Identification of cis-acting regulatory elements conferring differential IRAK-2C expression. (A) Genomic DNA or cDNA from F1(C57BL/6JxMOLF/Ei) macrophages was PCR amplified and analyzed by sequence analysis to determine allelic bias in IRAK-2C transcript. (B) Sequence alignment of the genomic region of IRAK-2C from three wild-derived strains (top three lanes) and C657BL/6J (bottom lane). Positions of the elements are given in base pairs with respect to the transcription initiation site (at 0 bp). Red boxes, mutations common for all wild-derived strains; Blue box, splice acceptor site for IRAK-2A isoforms; red text, NF-κB consensus and translation start of the IRAK-2C protein. Consensus sites were identified using MatInspector software (Genomatix Software GmbH). (C) EMSA was performed using nuclear extracts of LPS-activated RAW cells and a double-stranded 32P-labeled oligonucleotide corresponding to the sequence containing the GATA-binding site. Cold competition assay was performed with 5×-, 25×-, or 100×-fold excess of either B6 or MOLF unlabeled oligonucleotide. (D) Nuclear extracts from LPS-activated RAW cells were incubated with radiolabeled probe derived from C57BL/6J sequence or a sequence with an (A/T) substitution in the CACC-binding site (mutant). Alternatively, a MOLF-derived probe from the region corresponding to the 10-bp deletion was used. (E) RAW264.7 cells were stably transfected with constructs containing the indicated IRAK-2C promoter regions from MOLF/Ei or C57BL/6J fused to luciferase. Equivalent levels of transfection and integration were confirmed using real-time PCR analysis of genomic DNA (not depicted). Cells were stimulated with 100 ng/ml LPS for the indicated times and luciferase was quantified as a measure of promoter activity. Values are expressed relative to unstimulated MOLF/Ei promoter constructs and are shown as mean ± SEM of three wells. Data are representative of three independent experiments.
