Identification of highly conserved non-coding sequences in the genomic region bordering GATA-2
Pipmaker software was used to identify conserved non-coding sequences in genomic sequences flanking GATA-2 in mouse, rat, human and fugu and that of the zebrafish genome [34–36]. A 158 kbp zebrafish genomic sequence (Sanger Institute accession number: AL928619, Figure 1) was compared to orthologous regions of the other genomes. The genomic sequences from other species used for comparison were: rat chromosome 4 fragment 122,079,753 to 122,488,528 (~408 kbp); mouse chromosome 6 fragment 88,429,180 to 88,842,527 (~413 kbp); human chromosome 3 fragment 129,480,974 to 129,894,726 (~413 kbp) and fugu scaffold_374 (~189 kbp).
Construction of Tol2 transposon vectors
Highly conserved zebrafish motifs (Up1, and Up2) identified by comparative genomics were amplified by PCR. The primers used in the PCR reactions were:
Up1 5' primer: 5'-ATCCGCTCGAG TTTGACCTTTTCGGAAAACGAGCT-3'
Up1 3' primer: 5'-CGGAATTC TCTGATGGTCTCCACGGCAA-3'
Up2 5' primer: 5'-ATCCGCTCGA G ATCTTCCCCCTCAAATGATGCA-3'
Up2 3' primer: 5'-CGGAATTC GGGGGTAATATTTCATCGGCGT-3'
Up2a 3' primer: 5'-CGGAATTC TGGAAACAGGGTCCAAAGT-3'
Up2b 5' primer: 5'-ATCCGCTCG AG CCTCGCTCGGTCCTGACA-3'
The underlined bases correspond to restriction enzyme sites for Xho I (CTCGAG) or EcoR I (GAATTC); italicized bases are zebrafish genomic sequences. PCR products were digested with restriction enzymes Xho I and EcoR I and inserted proximal to a GATA-2 minimal promoter (mp)/GFP reporter gene contained in a Tol2 transposon vector (Figure 2G~L). The GATA-2 minimal promoter used is a 249 bp genomic sequence 5' proximal to the GATA-2 transcription start site. The Tol2 vector, kindly provided by Kochi Kawakami, was the original construct previously described [37].
Base change or deletion mutations were introduced into the putative binding sites of Up2 for transcription factors E2F-1, ARP-1, AML-1, LMO2 and HOXA3, respectively, using the mutagenesis kit from Strategene, Inc. (Catalog No. 200521). The primers used for the site-directed mutagenesis were:
AML-1 mF: 5'-ATTGAATGTATTTGCGAGAGCGGTTTGAAAAGGCGGCGA-3'
AML-1 mR: 5'-CCGCCTTTTCAAACCGCTCTCGCAAATACATTCAATGCA-3'
ARP-1 mF: 5'-CACCAATTTATTCAGCGCCACGGGAGACCCTGTTTCCAGCCT-3'
ARP-1 mR: 5'-AGGCTGGAAACAGGGTCTCCCGTGGCGCTGAATAAATTGGT-3'
E2F-1 mF: 5'-CACCAATTTATTCAGC__ACTTTGGACCCTGTTTCC-3'
E2F-1 mR: 5'-GGAAACAGGGTCCAAAGT__GCTGAATAAATTGGTG-3'
LMO2 mF: 5'-CCAGCGGTAAGCTACGCACGATTAGGCCATTTATTATCT-3'
LMO2 mR:5'-GTGAAGATAATAAATGGCCTAATCGTGCGTAGCTTACCG-3'
HOXA3-A mF: 5'-TTTGAAAAGGCGGCGATGGCCTGGGGGAGAGAGATAAG-3'
HOXA3-A mR: 5'-TTATCTCTCTCCCCCAGGCCATCGCCGCCTTTTCAAACC-3'
HOXA3-B mF: 5'-CCAGCGGTAAGCTGATAACGACCCGGCCATTTATTATCT-3'
HOXA3-B mR: 5'-GTGAAGATAATAAATGGCCGGGTCGTTATCAGCTTACC-3'
The underlined bases were the mutated sites. Three bases were deleted from E2F-1 binding site (shown as a line). Constructs carrying site-specific mutations were injected into wild type zebrafish embryos at one cell stage. The transient hematopoietic GFP expression was observed under fluorescent microscope after 24 hpf to calculate the percentage of hematopoietic GFP expression of each mutated construct.
Construction of modified BAC constructs
BACs containing zebrafish GATA-2 identified in previous studies were used in the construction of modified BAC vectors[13, 38]. Deletions of 5' upstream elements of GATA-2 were preformed using the shuttle vector pLD53.SCA_E_B as previously described [11, 16]. In brief, 300~500 bp flanking sequences of Up1, or Up2, respectively, were amplified by PCR. The primers used in deletions of elements Up1 or Up2 or both were:
Up1 A box 5' primer: 5'-GCATGGCGCG CC CCCCAAAAGTCACATCT-3'
Up1 A box 3' primer: 5'-CCGGTTAATTAA TTCCGAAAAGGTCAAACACA-3'
Up1 B box 5' primer: 5'-CCGGTTAATTA A GAGAGCGCGCGCCCGGTGAAGAT-3'
Up1 B box 3' primer: 5'-CCGGATGGCCGGC C TACTTAACAACTCCAACCCA-3'
Up2 A box 5' primer: 5'-GCATGGCGCG CC ACTGGATAAAGGAGGCAA-3'
Up2 A box 3' primer: 5'-ACGTTTAATTAA GATCGCATGGGGTGATAGAGACAG-3'
Up2 B box 5' primer: 5'-CCGGTTAATT AA AGAGAGGGCCCTGGCATCT-3'
Up2 B box 3' primer: 5'-CCGGATGGCCGGC C GTTCAATGGACTCAAAAGGT-3'
The underlined bases correspond to restriction enzyme sites for Asc I (GGCGCGCC), Pac I (TTAATTAA), or Fse I (GGCCGGCC), and italicized bases are zebrafish genomic sequences. PCR products were digested with restriction enzymes, and ligated into pLD53.SCA_E_B lacking the GFP reporter gene. The 5' flanking sequences were termed A boxes while the 3' flanking sequences as B boxes. After modification, the entire shuttle vector was integrated into the GATA-2 BAC clone by homologous recombination through one of the two homologous arms (A box or B box). Selection in LB media containing 5.5% sucrose resulted in deletions of Up1 or Up2 or both in approximately 50% of the BAC clones through recombination with the second homologous arm (B box or A box). Deletions were confirmed by sequencing.
The insertion of GFP into deletion and wild type GATA-2 BAC clones was carried out essentially as previously described [39]. In brief, a 377 bp PCR product, (A box) based on genomic sequence adjacent to and upstream of the zebrafish GATA-2 start codon, was inserted into the targeting vector pLD53.SC2 adjacent to a GFP reporter gene. The primers used in PCR amplification were:
5' primer: 5'-GATCGGCGCG CC CGGTAGTTATTTGAAATTGCGA-3'
3' primer: 5'-CTCAAGTGTCCGCGCTTAGAAA-3'
Underlined bases in the 5' primer correspond to restriction enzyme site for Asc I (GGCGCGCC) and italicized bases are zebrafish genomic sequences. In the presence of transiently expressed RecA, homologous recombination took place between the A box in the vector and the A box in the BAC clone resulting in integration of the vector insert into the BAC clone, placing the GFP reporter gene under control of the GATA-2 promoter within the clone (Figure 2B, D~F).
Modified GATA-2 BAC clones were identified by PCR using a forward primer (5'-CACGGGAAAAATAAACGCAGGA-3') upstream of the A box, and a reverse primer located in the GFP sequence (5'-GCTGCTTCATGTGGTCGGGGTA-3'). PCR positive clones were further confirmed by comparison of digestion patterns with that of the original BACs, or sequencing, or both.
Gel mobility shift analysis
EST clone BC076469 (cDNA clone MGC: 91782, IMAGE: 7039846), encoding zebrafish HOXA3, was purchased from Open Biosystems, Inc. The ORF of HOXA3 was PCR amplified with primers:
5' primer: 5'-ATCGAGGATCCACATTGGGAAACGGCGAG AT-3'
3' primer: 5'-AACAAAATACACTGCGCCA-3'
The underlined bases in the 5' primer contained the BamH I restriction site. The PCR product was digested with BamH I and cloned into the vector GEX-2T digested with enzyme BamH I and Sma I to generate a HOXA3 in frame fusion protein with GST. Plasmid containing the GST-HOXA3 fusion protein was expressed in BL21(DE3)plyYs. The construct containing fusion protein of GST and human E2F-1 was a gift from Dr. Nevin's lab at Duke University[40]. Protein expression and purification were preformed as previously described[41].
Electrophoretic Mobility Shift Analysis (EMSA) was preformed to confirm the binding affinity of the transcriptional factors with Up2 sequence with non-radioactive gel shift kit (Roche Applied Sciences, Inc. Catalog number: 03353591910). Complementary strand DNA oligoes containing transcription factor E2F-1 and HOXA3-A binding site, respectively, were annealed and DIG labeled. The sequences are as follow:
HOXA3-A probe: 5'-GGTTTGAAAAGGCGGCGATAATCTGGGGGAGAGAGATAA-3'
E2F1 probe: 5'-ACACCAATTTATTCAGCGCCACTTTGGACCCTGTTTCCA-3'
Mouse monoclonal antibody KH95 (Abcom, Inc) was used for super shift analysis and chicken anti-red fluorescent protein (RFP) polyclonal antibody (Chemicon International, Inc. Catalog No. AB3528) was used as non-specific antibody.
Generation of transgenic zebrafish
Tol2 vector-based plasmids were extracted and purified using a Qiaprep Spin Miniprep Kit (Qiagen). Modified BAC clone DNA was extracted using Hi-speed Maxi Prep Kit (Qiagen). BAC DNA was digested with restriction enzyme Not I and dialyzed in 0.5 × TE [13, 14]. DNA concentrations were adjusted to ~100 ng/ul in 0.1 M KCl.
BAC construct DNA was microinjected into wild type zebrafish fertilized eggs as previously described [38], resulting 4–7% germline transmission. Tol2 constructs were injected into transgenic zebrafish fertilized eggs containing maternally expressed Tol2 transposase [37] driven by the zebrafish vasa promoter [42]. F1 generation transgenic zebrafish embryos were identified by PCR. Fluorescent expression patterns were observed using a compound fluorescent microscope (Axioplan Imaging, Zeiss). Photographs were captured using an AxioCam digital camera (Zeiss), and Openlab software (Improvision).
Analysis of GFP reporter gene expression in mammalian cells lines
Transient transfection assays were performed in human myeloid leukemia cell lines, KG1 and K562, and in mouse fibroblasts NIH3T3. KG1 and K562 cells were cultured in Iscoves media containing 10% fetal bovine serum (FBS), 2 mM/L L-glutamine, 100 U/ml Penicillin and 100 mg/mL Streptomycin. NIH3T3 and 293T cells were cultured in DMEM supplemented with 10% FBS, 2 mM/L L-glutamine, 100 U/ml Penicillin and 100 mg/mL Streptomycin. Fifteen micrograms of GATA-2/GFP fusion constructs were co-transfected with 5 ug of the CMV-β-galactosidase plasmids into mammalian cells by electroporation as previously described[43, 44]. Forty-eight hours after transfection, cells were harvested and β-galactosidase activity was measured using the Promega assay kit (Promega). GFP expression levels were determined using flow cytometry. 106 Cells were harvested, washed, and resuspended in phosphate-buffered saline (PBS) and analyzed using a FACS-SCAN cytometer (Beckton-Dickinson). The percentage of GFP positive cells was calculated using cell quest software and expression levels were normalized using CMV-β-galactosidase activity. P values were calculated using the Student-T test method and Jump In software.