled (using polynucleotide kinase) with [-³²P]dCTP was heat denatured and run along with the DNase-treated samples as a size marker, and alignment with the ladder identified the protected nucleotide within the known sequence of the 250-bp probe.

EMSA and supershift assay EMSAs were performed using a Panomics kit. Biotin-labeled probes and unlabeled competitors for DR5, DR2, DR1, EGR1, Sp1, NFATc, NF1, CREB, and Pbx1 were also purchased from Panomics. Oligonucleotides containing the GT box element or the putative RAR-RXR binding sites in the blr1 promoter (identified by a TransFac database analysis) were synthesized by Qiagen Operon Inc. and end labeled with biotin before annealing to make double-stranded probes according to the manufacturer's instructions. The probes and competitors for EMSA are given in Table 2.

PCR-based mutagenesis Block or single-base deletions of core sequences of putative cis-acting elements in the blr1 promoter were done by PCR using a Stratagene QuikChange XL mutagenesis kit or QuikChange mutagenesis kit, respectively. A pair of primers (for block deletion) or a single primer (for single-base deletion) was designed for each deletion. The sequences of the primers lacking the underlined deleted portions are indicated in Table 3. The mutant sequence in the BLR1-Luc plasmid was verified by nucleotide sequence analysis. All primers were PAGE purified and phosphorylated at the 5' end with T4 polynucleotide kinase. Cycling parameters were adjusted on the basis of the thermal denaturation values of the primers.

ChIP assay ChIP assays were performed with a kit and a procedure modified from the manufacturer's instructions. Briefly, HL-60 cells (treated with or without RA for 12, 24, or 48 h) were cross-linked by formaldehyde at a final concentration of 1% and incubated for 15 min at 37°C. The cells were centrifuged to form a pellet and washed twice with ice-cold PBS containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µg of aprotinin/ml, and 1 µg of pepstatin A/ml). After centrifugation at 300 x g for 5 min, the cell pellet was resuspended in prewarmed SDS lysis buffer containing the protease inhibitors and incubated for 10 min on ice. The HL-60 cell lysate was sonicated, using an 80-watt model ultrasonic processor (SmithKline, Shelton, Conn.) (with 40 sets of 30-s pulses), on an ice bath to shear DNA to a length of approximately 1 to 2 kb. Samples were centrifuged for 10 min at 13,000 x g at 4°C. The sonicated cell supernatant was diluted 10-fold in ChIP dilution buffer containing the protease inhibitors.

After preclearing the diluted cell supernatant (2 ml) with salmon sperm DNA-protein A agarose-50% slurry (100 µl) for 30 min at 4°C with agitation, the agarose was precipitated by brief centrifugation and the supernatant fraction was collected. Antibodies specific for each of RAR, RXR, Oct1, NFATc3, and CREB2 were added and the suspension was incubated overnight at 4°C with rotation. Salmon sperm DNA-protein A agarose-50% slurry was added followed by incubation for 3 h at 4°C with rotation to collect the antibody-bound complex. After gentle centrifugation, the supernatant containing unbound, nonspecific DNA was carefully removed. The antibody-bound complex was subjected to a series of 5-min washes on a rotating platform with the buffers supplied by the manufacturer. The complex was eluted from the antibody with elution buffer (1% SDS, 0.1 M NaHCO₃). The cross-linked portion was dissociated by adding 5 M NaCl and heating at 65°C for 5 h. The fragmented DNA was treated with proteinase K at 45°C for 2 h and purified with a Qiagen quick-spin column. The DNA was analyzed by PCR (100 pmol of each primer set, 1 U of Pfu Hotstart polymerase [Stratagene], 0.01 µCi of [³²P]dCTP, 40 cycles). Primer sets for amplifying the blr1 promoter region from fragmented genomic DNA consisted of the sequences from -1096 to -1078 and +185 to +205. Primer sets for amplifying the blr1 promoter region from transfected plasmid pBLR1-Luc are located in the vector backbone sequence directly linking to the two ends of the blr1 promoter fragment. PCR products were resolved on PAGE and exposed to phosphor luminescence plates for 24 h to 48 h. Each experiment was repeated, and the same results were obtained.

	RESULT

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RA-induced blr1 expression is dose and time dependent and actinomycin D sensitive RA causes a dose- and time-dependent induction of blr1 expression in HL-60 cells, in which treatment with 1 µM all-trans-RA for 24 to 48 h elicits a largely maximized response. HL-60 cells (treated with various concentrations of all-trans-RA from 0.01 to 10 µM for 24 and 48 h) were harvested using total RNA for Northern analysis of blr1 expression. Figure 1A shows the results. Expression in untreated cells was undetectable. After 24 h, the response was largely maximized by 1 µM all-trans-RA. Even a 10-fold-higher concentration had no significant further effect. After 48 h, a concentration of 1 µM still elicited a largely maximized response. This identifies 1 µM as an effective dose. Using this dose, the kinetics of RA-induced blr1 expression over time was determined. HL-60 cells were treated with 1 µM all-trans-RA and harvested for Northern analysis at sequential times up to 96 h. Figure 1B shows the results. Induced expression maximized after 24 h, although it was first detectable at 9 h and weakly evident at 12 h. The induced expression was maximal from 24 to 60 h and decreased by approximately twofold every 12 h thereafter. This identifies treatment at 1 µM for 24 or 48 h as effective in eliciting largely maximized blr1 expression. These will be adopted as standardized treatment conditions in subsequent experiments.

View larger version (31K): [in this window] [in a new window]   FIG. 1. Effect of all-trans-RA on BLR1 mRNA expression. Total RNA isolated from HL-60 cells treated with RA was analyzed by Northern blotting (middle panels) using a blr1 cDNA-specific probe. Ethidium bromide-stained 28S and 18S rRNA bands are shown (lower panels) to indicate the uniformity of RNA loading per lane. The upper charts show the relative Northern blot band intensities (as measured by a PhosphorImager). The values were normalized using 18S rRNA band intensity. (A) Effect of all-trans-RA concentration on blr1 mRNA expression. HL-60 cells were treated for 24 or 48 h with the indicated concentrations of RA. The highest blr1 mRNA expression levels observed were in cells treated with 1 to 10 µM RA for 24 h and 0.5 to 10 µM RA for 48 h. (B) Time course of RA-induced blr1 mRNA expression. HL-60 cells were treated with 1 µM RA for the indicated times. The maximum increase in blr1 mRNA expression occurred at 24 to 60 h.

To confirm transcriptional regulation of blr1 expression in all-trans-RA-treated cells, cells were treated with actinomycin D, which caused a loss of blr1 mRNA in RA-treated cells. HL-60 cells were treated with 1 µM all-trans-RA for 24 h to elicit blr1 expression and then with various concentrations of actinomycin D for a further 24 h before Northern analysis. Figure 2 shows the results. Actinomycin D caused a dose-dependent loss of blr1 mRNA expression, with a concentration of 1 µM causing almost total loss. This concentration of drug did not inhibit HL-60 cell proliferation; hence, the mRNA is labile and was lost within the duration of one cell cycle when synthesis was inhibited. Interestingly, the mRNA of important regulatory molecules is often labile. Taken with previously reported results showing that RA-induced blr1 expression was cycloheximide insensitive (9), the data are consistent with the hypothesis of RA-induced primary transcriptional regulation of blr1.

View larger version (54K): [in this window] [in a new window]   FIG. 2. Inhibition of RA-induced BLR1 mRNA expression by actinomycin D. HL-60 cells were treated with the indicated concentrations of actinomycin D for 24 h following pretreatment with 1 µM all-trans-RA for 24 h. (Middle panel) Total RNA isolated from the treated cells was analyzed by Northern blotting using a blr1 cDNA-specific probe. (Lower panel) Ethidium bromide-stained 28S and 18S rRNA bands are shown to demonstrate uniform loading of RNA per lane. (Upper panel) The chart shows the relative intensities of Northern blot bands (as measured with a PhosphorImager). The values were normalized using 18S rRNA band intensity.

Activation of RAR and RXR expressed in HL-60 cells induces blr1 expression

View larger version (30K): [in this window] [in a new window]   FIG. 3. Expression of RARs and RXRs in HL-60 cells. Total RNA (isolated from HL-60 cells that were left untreated [RA-] or treated with 1 µM RA for 48 h [RA+]) was analyzed by Northern blotting. RAR and RXR subtype-specific primers located in the 3' ends of their corresponding cDNAs were used so that for each receptor subtype, all possible isoforms derived from alternative in-frame translational start codons would be detected. Except for the primer for RXR (which was amplified from a plasmid), all other specific primers were prepared by RT-PCR from HL-60 cells. The probes were [-32P]dCTP labeled. Ethidium bromide-stained 28S and 18S rRNA bands are shown to serve as size markers. Constitutively expressed transcripts of RAR, RAR, RAR2, RXR, and RXRß were observed. RAR and RXRß showed two isoforms. RARß (detected only after RA treatment) showed four isoforms. RXR was not detectable in untreated or RA-treated cells.

Activation of RAR plus RXR induced blr1 expression, but additional activation of other RAR or RXR subtypes did not substantially augment the response. Activation of an RAR or RXR alone failed to induce blr1 expression. RAR- or RXR-selective ligands were used to identify which RARs and RXRs could regulate blr1 expression. HL-60 cells were treated for 48 h with 1 uM all-trans-RA, 1 µM Ro 40-6055 (an RAR-selective agonist), 0.5 µM AM80 (an RAR- and RARß-selective agonist), 0.5 µM AGN190168 (an RARß- and RAR-selective agonist), 1 µM Ro25-7386 (an RXR-selective agonist), or 0.3 µM AGN194204 (an RXR panagonist) used either singly or in combinations. The final concentrations used were chosen to optimize response and minimize toxicity. Total RNA from the treated cells was harvested for Northern analysis of blr1 expression. Figure 4 shows the resulting Northern blot. Untreated cells showed no expression, whereas all-trans-RA-treated cells showed prominent expression. Cells treated with agonists selective for just RARs or just RXRs also showed no expression, indicating that activation of just RARs or just RXRs was insufficient to activate blr1. Treatment with an RAR- plus an RXR-selective agonist elicited strong expression (comparable to that seen with all-trans-RA), indicating that activation of RAR plus RXR was needed to activate blr1.

View larger version (34K): [in this window] [in a new window]   FIG. 4. Effect of RAR- and RXR-selective agonists on BLR1 mRNA. HL-60 cells were treated with the indicated retinoid ligands for 48 h, after which total cellular RNA was isolated and analyzed by Northern blotting using a blr1 cDNA-specific probe (middle panel). Ethidium bromide-stained 28S and 18S rRNA bands are shown to indicate the uniformity of RNA loading per lane (lower panel). The concentrations of the compounds used are as follows: RA, 1 µM; Ro 40-6055 (RAR-selective agonist), 1 µM; AM80 (RAR- and RARß-selective agonist), 0.5 µM; AGN190168 (RARß- and RARß-selective agonist), 0.5 µM; Ro25-7386 (RXR-selective agonist), 1 µM; AGN194204 (RXR panagonist), 0.3 µM. The upper chart shows the relative intensities of bands on the Northern blot (as measured by a PhosphorImager). The values were normalized using 18S rRNA band intensity.

A 1,360-bp 5'-flanking region of the blr1 gene confers RA response and contains a distal 205-bp sequence necessary for response A 5'-flanking region extending from -1096 to the 5' end of the first exon at +266 of the blr1 gene confers response to RA. A Transfac database (http://www.gene-regulation.com/pub/databases.html#transfac) (53) search for putative TF binding sites in the 5' region of the blr1 gene showed that the 1,360 bp 5'-flanking region before the first exon was rich with putative sites potentially including two single putative RAR-RXR half-sites at -785 to -780 and -47 to -42. This region extended from -1096 through the transcriptional start (+1) to +266, which is the beginning of the first coding exon. This fragment was isolated by PCR and cloned into pGL3-basic, the promoterless reporter plasmid, to create a blr1 promoter-luciferase reporter construct, BLR1-Luc. HL-60 cells were transiently transfected and then regrown in medium with 1 µM all-trans-RA for 24, 48, 72, or 96 h. Luciferase activity was then fluorometrically measured. Figure 5 shows the reporter activity after 48 h. The all-trans-RA-induced expression level of BLR1-Luc was 75 to 80 times higher than the expression level of pGL3-basic vector and 50 to 55 times higher than the non-RA-induced expression level of the BLR1-Luc construct. Similar results were observed after 24 h of all-trans-RA treatment (data not shown). Luciferase expression mirrored the kinetics of RA-induced blr1 expression, with comparable maximal activity seen at 24 and 48 h which progressively decreased at 72 h and was hardly detectable at 96 h. The blr1 promoter activation pattern in the transient experiment thus mimicked that observed by Northern analysis for the in vivo pattern (data not shown), indicating that the RA-induced reporter activity bore kinetic fidelity to RA-induced blr1 expression and suggesting that this 1,360-bp 5'-flanking region harbors complete cis elements required for the full activity of the blr1 promoter for RA responsiveness.

View larger version (19K): [in this window] [in a new window]   FIG. 5. Mapping of the RA-regulatory elements in the BLR1 promoter. Reporter constructs resulting from 5' deletions from the 1,360 bp of the BLR1 5'-flanking region were prepared by PCR and transiently transfected into HL-60 cells as described in Materials and Methods. The lines and numbers on the left side of the figure indicate the blr1 promoter sequence contained in each reporter construct (+1 denotes the start of transcription). The chart on the right side indicates relative luciferase activity levels of the reporter constructs. All transient transfection studies were conducted in triplicate on three separate occasions, with similar results. The data reported in the chart represent the means of three replicate experiments.

The distal 205-bp sequence contains two GT boxes constituting a novel RARE RA induces the binding of a nuclear protein complex to an element containing two GT boxes within the critical 205-bp distal sequence from -1096 to -891 that is necessary for RA response. To determine whether RA induces TFs to bind within the 205-bp sequence identified above, DNase I footprinting was performed using nuclear extracts from RA-treated and untreated cells. The probe was a dsDNA fragment of 250 bp that contained the sequence spanning -1096 to -879 from the blr1 promoter plus 25 bp at the 5'end from the BLR1-Luc plasmid backbone and 8 nt from the incorporated EcoRI and PstI sites at the 5' and 3' ends of the fragment, respectively. The probe was generated by PCR, digested with the two restriction enzymes, [-³²P]dATP and [-³²P]dTTP labeled with Klenow on the 3' recessed end created by EcoRI digestion, and used for DNase I footprinting. Figure 6 shows the resulting sequencing gel. Nuclear lysate from all-trans-RA-treated cells protected a region of the probe from nt 74 to 58, according to the sequencing ladder, which corresponds to an element between -1071 and -1055 in the blr1 promoter. The sequence of the 17-nt protected element (deduced from the known sequence of the blr1 5'-flanking region) is shown in Fig. 6 and includes two truncated GT boxes located at -1068 to -1065 and -1062 to -1059. Interestingly, this element has no sequence homology with known RAR-RXR consensus sequences.

View larger version (37K): [in this window] [in a new window]   FIG. 6. RA-induced nuclear extracts protect sequences in the distal region of the BLR1 promoter. A dsDNA fragment of 250 bp (spanning 217 bp [-1096 to -879] in the BLR1 promoter plus 25 bp at the 5' end from the plasmid backbone sequence in the pBLR1-Luc promoter-reporter construct and 8 nt from the incorporated EcoRI and PstI site) was prepared by PCR. After digestion with EcoRI and PstI, the amplified fragment was [-32P]dATP and [-32P]dTTP end labeled at the 3' recessed end with the Klenow fragment of Escherichia coli DNA polymerase I and used in the DNase I footprinting assay with nuclear extracts from HL-60 cells that were either left untreated (RA-) or treated (RA+) with all-trans-RA for 48 h. A DNA sequencing ladder (10 bp) was end labeled (using T4 polynucleotide kinase) with [-32P]ATP, heat denatured, and corun with the DNase I-treated samples as a size marker. The nucleotide sequence of the DNase I-protected site was determined by alignment of the protected region with the sequencing ladder. An approximately 17-bp region (-1071 to -1055) with the indicated sequence was specifically protected from DNase I digestion in the nuclear extracts from RA-treated cells. No footprint was visible with nuclear extracts from untreated cells. An autoradiograph of the DNA footprint is shown. The sizes of the denatured DNA sequence markers that were corun with the samples are indicated with arrows on the left side of the right panel. The 5' and 3' ends of the DNA probe used in the footprinting assay are indicated by arrows pointing up and down. The nucleotide sequence of the DNA footprint is shown on the right. Numbers indicate the positions of start and end points of the protection region relative to +1, the transcriptional initiation site.

The GT box-containing element binds recombinant RAR and RXR in vitro independently of RA

View larger version (28K): [in this window] [in a new window]   FIG. 7. Retinoid receptors specifically bind to the GT box element in the blr1 promoter. Electrophoretic mobility shift assays were conducted using the in vitro translation products of RAR and RXR or nuclear extracts of HL-60 cells with (+) or without (-) 1 µM RA treatment and the GT box element (identified in the blr1 promoter as a biotin-labeled double-stranded oligonucleotide probe as described under Materials and Methods). DNA on the membrane was visualized and quantified on a PhosphorImager. The upper charts indicate the relative binding affinities of the DNA-protein complex and supershifted complex. (A) Lane 1 (first lane from the left), free biotin-labeled GT box element probe; lanes 2, 3, and 8, RAR-incubated probe; lanes 4, 5, and 9, RXR-incubated probe; lanes 6, 10, 11, and 12, RAR- and RXR-coincubated probe; lanes 3, 5, and 7, probe in the presence of a 100-fold excess of unlabeled GT box competitor; lanes 8 and 10, probe in the presence of RAR antibody; lane 9 and 11, probe in the presence of RXR antibody; lane 12, probe in the presence of anti-RAR IgG and anti-RXR IgG. (B) Lanes 1, 3, and 5, GT box probe and in vitro-translated RAR without RA; lanes 2, 4, and 6, GT box probe and in vitro-translated RAR incubated with 1 µM RA for 24 h at 37°C; lane 7, GT box probe and nuclear extracts from non-RA-treated HL-60 cells incubated without RA; lane 8, GT box probe and nuclear extracts from non-RA-treated HL-60 cells incubated with 1 µM RA for 24 h at 37°C; lanes 9 and 10, GT box probe and nuclear extracts from RA-treated HL-60 cells incubated without or with 1 µM RA for 24 h at 37°C.

RA induces binding of a TF complex to the GT box-containing element which could be competed off with DR2 oligonucleotides If, as the data thus far suggest, the presence of this GT box-containing element is critical to all-trans-RA-induced activation of the blr1 promoter, then one might anticipate that RA induces the binding of a nuclear protein complex to it. Furthermore, if it contains RAR-RXR, then DR1, DR2, or DR5 oligonucleotides, the idealized consensus sequences for RAR-RXR binding, should compete for binding of the complex to the element. EMSAs were performed using the 17-nt GT box-containing element as a probe for in vitro-translated RAR-RXR and nuclear extracts, respectively. The nuclear extracts were from untreated cells and cells treated with all-trans-RA for 48 h, respectively. Figure 8A shows results demonstrating that both DR2 and DR5 compete with the GT box sequence for binding in vitro-translated RARs (RAR, RXR, and RAR-RXR) but that DR1 failed to compete. Interestingly, Fig. 8B shows that compared to the binding of in vitro-translated receptors, the binding of the nuclear protein complex from nuclear extracts was not as susceptible to competition. Without RA, there was no protein complex binding to the probe. RA induced the binding of a nuclear protein complex which increased as the amount of lysate was increased from 5 to 20 to 80 µg. The binding was progressively competed off by increasing the amounts of cold probe. DR2 oligonucleotide also diminished binding but DR1 and DR5 oligonucleotides did not (Fig. 8B), suggesting that the GT box has a DR2-like binding motif character. This again suggested that the nuclear protein complex has more factors than just RAR and RXR which may be involved in the binding to the GT box motif and that the presumed multiprotein complex has much higher binding affinity for the GT box sequence than for DR2 and DR5.

View larger version (27K): [in this window] [in a new window]   FIG. 8. DR2 oligonucleotides compete with the GT box element for binding nuclear proteins of RA-treated (+) HL-60 cells. EMSAs with competing test oligonucleotides were performed using in vitro-translated RARs and nuclear extracts from untreated (-) and all-trans-RA (1 µM)-treated HL-60 cells. The probe was the wild-type GT box element. The numbers above the blots (lower panels) indicate the severalfold excesses of the competing oligonucleotides added. The arrows indicate the specific GT box element-nuclear protein binding complex (upper part of the gel) and the free GT box probe (bottom of the gel). The upper chart indicates the relative binding levels of the DNA-protein complex. (A) Lanes 1, 2, and 3 (lane 1 is the first lane from the left), GT box probe and in vitro-translated RAR and RXR receptors; lanes 4, 7, and 10, GT box probe and in vitro-translated RARs incubated with DR1; lanes 5, 8, and 11, GT box probe and in vitro-translated RARs incubated with DR2; lanes 6, 9, and 12, GT box probe and in vitro-translated RARs incubated with DR5. The EMSAs with in vitro-translated RARs were repeated, and the same results were obtained. (B) GT box probe was incubated with nuclear extracts from 48-h RA (1 µM)-treated HL-60 cells (lanes 1 and 5 to 10, 80 µg each; lanes 2, 3, and 4, respectively, 5, 20, and 80 µg). Unlabeled synthetic oligonucleotides for typical RAR binding DR5, DR2, and DR1 sequences, as well as the Sp1 DNA binding consensus sequence, were used as competitors for GT box element binding. The assays repeated using nuclear extracts from cells treated with 1 µM RA for 24 h yielded similar results.

Assembly of the TF complex on the GT box-containing element depends on both GT boxes Whereas a cold GT box-containing element could compete with the GT box-containing element probe, deletion mutants of either GT box could not compete with the GT box-containing element probe. EMSAs using the 17-nt GT box-containing element as a probe of nuclear extracts from all-trans-RA-treated and untreated cells were performed as described above. The ability of the unlabeled GT box-containing element or the element mutated in either the first or the second or both GT boxes (the mutants are shown in Fig. 9B) to compete for binding of the nuclear protein complex was tested. Figure 9A shows the results. Mutating either or both of the GT boxes resulted in the loss of the ability to compete for binding. Binding of the TF complex to the GT box-containing element thus depended on both GT boxes being intact.

View larger version (23K): [in this window] [in a new window]   FIG. 9. Mutation of the GT box element diminishes binding of the RA-induced complex to the sequence motif and abolishes activation of the blr1 promoter following RA induction. (A) EMSAs were done with nuclear extracts of HL-60 cells with (+) or without (-) RA treatment and the biotin-labeled 17-nt wild-type GT box element (GTbox) from the blr1 promoter and its mutants with either or both GT boxes deleted (13-bp fragment, GTm1 and Gtm2; 9-bp fragment, GTm12). Binding was detected with wild-type GT boxes; this binding was not detected with either of the GT boxes mutated. The EMSA was repeated, and the same result was achieved. (B) Mutation of GT boxes within the wild-type (W.T.) GT box element used in the BLR1-Luc promoter reporter construct was performed using PCR with three pairs of primers as described in Materials and Methods. The BLR1-Luc reporter constructs containing the indicated mutations in either one or both of the GT boxes were cotransfected with pRL-TK into HL-60 cells. Cells were collected after 48 h for assaying luciferase levels. The chart at the right represents the activity of the BLR1-Luc reporters relative to that of the promoterless reporter control. The transient transfection with the mutated constructs was repeated twice with similar results.

In sum, the distal portion of the 5'-flanking region supporting RA responsiveness in the BLR1 promoter contains an element with two GT boxes. This element binds an RA-induced TF complex. The element binds in vitro both RAR and RXR of high concentration. Assembly of the TF complex on this element depends on both GT boxes. Consistent with this, transcriptional activation in response to RA also depends on both GT boxes. The data suggest that this GT box-containing element binds a TF complex containing RAR and RXR (in response to the presence of RA) to transcriptionally activate blr1.

RA responsiveness conferred by the distal element depends on downstream sequences containing critical Oct1, NFATc, and CREB sites The ability of the TF complex-bound distal promoter element to confer RA responsiveness depends on downstream sequences. In particular, mutation of downstream Oct1, NFATc, and CREB sites (but, interestingly, not mutation of either of the two RAR-RXR half-sites) caused loss of RA responsiveness. Deletion mutants of the BLR1-Luc promoter reporter described above were created by deletion of -891 to +1 or +1 to +266 and transiently transfected into cells, and their luciferase activity levels were assayed after 48 h of all-trans-RA treatment as described earlier. Figure 10 shows the results. With the loss of either segment, the distal sequence from -1096 to -891 containing the GT box sequence was no longer able to confer responsiveness to RA. There were thus sequences within the deleted segments that were essential for the distal domain to activate transcription in response to RA.

View larger version (12K): [in this window] [in a new window]   FIG. 10. RA responsiveness by the GT box element RARE depends on the downstream context in the blr1 promoter. Promoter-reporter constructs from which 5' sequences of -891 to +1 or +1 to +266 in the 1,360 bp of the BLR1 5'-flanking region were deleted were prepared by PCR and transiently transfected into HL-60 cells which were then treated with all-trans-RA for 48 h and assayed for luciferase activity as described in Materials and Methods. The lines and numbers indicate the blr1 promoter sequence contained in each promoter-reporter construct. The chart on the right side indicates the relative luciferase activity level for each promoter-reporter construct. With the loss of either segment, the distal sequence from -1096 to -891 containing the GT box element was no longer able to confer responsiveness to all-trans-RA. All transient transfection studies were conducted in triplicate on three separate occasions with similar results. The chart reports the means of three experiments.

View larger version (27K): [in this window] [in a new window]   FIG. 11. The putative RAR-RXR half-sites identified by TransFac database analysis do not control blr1 response to RA. Mutations within either or both of two putative RAR-RXR half-sites at -783 to -782 and -45 to -44 in the blr1 promoter identified by TransFac database analysis were created in the BLR1-Luc construct with two pairs of primers specific to these putative sites as described in Materials and Methods. The wild-type (W.T.) BLR1-Luc reporter and its mutants were cotransfected with pRL-TK into HL-60 cells. The cells were treated with 1 µM all-trans-RA for 48 h and harvested to assay luciferase activity. The values represent the activity of the BLR1-Luc reporters relative to that of the promoterless reporter control. Transient transfections with these mutated constructs were repeated twice, and the same results were obtained.

View larger version (38K): [in this window] [in a new window]   FIG. 12. Mutation of Oct1, NFATc, or CREB motifs downstream of the GT box element abolishes RA inducibility of the BLR1 promoter. Deletion mutagenesis targeting the three Oct1, four NFATc, and one CREB consensus sequences in the blr1 promoter was performed on the BLR1-Luc construct as described in Materials and Methods. Bases in lowercase are deleted. The consensus sequence (ATTC) in italics is located in the antisense strand of DNA. The BLR1-Luc reporter constructs containing the indicated mutations were cotransfected with pRL-TK into HL-60 cells, which were treated for 48 h with all-trans-RA and then collected to assay luciferase activity levels. The values represent the activity of the BLR1-Luc reporters relative to that of the promoterless reporter control. Transient transfections with the mutated constructs were repeated twice, and the same results were obtained.

Finally, the TransFac database search indicated a potential CREB site at +178 to +181. Testing (using BLR1-Luc transfection) the functional significance of this site indicated that mutation of the site caused loss of RA responsiveness. (Figure 12 shows the results of the luciferase activity assay.) The CREB site thus appears to be necessary for RA responsiveness.

To confirm that the TransFac database-identified, functional NFATc, Oct1 and CREB sites in the blr1 promoter bind their cognate factors, supershift EMSAs were performed. The oligonucleotide sequences corresponding to the functional binding sites (for Oct1, TGAATAAAAAATTG -707 to -694; for NFATc, GTGAGGAAAATG -212 to -201; for CREB, GAGCTGACGGCT +174 to +185) in the blr1 promoter were synthesized and used as probes. Nuclear extracts from HL-60 cells were incubated with the biotin-labeled probe, and antibodies specific to each of the individual factors were added to induce supershifts. Since Oct1 and Oct2 recognize very similar binding sites, EMSAs with Oct1- or Oct2-specific antibodies were used to detect which actually bound the blr1 promoter sequences. The results are shown in Fig. 13. Anti-Oct1, but not anti-Oct2, caused a supershifted band (Fig. 13, left panel), suggesting that Oct1 binds the putative binding site in the blr1 promoter. NFATc1-, NFATc2-, NFATc4-, and NFATc5-specific antibodies did not produce super shifts, but NFATc3 did (Fig. 13, center panel), suggesting that NFATc3 is the factor binding to the putative site in the blr1 promoter. While CREB1-specific antibody did not cause a supershift, the CREB2-specific antibody did (Fig. 13, right panel), suggesting that CREB2 but not CREB1 binds the putative site in the blr1 promoter. The data thus demonstrate that the sequences identified in silica and shown genetically to be functional do biochemically bind their corresponding TFs.

View larger version (27K): [in this window] [in a new window]   FIG. 13. Oct1, NFATc3, and CREB2 bind to their corresponding consensus sites in the blr1 promoter. EMSAs were performed to confirm that the Oct1, NFATc, and CREB sites identified in the blr1 promoter by TransFac database analysis and functional mutagenesis experiments are actual consensus binding sites for these factors. The oligonucleotide sequences harboring the database-deduced binding sites (Oct1, TGAATAAAAAATTG [-707 to -694]; NFATc, GTGAGGAAAATG [-212 to -201]; CREB, GAGCTGACGGCT [+174 to +185]) were synthesized and used as probes. Nuclear extracts (NE) from non-RA-treated HL-60 cells were incubated with the biotin-labeled probe. Antibodies specific to the individual factors were added to induce supershifts. Supershifts were observed with anti-Oct1 (left panel), anti-NFATc3 (center panel), and anti-CREB2 (right panel), suggesting that Oct1, NFATc3, and CREB2 bind their putative consensus binding sites in the blr1 promoter.

The RA-induced TF complex assembled at the RAR-RXR binding GT box-containing element includes at least Oct1 and NFATc3