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. 2012 Oct;52(3-4):242-8.
doi: 10.1016/j.molimm.2012.06.005. Epub 2012 Jun 29.

Oct-1 acts as a transcriptional repressor on the C-reactive protein promoter

Bhavya Voleti  1 , David J Hammond Jr, Avinash Thirumalai, Alok Agrawal
Affiliations

Oct-1 acts as a transcriptional repressor on the C-reactive protein promoter

Bhavya Voleti et al. Mol Immunol. 2012 Oct.

Abstract

C-reactive protein (CRP), a plasma protein of the innate immune system, is produced by hepatocytes. A critical regulatory region (-42 to -57) on the CRP promoter contains binding site for the IL-6-activated transcription factor C/EBPβ. The IL-1β-activated transcription factor NF-κB binds to a κB site located nearby (-63 to -74). The κB site overlaps an octamer motif (-59 to -66) which is the binding site for the constitutively active transcription factor Oct-1. Oct-1 is known to function both as a transcriptional repressor and as an activator depending upon the promoter context. Also, Oct-1 can regulate gene expression either by binding directly to the promoter or by interacting with other transcription factors bound to the promoter. The aim of this study was to investigate the functions of Oct-1 in regulating CRP expression. In luciferase transactivation assays, overexpressed Oct-1 inhibited (IL-6+IL-1β)-induced CRP expression in Hep3B cells. Deletion of the Oct-1 site from the promoter drastically reduced the cytokine response because the κB site was altered as a consequence of deleting the Oct-1 site. Surprisingly, overexpressed Oct-1 inhibited the residual (IL-6+IL-1β)-induced CRP expression through the promoter lacking the Oct-1 site. Similarly, deletion of the Oct-1 site reduced the induction of CRP expression in response to overexpressed C/EBPβ, and overexpressed Oct-1 inhibited C/EBPβ-induced CRP expression through the promoter lacking the Oct-1 site. We conclude that Oct-1 acts as a transcriptional repressor of CRP expression and it does so by occupying its cognate site on the promoter and also via other transcription factors by an as yet undefined mechanism.

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Figures

Fig. 1
Fig. 1
The CRP promoter and the oligos used in this study. (A) The −300 to +3 region of the CRP gene is shown. The binding sites of various transcription factors on the promoter are boxed. (B) Sequences of the oligos derived from the CRP promoter and used as probes in EMSA. (C) Sequences of the mutagenic oligos used for mutagenesis of the CRP promoter to construct the m-Oct promoter.
Fig. 2
Fig. 2
Oct-1 binds to its site on the CRP promoter and competes with the binding of NF-κB to the overlapping κB site. A representative of three EMSA is shown. Radiolabelled WT oligo (−38 to −78) was used as the probe and nuclear extract from IL-β-treated Hep3B cells was used as the source of NF-βB. Self oligo competitor (unlabelled WT oligo) and antibodies were added to nuclear extract before the addition of the probe. DNA probe-protein complexes were visualized by using a phosphorimager. Arrows point to the complexes formed on the probe. The mobility of the free probe is not shown.
Fig. 3
Fig. 3
Oct-1 does not bind to CRP promoter in the absence of the Oct-1 site. A representative of two EMSA is shown. Radiolabelled WT oligo (lanes 1–5) and m-Oct oligo (8 bp Oct-1 site deleted, lanes 6–10) were used as probes. Nuclear extract from untreated Hep3B cells was used as the source of constitutively active transcription factors. Self oligo competitors (unlabelled WT and m-Oct oligos) and antibodies were added to nuclear extract before the addition of the probe. DNA probe-protein complexes were visualized by using a phosphorimager. Arrows point to the complexes formed on the WT probe. The mobility of the free probe is not shown.
Fig. 4
Fig. 4
Oct-1 inhibits (IL-6+IL-1β)-induced CRP promoter-driven Luc activity. Luc-157 WT and Luc-300 WT CRP promoter constructs were transfected into Hep3B cells along with increasing amounts of the expression vector encoding Oct-1. After 16 h, cells were treated with IL-6 and IL-1β for 24 h. CRP expression was measured as Luc activity. Percent inhibition of Luc activity is plotted on the y-axis. Average ± S.E.M. of three experiments are shown. For some data points, error bars are not visible because the S.E.M. was low.
Fig. 5
Fig. 5
Effects of deleting the Oct-1 site on basal and (IL-6+IL-1β)-induced CRP expression. (A) Hep3B cells were transfected with Luc-157 WT, Luc-157 m-Oct, Luc-300 WT and Luc-300 m-Oct CRP promoter constructs. After 40 h, CRP transcription was measured as Luc activity and plotted on the y-axis. Average ± S.E.M. of four experiments are shown. Unpaired two-tailed Students t-test was used to calculate p values. (B) Hep3B cells were transfected with Luc-157 WT, Luc-157 m-Oct, Luc-300 WT and Luc-300 m-Oct CRP promoter constructs. After 16 h, cells were either treated with IL-6 and IL-1β or left untreated for 24 h. CRP expression was measured as Luc activity. Basal Luc activity was taken as 1 and (IL-6+IL-1β)-induced Luc activity was plotted on the y-axis as fold over basal Luc activity. Average ± S.E.M. of five experiments are shown. Unpaired two-tailed Students t-test was used to calculate p values.
Fig. 6
Fig. 6
Oct-1 inhibits (IL-6+IL-1β)-induced CRP expression even if the Oct-1 site is deleted from the promoter. Hep3B cells were transfected with Luc-157 WT, Luc-157 m-Oct, Luc-300 WT and Luc-300 m-Oct CRP promoter constructs. One set of cells were co-transfected with the expression vector encoding Oct-1 (1 μg). After 16 h, cells were either treated with IL-6 and IL-1β or left untreated for 24 h. CRP expression was measured as Luc activity. Luc activity in the absence of Oct-1 was taken as 100%. Average ± S.E.M. of four experiments are shown. The p values for the difference between (IL-6+IL-1β) and (IL-6+IL-1β+Oct-1) groups were calculated by using paired two-tailed Students t-test (*p < 0.05, **p < 0.005). The p values for the difference between WT and corresponding m-Oct groups were calculated by using unpaired two-tailed Students t-test.
Fig. 7
Fig. 7
Oct-1 inhibits C/EBPβ-induced CRP expression regardless of the Oct-1 site. (A) Hep3B cells were transfected with Luc-157 WT, Luc-157 m-Oct, Luc-300 WT and Luc-300 m-Oct CRP promoter constructs. One set of cells were co-transfected with the expression vector encoding C/EBPβ (50 ng). After 40 h, CRP expression was measured as luciferase activity. Basal Luc activity was taken as 1 and C/EBPβ-induced Luc activity was plotted on the y-axis as fold over basal Luc activity. Average ± S.E.M. of four experiments are shown. Unpaired two-tailed Students t-test was used to calculate p values. (B) Hep3B cells were transfected with Luc-157 WT, Luc-157 m-Oct, Luc-300 WT and Luc-300 m-Oct CRP promoter constructs. One set of cells were co-transfected with the expression vector encoding C/EBPβ (50 ng). Another set of cells were co-transfected with the expression vectors encoding C/EBPβ (50 ng) and Oct-1 (1 μg). After 40 h, CRP expression was measured as Luc activity. Luc activity in the absence of Oct-1 was taken as 100%. Average ± S.E.M. of four experiments are shown. The p values for the difference between C/EBPβ and (C/EBPβ + Oct-1) groups were calculated by using paired two-tailed Students t-test (**p < 0.005). The p values for the difference between WT and corresponding m-Oct groups were calculated by using unpaired two-tailed Students t-test.
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