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Figure 2 | BMC Molecular Biology

Figure 2

From: Interaction of C/EBP-beta and NF-Y factors constrains activity levels of the nutritionally controlled promoter IA expressing the acetyl-CoA carboxylase-alpha gene in cattle

Figure 2

NF-Y and C/EBP binding sites in element A and C respectively in the distal region are involved in PIA repression. (A) Genomic organization of PIA. The distal region includes three putative repressive elements, as revealed by coarse mapping analyses involving Stu I and Nde I deletions, respectively (filled boxes, element A, B, C). Element A harbors a NF-Y attachment site (filled circle), and element C contains a C/EBP binding site (filled square). The core binding sequences of those factors and mutational derivatives are shown. The proximal driving region features a NF-Y site (open circle) and a C/EBP binding site (open square). ‘+1’ represents the tsp. (B) Abrogation of NF-Y binding and C/EBP binding in the distal PIA relieves repression in reporter gene assay. Shown are mean values (+ S.E.M.) of at least three independent experiments, each assayed in triplicate. Significance of difference relative to construct −1045 (WT) is indicated (** P < 0.01). (C) EMSA analysis of transcription factors binding to element A. The labeled probe A_WT (Additional file 1: Table S4) is bound by NF-Y factors from nuclear extracts of HC-11, pbMEC and bMG (“S”) and is supershifted (SS) by the addition of antibodies against NF-YA (αA). Lanes: FP, free probe; C and Y, competition with 100-fold molar excess of the unlabeled A_WT probe and a probe harboring an NF-Y consensus site respectively (Additional file 1: Table S4). (D) EMSA validation that C/EBP factors bind to element C. Nuclear extracts from HC-11 cells were incubated with the radioactively labeled probe C_WT (Additional file 1: Table S4). Lane: C, competition with 100-fold molar excess of unlabeled C_WT; S, shift; αB, supershift (“SS”) with anti-C/EBP antibodies; Cm2, competition with 100-fold molar excess of the unlabeled probe mutated as shown in Figure 2A.

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