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. 2022 Nov 29;66:101646. doi: 10.1016/j.molmet.2022.101646

Figure 5.

Figure 5

Promoters of key regulatory genes for islet development contain THR and ChREBP binding sites. A. ChREBP and THRB binding sites in human selected genes. Each panel is arranged as follows. The ideogram of the gene with its chromosomal location from the UCSC genome browser is shown. The representation displays exons (dark blue boxes) and introns (dark blue lines with arrowheads pointing to the direction of transcription). The promoter region (TSS ± 2,500 bp) is shown as a transparent red arrow. For the ChREBP gene, the position of the additional exon 1b is marked with a purple box and the intron between exons 1b and 1a is marked with a purple line with arrowheads oriented as for the rest of the gene. Blue and red upward arrowheads identify the center of ChREBP and THRB binding sites. ChREBP binding sites have been scored with the HOMER package (see material and methods) by using the frequency matrix of Supplementary Fig. 9, except for three sites that have been experimentally validated and are marked with asterisks near the respective arrowheads. The two ChREBP binding sites experimentally validated within exon 1b of the ChREBP gene have been tested by our lab. The single ChREBP binding site upstream of the PCK1 promoter has been tested by Jeong et al. [76]. THRB sites have been extracted from the ReMap2022 database. Supplementary Table 3 provides the coordinates of both ChREBP and THRB sites displayed. B. Chromatin Immunoprecipitation in Ins-1 cells grown in RPMI (11 mM glucose) supplemented with regular FCS. ChIP was performed using ChREBP and THR (alpha and beta, [30,67]) antibodies to detect binding on ChREBP promoter area and actin coding area (C). D. Proximity ligation assay for ChREBP and THR in Ins-1 cells was performed as described in materials and methods. Cells were growing low and high glucose, in the presence or absence of T3. Bottom panel-quantification of cells showing positive proximity ligation signal. ∗P < 0.05; ∗∗∗P < 0.01; ∗∗∗∗P < 0.005 using one-way ANOVA. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)