Figure 1.
Figure 1. Survey of conserved noncoding sequences as potential regulatory regions of human endoglin. (A) Comparative sequence analysis reveals multiple conserved noncoding segments (E1-E9) both upstream and downstream of exon1 (asterisk) of endoglin. A SynPlot graphical representation of mammalian endoglin loci aligned using the multi-Lagan alignment program with sequences from Hs (Homo sapiens), Cf (Canis familiaris), Mm (Mus musculus), and Md (Monodelphis domestica). Segments within noncoding regions (green rectangles numbered E1-E9) with more than 50% sequence identity were selected for further study. The coding exons are represented as red rectangles along the sequence line and the untranslated regions and repetitive elements are marked with beige and blue rectangles, respectively. The base-pair numbering along the horizontal axis includes gaps introduced by the alignment program. The endoglin locus is flanked by AK1 (adenylate kinase 1) and FPGS (folylpolyglutamate synthetase) genes that are partially represented in the diagram. (B) Histone acetylation status determined by a chromatin immunoprecipitation assay comparing the endoglin expressing endothelial cell line, MS1 (▪), with nonexpressing fibroblasts, NIH3T3 (□). Histone acetylation status was used as a surrogate marker of chromatin accessibility and the results for each segment are expressed relative to acetylated histones around the promoter of a nonexpressed gene (alpha-fetoprotein). Segment E3 corresponds to the promoter region of endoglin and had the highest level of acetylation in MS1 cells. The enrichment of acetylated histones around segment E1 was second highest and more than 2-fold higher than any of the other segments. There was no significant enrichment in any segment in NIH3T3 cells. (C) Stable transfection assays in MS1 (▪) and NIH3T3 (□) cells to assess enhancer activity of the selected segments. E3, a 484-bp fragment that corresponds to the endoglin promoter, enhances luciferase activity over the promoterless vector, pGL2basic, by approximately 400-fold in MS1 cells and is significantly more active in MS1 cells than in NIH3T3 cells. E1, a 329-bp fragment, approximately 8-kb 5′ of exon 1, was the most active of the other segments with approximately 8-fold enhancement of luciferase activity over the pGL2 (SV) promoter vector in MS1 cells. Error bars indicate standard deviation

Survey of conserved noncoding sequences as potential regulatory regions of human endoglin. (A) Comparative sequence analysis reveals multiple conserved noncoding segments (E1-E9) both upstream and downstream of exon1 (asterisk) of endoglin. A SynPlot graphical representation of mammalian endoglin loci aligned using the multi-Lagan alignment program with sequences from Hs (Homo sapiens), Cf (Canis familiaris), Mm (Mus musculus), and Md (Monodelphis domestica). Segments within noncoding regions (green rectangles numbered E1-E9) with more than 50% sequence identity were selected for further study. The coding exons are represented as red rectangles along the sequence line and the untranslated regions and repetitive elements are marked with beige and blue rectangles, respectively. The base-pair numbering along the horizontal axis includes gaps introduced by the alignment program. The endoglin locus is flanked by AK1 (adenylate kinase 1) and FPGS (folylpolyglutamate synthetase) genes that are partially represented in the diagram. (B) Histone acetylation status determined by a chromatin immunoprecipitation assay comparing the endoglin expressing endothelial cell line, MS1 (▪), with nonexpressing fibroblasts, NIH3T3 (□). Histone acetylation status was used as a surrogate marker of chromatin accessibility and the results for each segment are expressed relative to acetylated histones around the promoter of a nonexpressed gene (alpha-fetoprotein). Segment E3 corresponds to the promoter region of endoglin and had the highest level of acetylation in MS1 cells. The enrichment of acetylated histones around segment E1 was second highest and more than 2-fold higher than any of the other segments. There was no significant enrichment in any segment in NIH3T3 cells. (C) Stable transfection assays in MS1 (▪) and NIH3T3 (□) cells to assess enhancer activity of the selected segments. E3, a 484-bp fragment that corresponds to the endoglin promoter, enhances luciferase activity over the promoterless vector, pGL2basic, by approximately 400-fold in MS1 cells and is significantly more active in MS1 cells than in NIH3T3 cells. E1, a 329-bp fragment, approximately 8-kb 5′ of exon 1, was the most active of the other segments with approximately 8-fold enhancement of luciferase activity over the pGL2 (SV) promoter vector in MS1 cells. Error bars indicate standard deviation

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