Skip to main content
Figure 4 | BMC Molecular Biology

Figure 4

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 4

Comparison of the binding affinities of the distal and proximal NF-Y and C/EBP sites and influence of these factors on the PIA activity. (A) Affinity comparison of the proximal and distal NF-Y binding sites of PIA. Nuclear extracts were prepared from HEK293 cells jointly over-expressing the three bovine NF-Y factors (A, B, C). Sequences of the labeled NF-Y consensus probe and of the unlabeled competitor of the distal NF-Y (A_WT) and proximal NF-Y sites (pNF_WT) are shown in Additional file 1: Table S4 cf (Figures 2A and Figure 3A). Lane: FP, free probe; C, competition with 100-fold molar excess of unlabeled probe (self); S, shift. The fold molar excess of the respective unlabeled competitors added to the reactions, as resolved in lanes 4–8 or lanes 9–13 were 10, 25, 50, 100, 200-fold of the labeled probe. (B) Same as A, but evaluating the C/EBP binding sites. These samples were run out on the same gel. Nuclear extracts were prepared from HC-11 cells over-expressing the DNA binding domain of the bovine C/EBPβ factor. Sequences of the labeled C/EBP consensus probe, the unlabeled oligonucleotide competitors of C/EBP distal (C_WT) and C/EBP proximal (pCE_WT) are shown in Additional file 1: Table S4 cf (Figure 2A and Figure 3A). (C) Effects of NF-Y and C/EBPβ factors on the proximal PIA activity. The proximal PIA reporter construct −127 was transfected into HC-11 cells, together with the empty cloning vector (EV) or with constructs expressing C/EBPβ or NF-Y subunits, as indicated. The functional NF-Y trimer can quench this effect. Results (±S.E.M.) are representative for two experiments, each assayed in triplicate. Statistical significance of the differences was calculated relative to the control having been cotransfected only with empty vector (grey column), **P <0.01. (D) NF-Y factors interact with C/EBPβ having bound to PIA. HEK293 cells were co-transfected with the −127 PIA reporter gene and expression constructs for the factors C/EBPβ (tagged with a flag), NF-YAm29, -YB, -YC. Nuclei were used for ChIP assays using antibodies against C/EBPβ or NF-YA or control IgG. Data are mean values (±S.E.M.) of four qPCR assays of the amount of recovered PIA promoter molecules (**P <0.01, as compared to control IgG). (E) A master control unit modulates PIA activity. NF-Y and C/EBP factors (black) having bound to the distal repressive factor binding sites may be looped back to physically contact the respective other factor (open) having bound to the proximal driver region. This interaction lowers PIA activity. The map is not drawn to scale. “+1” represents the tsp.

Back to article page