Genome-wide analysis of <i>in vivo</i> TRF1 binding to chromatin restricts its location exclusively to telomeric repeats

<div><p>Telomeres are nucleoprotein structures at the ends of eukaryotic chromosomes that protect them from degradation, end-to-end fusions, and fragility. In mammals, telomeres are composed of TTAGGG tandem repeats bound by a protein complex called shelterin, which has fundamental roles in the regulation of telomere protection and length. The telomeric repeat binding factor 1 (TERF1 or TRF1) is one of the components of shelterin and has been shown to be essential for telomere protection. Telomeric repeats can also be found throughout the genome, as Internal or Interstitial Telomeric Sequences (ITSs). Some of the components of shelterin have been described to bind to ITSs as well as other extra-telomeric regions, which in the case of RAP1 exert a key role in transcriptional regulation. Here, we set to address whether TRF1 can be found at extra-telomeric sites both under normal conditions and upon induction of telomere shortening. In particular, we performed a ChIP-sequencing technique to map TRF1 binding sites in MEFs wild-type and deficient for the telomerase RNA component (<i>Terc^−/−</i>), with increasingly short telomeres. Our findings indicate that TRF1 is exclusively located at telomeres both under normal conditions, as well as under extreme telomere shortening. These results indicate that in mice not all members of shelterin have extra-telomeric roles as it was described for RAP1.</p></div

Summary of mutations found in the <i>ARID1A</i> resequencing study.

<p>Non-synonymous mutations found at a frequency ≥10% that were confirmed by Sanger sequencing are shown here.</p>*<p>denotes a truncating mutation.</p

Loss of ARID1A expression is associated with more aggressive UBC and with patient outcome.

<p>ARID1A expression was assessed by IHC on tissue microarrays. Patients (n = 84) were followed-up as indicated in Methods and classified as having “recurred”, “progressed”, or being free of disease. Patients with high ARID1A-expresssing tumors display a lower risk of recurrence and a higher risk of progression indicating a more aggressive clinical course.</p

Characteristics of the patients and tumors included in series 2 (tissue microarray).

<p>Characteristics of the patients and tumors included in series 2 (tissue microarray).</p

Loss of ARID1A expression is associated with more aggressive UBC.

<p>UBC cases were classified in three categories: low grade NMI (TaG1 and TaG2 tumors), high grade NMI (TaG3 and T1G3 tumors), and MI (≥T2 tumors). <i>Panel A</i>. ARID1A immunohistochemical score is significantly lower in more aggressive, advanced tumors. FGFR3 immunohistochemical score, which is directly associated with <i>FGFR3</i> mutations, is also significantly lower in more aggressive tumors. By contrast, p53 score is higher in more aggressive tumors. <i>Panel B</i>. Differential expression of <i>ARID1A, FGFR3</i> and <i>TP53</i> at the mRNA level is observed in two different, independent UBC microarray series: the mRNA levels of all 3 genes are significantly lower in MIBC. *denotes an FDR adjusted <i>P</i>-value <0.5.</p

Characteristics of the patients from whom fresh tumor was used for <i>ARID1A</i> sequence analysis.

<p>Characteristics of the patients from whom fresh tumor was used for <i>ARID1A</i> sequence analysis.</p

Relationship between ARID1A and cell differentiation markers, as detected using immunohistochemistry in tumor tissue microarrays.

<p>UBC cases were classified in three categories: LG-NMIBC (TaG1 and TaG2 tumors), HG-NMIBC (TaG3 and T1G3 tumors), and MI (≥T2 tumors). Non-hierarchical clustering of IHC scores for ARID1A, FGFR3, KRT5/6, KRT14, and KRT20 was performed. IHC scores are shown in a green-red color code. Color bars below the dendogram include information about tumor stage and grade (tones of blue) and <i>FGFR3</i> mutational status (grey/black) when known. White squares indicate that information for that parameter is not available.</p

<i>ARID1A</i> mutations and expression in UBC.

<p><i>Panel A</i>. A G>C transversion identified through Solexa resequencing, confirmed by Sanger sequencing of independent PCR products, leading to a predicted Q2210H substitution in VMCUB-3 cells. <i>Panel B</i>. Western blotting analysis in a panel of UBC cell lines identifies a subset with undetectable expression, including VMCUB-3. mRNA expression was analyzed by RT-qPCR; results are shown as values normalized with respect to the housekeeping gene <i>HPRT</i>. <i>Panel C</i>. A C>T mutation in codon 403, leading to a premature stop codon, was identified in a primary T1G3 tumor. The mutation was absent from matched normal leukocyte DNA. Lack of protein expression in the corresponding tumor tissue was confirmed using immunohistochemistry. The red arrowhead points to a tumor cell lacking ARID1A staining, whereas the black arrowhead indicates a positive stromal cell. For comparison, a TaG1 tumor with wild type <i>ARID1A</i> sequence is shown.</p

Effects of <i>ARID1A</i> knockdown in UBC cell lines.

<p><i>Panel A</i>. <i>ARID1A</i> was knocked-down using three different shRNAs in the RT112 and VMCUB-3 cells. The knock-down was efficient at both the protein and mRNA levels. The bars represent the relative quantification of ARID1A mRNA levels taking non-targeting shRNA interfered cells as controls. <i>Panel B</i>. The quantification colony formation is shown, with error intervals of results from triplicate experiments denoting SEM. In RT112 cells, ARID1A knockdown was associated with reduced colony formation. By contrast, no major effects were observed in VMCUB-3 cells harboring an <i>ARID1A</i> mutation. Representative morphological changes in cells interfered with control shNT (scrambled shRNA) and with one of the shRNAs targeting <i>ARID1A</i> are shown.</p

Clinical features, evolution and biological characteristics of the ten CLL patients studied.

<p>Summary of the prognosis factors considered in the clinic and genetic lesions that the ten patients harbor. ND = not done, del = deletion.</p