TRF2 as a mediator of telomere looping between the h<i>TERT</i> and sub-telomeric 5p in cells with long telomeres.

<p>(<b>A</b>) The number of (TTAGGG)₂ repeats was analyzed on a small series of mammalian genomes. The <i>TERT</i> locus flanked by 350kb was examined. The (TTAGGG)₂ repeats are depicted as a single black box. Multiple (TTAGGG)₂ repeats at a locus are depicted by stacked black boxes. Transcription orientation of <i>TERT</i> and location of telomere are shown in figure. (<b>B</b>) 3C analyses show distal genomic interactions between the 5p telomere and h<i>TERT</i>. 3C libraries were generated from BJ cells treated with siRNA against TRF2, followed by ddPCR amplification to determine genomic interactions. <i>t</i> test revealed a significant effect. *<i>p</i> < 0.05, **<i>p</i> < 0.01 (<b>C</b>) ChIP shows enrichment of TRF2 protein on h<i>TERT</i>-ITS. The 1.153 Mbp to 1.154 Mbp region from the end of the chromosome 5p was examined. Chromatin extracts from BJ cell at PD25 and PD50 were prepared and pulled down with an antibody against TRF2. TTAGGG repeats are depicted as a red box. (<b>D</b>) A schematic map of the human chromosome 5 at the 1,153,167 bp to 1,353,167 bp region from the p arm terminus. Arrows indicate location of HindIII restriction enzyme sites. Red boxes indicate location of TTAGGGTTAGGG repeats along the genome. (<b>E</b>) 3C analysis shows distal genomic interactions between 5`end of h<i>TERT</i> locus and each HindIII site. 3C libraries were generated from BJ cells at PD25 and PD70, followed by ddPCR amplification to determine genomic interactions. <i>t</i> test revealed a significant effect. *<i>p</i> < 0.05, **<i>p</i> < 0.01. (<b>F</b>) ChIP shows enrichment of TRF2 protein on the h<i>TERT</i> promoter. Chromatin extracts from BJ cell at PD25 and PD50 were prepared and pulled down with an antibody against TRF2. The h<i>TERT</i> promoter region from -500bp to +200bp was analyzed by real-time-qPCR. <i>t</i> test revealed a significant effect. *<i>p</i> < 0.05, **<i>p</i> < 0.05, n.s. = no significance. (<b>G</b>) Model of genomic folding at h<i>TERT</i> locus based on 3C analyses. Blue boxes indicate the location of genes on chromosome 5p. Red boxes indicate the location of h<i>TERT</i>-ITS. Blue and red dashes indicate chromosome 5p and telomere of chromosome 5p, respectively. (<b>H</b>) BJ cells at PD 17 were transfected in individual experiments with a siRNA against TRF2, CTCF, and LDB1. Three days after transfection, cells were fixed for 3D-FISH analysis. Percentage of adjacent allele (A) pairs versus separated allele (S) pairs was determined by 3D-FISH. (<b>I</b>) Western blotting analysis shows knockdown efficiency of the siRNA against TRF2, CTCF, and LDB1. Histone H3 was used as a loading control. Data associated with this figure can be found in the supplemental data file (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000016#pbio.2000016.s007" target="_blank">S1 Data</a>).