Figure 3. NR2F1 supresses MTP expression in undifferentiated cells.

(A) Illustration of HNF1, HNF4, and DR1 elements in the 204-bp MTTP promoter. Activators and repressors that could bind to these elements were identified using MatInspector (Genomatix).

(B–C) Caco-2 cells were cross-linked (1% formaldehyde, 15 min, room temperature), sonicated, and incubated with specific antibodies against various DNA-binding proteins. The immunoprecipitated DNA fragments were amplified using primers targeted to MTTP, HNF4A, and APOB promoters and subjected to semiquantitative (B) or quantitative (C) PCR. Binding of different factors to MTTP promoter was normalized with their binding to apoB (C). Pol II, polymerase II; IgG, normal goat serum IgG; Input, 1% of total DNA

(D) Expression of repressors during differentiation was determined by measuring mRNAs using qRT-PCR.

(E, F) Undifferentiated Caco-2 cells were transfected with different siRNAs against NR2F1, NR2F2, and NR2F6. After 48 h, expressions of these repressors (E) and endogenous MTP (F) were determined.

(G) Caco-2 cells were co-transfected with pMTP-204 and pCMV-RL, a transfection control, along with pMT2-NR2F1 (plasmid expressing NR2F1) or pcDNA3.1 (control). Luciferase activities were measured 48 h later.

(H–I) ChIP followed by regular (inset) and qPCR determined NR2F1 binding to MTTP promoter in day 4 and 14 Caco-2 cells (H). Changes in acetylation of MTTP promoter were evaluated using Ac-H3 antibodies (sc-8655) (I).

(J) Immunoblotting evaluated nuclear NR2F1 protein in Caco-2 cell cultures before (D4) and after (D14) differentiation. Tubulin was a control.

(K) Undifferentiated Caco-2 cells stably transfected with pMTP-204 or the same plasmid carrying mutant DR1 element (DR1m) were transfected with different siRNAs.

Luciferase activities were measured using same amounts of protein. Data were normalized with siControl treated cells. Mean ± SEM; #, p < 0.05, ###, p < 0.001, One-way ANOVA; **, p < 0.01, ***, p < 0.001, Student t test.