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Fig. 4 | Genome Medicine

Fig. 4

From: A functional SNP associated with atopic dermatitis controls cell type-specific methylation of the VSTM1 gene locus

Fig. 4

rs612529 C/T controls the binding of PU.1/YY1 to the VSTM-1 promoter. a Allele-specific binding to rs612529. EMSA experiments with nuclear extracts from monocytes were carried out with radiolabeled probes representing the C allele (“C probe;” lanes 1–7) or the T allele of rs612529 (“T probe;” lanes 8–14). In a “cold” competition, the binding to the radiolabeled probes was competed with unlabeled C probes (lanes 2–4 and 9–11) or T probes (lanes 5–7 and 12–14). Bands 1 and 2 are two bands formed by allele-specific binding; “N.S.” indicates a non-specific band. b Competition with consensus binding motifs of predicted candidate factors. Binding to the T probe was competed with unlabeled probes representing consensus binding motifs for MZF-1, PU.1 and YY1 (PBX was used as control). Binding of each factor was competed with the optimal consensus sequence as well as a mutated variant to which the binding was abolished (sequences are listed in Additional file 7). c Super-shift assays. Super-shift assays were carried out with the T probe and antibodies against MZF-1 (lane 2), PU.1 (lane 3), Ets1 (lane 4), Sp1-B (lane 5), GATA1 (lane 7) CREB1 (lane 8), and YY1 (lane 9) or isotype-matched control antibodies (lanes 1 and 6). Super-shifted bands and bands lost by the antibody treatment are indicated by red arrows. d PU.1-ChIP assay. A ChIP assay with nuclear samples from monocytes of four CC and five TT donors (rs612529) was carried out with PU.1-specific antibodies (red dots) or isotype-matched control antibodies (blue dots). PU.1 occupancy, depicted as fold change compared to the isotype, was determined by quantitative PCR amplification of the respective region in the VSTM-1 promoter (Additional file 3)

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