Figure 2

Selective activation of MS4A12 by CDX2. (A) mRNA expression of CDX1, CDX2, and MS4A12 was quantified by real-time RT-PCR 48 h after transfection of LoVo and SW48 cells with 10 nM siRNA duplexes (Qiagen) using HiPerFect transfection reagent (Qiagen). siRNA duplexes targeted nucleotides 1054-1072 (siRNA1 sense 5'-r(CAG UAA GCC UGU UGG AUA A)dAdG-3', antisense 5'-r(UUA UCC AAC AGG CUU ACU G)dCdA-3') and 1675-1693 (siRNA2 sense 5'-r(GGA UGC AGC UUC AAG AAU A)dAdA-3', antisense 5'-r(UAU UCU UGA AGC UGC AUC C)dAdA-3') of the CDX1 mRNA sequence (NM_001804.2), and nucleotides 1492-1510 (siRNA1 sense 5'-r(GAG AGG GAC UCA AGG GAA A)dGdG-3', antisense 5'-r(UUU CCC UUG AGU CCC TCU C)dTdC-3') and 1263-1281 (siRNA2 sense 5'-r(GAG UAA GAC AAG UGG GAU U)dTdC-3', antisense 5'-r(AAU CCC ACU UGU CUU ACU C)dCdT-3') of the CDX2 mRNA sequence (NM_001265.2). As control a non-silencing (ns) siRNA duplex (sense 5'-r(UAA CUG UAU AAU CGA CUA G)dTdT-5', antisense 5'-r(CUA GUC GAU UAU ACA GUU A)dGdA-3') was used. RNA extraction, first-strand cDNA synthesis and real-time reverse transcription-PCR (RT-PCR) were performed as previously described [4]. Real-time quantitative expression analysis was performed in triplicates in a 40 cycle RT-PCR. After normalization to HPRT (sense 5'-TGA CAC TGG CAA AAC AAT GCA-3'; antisense 5'-GGT CCT TTT CAC CAG CAA GCT-3', 62°C annealing) expression of CDX1 (sense 5'-CTC ACT GAA CGG CAG GTG AAG-3'; antisense 5'-TAG GTG ACT GTC CAC CAT GTC-3', 60°C annealing), CDX2 (sense 5'- GCT TCT GGG CTG CTG CAA ACG-3'; antisense 5'-CTT TCG TCC TGG TGG TTT TCA CTT GG-3', 62°C annealing), and MS4A12 (sense 5'- GAG CTT TCC CGT TGT CTG GTG-3'; antisense 5'- GCT GAA GAA GAC GCT GGT GTC-3', 60°C annealing) was quantified using ΔΔCt calculation. Expression of each gene is shown in relation to expression levels in untreated control cells (100%). (B) 48 h after transfection with siRNA duplexes LoVo and SW48 cells were transfected with luciferase reporter genes linked to the wild type MS4A12 promoter domain (-952 to +12). Activation of the MS4A12 promoter was measured in luciferase activity assays after 24 h. Luciferase activity was normalized to fluorescence obtained by co-transfection with eGFP reporter plasmid. All assays were done in triplicates; mean values +/- STD are shown. (C) ChIP was performed using the Chromatin Immunoprecipitation (ChIP) Assay Kit (Millipore) according to the manufacturer's instructions with chromatin prepared from LoVo and SW48 cells. The promoter region containing the CDX2 binding site (-115/-273) and a region upstream to the promoter (-1078/-1295) as negative control were analyzed by PCR following immunoprecipitation with the antibodies indicated. Results of amplification of soluble chromatin prior to precipitation are shown as control (input).