Influence of BCL6 on the regulation of rat Oat1 and Oat3 promoters

Oat1 and Oat3 promoter constructs, all varying in their lengths of promoters and number of predicted BCL6 binding sites, were investigated with respect to their responsiveness to BCL6.

OK cells were transiently transfected with different promoter constructs, BCL6 expression vector pcDNA3-BCL6 or empty control vector pcDNA3, and pRL-TK. Cells were lysed 48 h after transfection, followed by quantification of firefly and Renilla luciferase activity. Firefly luciferase activity was normalized to Renilla and data are presented as the fold increase over pGL3-Enhancer.

The localization of predicted BCL6 binding sites of examined Oat1 promoter constructs, are shown in figure 3.12A. The promoter construct Oat1 (-1226/+113) included two, Oat1 (-1666/+113) four, and Oat1 (-2252/+113) also four predicted BCL6 binding sites, respectively. All three Oat1 promoter constructs were activated by BCL6 with a comparable induction, independent of their predicted number of response elements [figure 3.12B;

(Wegner et al., 2012)]. Comparing the localization of predicted binding sites with the fold increase of investigated Oat1 promoter constructs revealed that the first and second BCL6 binding site (-492 to -476 bp and -1143 to -1127 bp) might be responsible for BCL6-dependent activation of Oat1 promoter.

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A: Localization of BCL6 binding sites (indicated as black bars) in different Oat1 promoter constructs; +1:

transcriptional start site. B: OK cells were transiently transfected with indicated promoter constructs of Oat1, and with control vector pcDNA3 (white bars) or expression vector pcDNA3-BCL6 (black bars). Luciferase activity was measured and firefly luciferase was normalized to Renilla luciferase. Data are reported as the fold increase over pGL3-Enhancer and presented as mean ± S.E.M.; n = 4; **: p < 0.01; ***: p < 0.001, significantly different from control (pcDNA3) using the unpaired two-tailed t-test [(Wegner et al., 2012), modified].

Six point mutations in each of the two functional hypothesized BCL6 binding sites, located at -1143 to -1127 bp and -492 to -476 bp, were introduced into the promoter construct Oat1 (-1226/+113) (figure 3.13A). Mutation of indicated nucleotides lead to the abolishment of the in silico predicted binding sites.

The promoter construct Oat1 (-1226/+113) mut 1 contained mutations within the first BCL6 binding site, whereas the Oat1 (-1226/+113) mut 2 construct harbored mutations in the second BCL6 binding sites. Moreover, the Oat1 (-1226/+113) mut 1+2 construct was generated containing mutations in both BCL6 binding sites. Wild type and mutated Oat1 (-1226/+113) promoter constructs were transfected into OK cells, and their responsiveness to BCL6 was investigated (figure 3.13B). All three mutated Oat1 (-1226/+113) promoter constructs were still activated by BCL6. No significant differences between the mutated promoter constructs

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and the wild type construct were observed, because the ratios between pcDNA3-BCL6 and pcDNA3 of every mutated promoter construct with wild type were comparable (figure 3.13C).

Taken together, all Oat1 (-1226/+113) promoter constructs showed equal activations, unaffected by mutated BCL6 binding sites.

Figure 3.13: BCL6-dependent rat Oat1 promoter activity after binding sites mutation.

A: Predicted BCL6 binding sites in the promoter construct Oat1 (-1226/+113) were mutated; +1: transcriptional start site. Bold typed and underlined nucleotides indicate the differences between wild type (WT) and mutated (mut) BCL6 binding site. B: Promoter constructs with or without indicated mutated BCL6 binding sites and pcDNA3 control vector (white bars) or BCL6 expression vector pcDNA3-BCL6 (black bars) were transiently transfected into OK cells. Luciferase activity was measured and firefly luciferase was normalized to Renilla luciferase. Data are reported as the fold increase over pGL3-Enhancer and presented as mean ± S.E.M.; n = 4;

**: p < 0.01; ***: p < 0.001, significantly different from control (pcDNA3) using the unpaired two-tailed t-test.

C: The ratio between the fold increase of pcDNA3-BCL6 and pcDNA3 transfected cells was estimated for each promoter construct. Data are presented as mean ± S.E.M.; n = 4, n.s. not significant compared to wild type Oat1 (-1126/+113), by using one-way ANOVA with Dunnett´s multiple comparison test.

Subsequently, the influence of BCL6 on the activation of Oat3 promoter was examined. Three promoter constructs, Oat3 (-444/+12), Oat3 (-752/+12), and Oat3 (-256/+12) were investigated (Wegner et al., 2012). The localization of predicted BCL6 binding sites is given in figure 3.14A. The shortest Oat3 promoter construct (-444/+12) contained two predicted

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BCL6 binding sites, the longer one (-752/+12) three, and the longest one (-2567/+12) six predicted BCL6 binding sites, respectively. OK cells were transiently transfected with Oat3 promoter constructs, BCL6 expression vector pcDNA3-BCL6 or empty vector pcDNA3, and their luciferase activity was measured (figure 3.14B). All three tested promoter constructs showed activation by BCL6. Oat3 (-444/+12) and Oat3 (-752/+12) were activated in a significant manner, and the promoter construct of Oat3 (-2567/+12) showed a trend of activation (figure 3.14B). Adjustment of predicted BCL6 binding sites localization with the fold increase of investigated Oat3 promoter constructs revealed that the first and second BCL6 binding site (-279 to -263 bp and -439 to -423 bp) might be critical for Oat3 promoter activation by BCL6.

Figure 3.14: Impact of BCL6 on rat Oat3 promoter activity.

A: Localization of predicted BCL6 binding sites (indicated as black bars) in different Oat3 promoter constructs;

+1: transcriptional start site. B: OK cells were transiently transfected with indicated promoter constructs of Oat3, and with control vector pcDNA3 (white bars) or expression vector pcDNA3-BCL6 (black bars). Luciferase activity was measured and firefly luciferase was normalized to Renilla luciferase. Data are reported as the fold increase over pGL3-Enhancer and presented as mean ± S.E.M.; n = 3; n.s.: not significant; *: p < 0.5, significantly different from control (pcDNA3) using the unpaired two-tailed t-test [(Wegner et al., 2012), modified].

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Four to six point mutations within the BCL6 binding sites located at -279 to -263 bp and -439 to -423 bp were introduced and led to the loss of in silico predicted BCL6 binding (figure 3.15A). Three different mutated Oat3 (-444/+12) promoter constructs were generated.

Oat3 (-444/+12) mut 1 contained the first BCL6 binding site being mutated, Oat3 (-444/+12) mut 2 the second, and Oat3 (-444/+12) mut 1+2 included both mutated BCL6 binding sites.

Although predicted binding sites were mutated, all three Oat3 promoter constructs were still activated by BCL6 (figure 3.15B). The fold induction of mutated promoter constructs were not significantly different from that of wild type, calculated by comparing the ratios between pcDNA3-BCL6 and pcDNA3 of every mutated promoter construct with wild type (figure 3.15C).

These data showed that activation of Oat3 (-444/+12) promoter construct by BCL6 was independent of predicted BCL6 binding sites located at -279 to 263 bp and -439 to -423 bp.

Figure 3.15: Rat Oat3 promoter activity after mutation of BCL6 binding sites.

A: Both predicted BCL6 binding sites in the promoter of Oat3 were mutated; +1: transcriptional start site. Bold typed and underlined nucleotides indicate the differences between wild type (WT) and mutated (Mut) BCL6 binding site. B: Promoter constructs with or without indicated mutated BCL6 binding sites and pcDNA3 control vector (white bars) or BCL6 expression vector pcDNA3-BCL6 (black bars) were transiently transfected into OK cells. Luciferase activity was measured and firefly luciferase was normalized to Renilla luciferase. Data are reported as the fold increase over pGL3-Enhancer and presented as mean ± S.E.M.; n = 5; *: p < 0.05;

**: p < 0.01; ***: p < 0.001, significantly different from control (pcDNA3) using the unpaired two-tailed t-test.

C: The ratio between the fold increase of pcDNA3-BCL6, and pcDNA3 transfected cells was estimated for each promoter construct. Data are presented as mean ± S.E.M.; n = 5, n.s. not significant compared to wild type Oat3 (-444/+12), by using one-way ANOVA with Dunnett´s multiple comparison test.

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