Transcription Factor CEBPB Inhibits the Expression of the Human HTR1A by Binding to 5′ Regulatory Region In Vitro

Figures

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  Figure 1. Prediction of transcription factor in the human HTR1A based on JASPAR software and related literature, the effect of the transcription factor CEBPB on the HTR1A expression were evaluated. FEV and LMX1B transcription factors may target fragments adjacent to the CEBPB binding sequence. Numbers represent sequence location (ATG+1), and bases recognized by the transcription factors are underlined.

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  Figure 2. (a) EMSA identified CEBPB bound to the −1219 bp to −1209 bp fragment, (b) A synthesized 5′-end biotin-labeled probe was located at −1235 bp to −1196 bp. The unlabeled specific competitive sequence was consistent with the labeled probe, whereas the non-specific competitive sequences mut2, mut3 and mut4 were mutated according to the fragments identified by CEBPB, FEV and LMX1B, respectively. All lanes contained the probe. In addition to the first lane on the left, nuclear protein extracted from HEK-293 or SK-N-SH cells was loaded. The third to the sixth lanes showed probe mixed with the specific or non-specific competitive sequences. Protein hysteresis suggested that at least three proteins combined with this sequence in both cell lines. The fragment recognized by CEBPB might be located at −1219 bp to −1209 bp. (c) In the supershift assay, the components of the first to fifth lanes were identical to that of the Figure 2a,b. In particular, the sixth lane compared with the second lane additionally contained the CEBPB-specific antibody. A more delayed protein-DNA binding band was observed in HEK-293 cells. This suggests CEBPB was bound to −1219 bp to −1209 bp in the HEK-293 cells. The…

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  Figure 3. (a) Relative fluorescence intensity of pGL3-HTR1A-1235, pGL3-HTR1A-1227, pGL3-HTR1A-1208 and pGL3-HTR1A-1196, (b) In HEK-293 and SK-N-SH cells, the relative fluorescence intensity of the adjacent sequence showed a statistically significant difference. Relative fluorescence intensity of each sample was normalized as the mean ± standard deviation (SD), and the least significant difference (LSD) t-test was used to compare the two samples. * 0.02 < p < 0.05, ** 0.00 < p <= 0.02, *** p <= 0.001.

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  Figure 4. (a) Effect of CEBPB on the relative fluorescence intensity in two cell lines, (b) The rectangle and circle represent the sequence positions identified by CEBPB, LMX1B, respectively. In HEK-293 and SK-N-SH cells, CEBPB significantly inhibited the relative fluorescence intensity. When we compared the relative fluorescence intensity of pGL3-HTR1A+pEGFP-N1-Basic with pGL3-HTR1A+pEGFP-N1-CEBPB, different sequence lengths of the HTR1A showed statistical significance. However, the degree of inhibition was different, indicating that CEBPB might specifically act on −1219 bp to −1209 bp. * 0.02 < p < 0.05, ** 0.00 < p <= 0.02, *** p <= 0.001.

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  Figure 5. Effect of CEBPB on other fragments through the co-transfection of CEBPB with multiple target fragments in the two cell lines, we found that all sequences with the common region −722 bp–−372 bp strongly responded to CEBPB and were significantly inhibited by CEBPB. However, the inhibition degrees of the sequences were different. * 0.02 < p < 0.05, ** 0.00 < p <= 0.02, *** p <= 0.001.

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  Figure 6. (a) Insignificant inhibitory effect of CEBPB on the endogenous 5-HT1A receptors expression, (b) Western blot were performed with three groups including the two cell lines protein, the protein which were extracted from the cell transfected with the basic vector or the over-expressing CEBPB vector. The gray values for each sample were normalized using β-actin (relative protein expression and showed as the mean ± SD. Student’s t-test was used for differential comparison. The results showed that CEBPB did not inhibit endogenous 5-HT1A receptor expression in both cell lines.

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  Figure 7. Inhibition effect of CEBPB on the 5-HT1A receptor mRNA expression. The effect of CEBPB on the expression of endogenous 5-HT1A receptor mRNA was detected by quantitative real-time PCR (qRT-PCR) and used the 2−ΔΔCT method for statistics. The results showed that CEBPB significantly inhibited the mRNA expression in both cell lines. * 0.02 < p < 0.05.