Rogulski et al. 10.1073/pnas.0507902102. |
Fig. 6. Promoter sequences of genes listed in Table 3. Consensus and nonconsensus E-boxes and consensus motifs A, B, and C are highlighted in the same colors used in Fig. 2.
Supporting Materials and Methods
Purification of RNAs, Microarray Analysis, and Quantitative Real-Time RT-PCR Quantification.
The integrity of each RNA sample was confirmed by 1% agarose formaldehyde gel electrophoresis followed by ethidium bromide staining and visual inspection and by analysis on an Agilent Bioanalyzer 2100 (Agilent, Palo Alto, CA). Total RNA (5 mg) was used as a template to generate cDNA using a T7-oligo(dT)24 primer and Superscript II reverse transcriptase (Invitrogen) according to the manufacturer’s protocol. Double-stranded cDNA was synthesized in second strand buffer (Invitrogen) by the addition of 10 units of T4 DNA ligase, 40 units of E. coli DNA polymerase I, 200 mM dNTPs, and 2 units of RNase H at 16°C for 2 h. Ten units of T4 DNA polymerase were then added, and the reaction was continued for an additional 5 min. Reactions were stopped by the addition of EDTA to 30 mM. After phenol:chloroform extraction and ethanol precipitation, an aliquot of the double-stranded cDNA was used for the synthesis of biotinylated cRNA using a BioArray high-yield RNA transcript-labeling system (ENZO, Farmingdale, NY). Biotinylated cRNAs were purified by using an RNeasy kit (Qiagen, Valencia, CA). The product (20 mg) was then incubated at 94°C for 35 min in fragmentation buffer (40 mM Tris-acetate, pH 8.1/100 mM potassium acetate/30 mM magnesium acetate). Fragmented cRNA (15 mg) was added to 1´ hybridization mixture (Affymetrix, Santa Clara, CA) in a final volume of 300 ml and hybridized at 45°C overnight to the Affymetrix murine genome MG-U74Av2 GeneChip. In addition to ≈6,000 EST clusters, the GeneChip contains all sequences in the mouse UniGene database that have been functionally characterized. The quality of the cRNA and the efficiency of its labeling were also assessed by hybridization to a Test 3 Array GeneChip (Affymetrix) to ensure equivalent hybridization to the 5' and 3' oligonucleotides of housekeeping genes before hybridization with the MG-U74Av2 GeneChip. Washing and staining were done according to the recommendations for use of the Affymetrix Fluidics Station.MICROARRAY SUITE 5.0
software (Affymetrix) was used to extract image intensities and to normalize raw expression data to correct for differences in signal intensities across the microarray. Expression values were multiplied by a scaling factor to make the average intensity of the housekeeping gene set on each microarray equal to a target intensity of 150. Independent comparative analyses for each of three experiments were performed with 32D-neo and 32D-MT-MC1 fluorescence data as the baseline and experimental .cel files, respectively. Results were imported into Microsoft EXCEL for further analysis. Only probe sets exhibiting a signal log2 ratio of ³1.0 or no less than or equal to –1.0 in each of three replicate experiments were scored as "MT-MC1-regulated." Random comparative analyses of 32D-neo.cel and 32D-MT-MC1.cel files were performed to eliminate genes that could be regulated by chance. Genes were ranked by net change with increased (I), decreased (D), and no change (NC) calls assigned values of +1, –1, and 0, respectively. A net expression change (NEC) of ±6 was used as the cutoff. As a second means of validation, 32D-neo.cel and 32D-MT-MC1.cel files were analyzed with DCHIP 1.3 software (27). After normalization of signal intensities with the prefect match-based model, a batch comparative analysis was performed. Experimental (32D-MT-MC1) to baseline (32D-neo) ratios (E/B and B/E) of 1.2 were used. A 20% filtering option was used in the Absent vs. Present call. DCHIP default settings were selected for all remaining parameters. Identified gene information and accession numbers were confirmed by searches of LocusLink (www.ncbi.nlm.nih.gov/LocusLink). Genes were grouped into functional categories based on their known molecular and/or biological functions denoted in LocusLink. Affymetrix MICROARRAY SUITE 5.0-processed comparative analysis results of the three experiments have been published as Table 2.To verify the results of microarray analyses, we performed QRT-PCR analyses on triplicate independent samples of total RNAs purified from the indicated 32D cell lines. RNAs were treated with Turbo-free DNase (Ambion, Austin, TX) to remove residual DNA, diluted to 50 ng/ml, and stored in multiple small aliquots. Each QRT-PCR analysis was performed on 50 ng of total input RNA using a SYBR Green-based assay (QuantiTect, Qiagen) according to the directions of the supplier. Oligonucleotide primers for select MT-MC1 target genes were chosen so as to span intron–exon boundaries and were designed with the PRIMER 3 program). Primers were synthesized by Integrated DNA Technologies (Coralville, IA) and are listed in Table 1. Reverse transcription and PCR amplification and analyses were performed on a LightCycler 2 (Roche Diagnostics, Indianapolis, IN) under the conditions recommended by the supplier. Melting curves were performed on all PCR products to ensure specificity of the amplification process. All analyses used LIGHTCYCLER 4 relative quantification software. Standard errors on all samples were generally <2%. Ct values for each reaction were normalized to those for GAPDH reactions included in each set of QRT-PCR runs and are expressed relative to values obtained with RNAs from 32D-neo cells.
Plasmid Constructs.
The myc-epitope-tagged MT-MC1 deletion mutant del(2-32) was generated by standard PCR using the forward primer 5'-CGC CTC GAG GCA ATC GGA GGA TTT CTC TGG-3' and the reverse primer 5'-CGC GAG CTC TCA GGA ATC GGG AAA TGC CT-3'. In the first primer, the italicized codons correspond to an engineered XhoI site, which is followed by codon 33 (GCA) of MT-MC1. In the second primer, the italicized codons correspond to a SacI site, which is adjacent to the naturally occurring MT-MC1 translation termination codon. The amplified fragment was digested with XhoI+SacI and cloned directionally into the XhoI-SacI polylinker site of the pSVL(MT)-neo vector. Confirmation of the plasmid structure was done by automated DNA sequencing.To create the ERTM-MT-MC1 (ERTM, estrogen receptor responsive to tamoxifen) fusion protein expression vector, we first amplified the region encoding the hormone binding domain (amino acids 281–599) of the mutant estrogen receptor ERTM (1) from the pBabePuro-c-mycERTM vector using the forward primer: 5'-CGC AAG CTT ACC ATG CGA AAT GAA ATG GGT GCT TCA GGA-3' and the reverse primer: 5'-CGC GGA TCC GAT CGT GTT GGG GAA GCC CTC T-3' where italicized codons represent engineered HindIII and BamHI sites, respectively. Following digestion with these enzymes, the purified fragment was ligated into HindIII+BamHI-digested pcDNA3.1(+) (Invitrogen) to give the vector pcDNA-ERTM. We next amplified the myc-epitope-tagged MT-MC1 coding region using the pSVL(MT)MT-MC1-neo vector as template (25). The forward primer consisted of the sequence 5'-CGC GGA TCC ACC ATG GCA GAG GAG CAA AAG C-3' and the reverse primer 5'-CGC GAA TTC TCA GGA ATC GGG AAA TGC CTT T-3'. Italicized bases indicate BamHI and EcoRI sites, respectively. The purified BamHI + EcoRI-digested fragment was then cloned into the pcDNA-ERTM vector described above. The resulting vector, pcDNA-ERTM-MT-MT-MC1, was verified by DNA sequencing. Expression of the ≈64 kDa myc-epitope-tagged fusion protein was confirmed by transient and stable transfection in Cos7 and 32D cells, respectively. The latter was achieved by electroporation of linearized plasmid DNA and selection in G418 as described in refs. 2 and 3. To activate the fusion protein, cells were exposed to 250 nM of 4-hydroxytamoxifen in the presence of 10 mg/ml of cycloheximide (both from Sigma–Aldrich). Total RNAs were then collected after 8 h and processed for QRT-PCR analyses as described above.
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