A quantitative telomeric chromatin isolation protocol identifies different telomeric states

Abstract

Telomere composition changes during tumourigenesis, aging and in telomere syndromes in a poorly defined manner. Here we develop a quantitative telomeric chromatin isolation protocol (QTIP) for human cells, in which chromatin is cross-linked, immunopurified and analysed by mass spectrometry. QTIP involves stable isotope labelling by amino acids in cell culture (SILAC) to compare and identify quantitative differences in telomere protein composition of cells from various states. With QTIP, we specifically enrich telomeric DNA and all shelterin components. We validate the method characterizing changes at dysfunctional telomeres, and identify and validate known, as well as novel telomere-associated polypeptides including all THO subunits, SMCHD1 and LRIF1. We apply QTIP to long and short telomeres and detect increased density of SMCHD1 and LRIF1 and increased association of the shelterins TRF1, TIN2, TPP1 and POT1 with long telomeres. Our results validate QTIP to study telomeric states during normal development and in disease.

Facioscapulohumeral Muscular Dystrophy As a Model for Epigenetic Regulation and Disease

“…Knocking down SMCHD1 protein in FSHD1 myotubes led to increases in both DUX4-fl mRNA and DUX4 target gene expression, suggesting that the modifying role for SMCHD1 in determining FSHD1 severity was at the 4q35 locus (134). However, SMCHD1 regulates more than D4Z4 arrays; it is required for maintenance of DNA methylation on the inactive X chromosome, regulates autosomal genes with monoallelic expression, and has enhanced binding to long telomeres, where it likely plays a role in establishing silent chromatin (13,54,61,113). It is possible that a reduction in SMCHD1, in addition to mediating increased DUX4-fl expression, contributes to the extreme disease phenotype by affecting expression of many other genes not typically misexpressed in FSHD (e.g., imprinted genes).…”

The telomeric DNA damage response occurs in the absence of chromatin decompaction

“…1D). The long telomeres in Hela L were obtained upon overexpression of the catalytic subunit of telomerase hTERT together with RNA moiety hTR (Cristofari and Lingner 2006;Grolimund et al 2013). The average R g of HeLa S was 68 nm, and the average R g of HeLa L 88 nm (Fig.…”

“…The gel was dried for 2 h at 50°C, denatured with 0.8 M NaOH and 150 mM NaCl, neutralized with 0.5 M Tris-HCl (pH 7.0) and 1.5 M NaCl, prehybridized at 50°C in Church buffer (1% BSA, 1 mM EDTA, 0.5 M Na-phosphate buffer at pH 7.2, 7% SDS), and hybridized overnight at 50°C to a [ 32 P]-labeled telomeric probe as described (Grolimund et al 2013). After hybridization, the gel was rinsed in 4× SSC followed by successive 1-h washes at 50°C in 4× SSC, 4× SSC, 0.5% SDS, 2× SSC, and 0.5% SDS.…”

“…HeLa cell lines harboring 11-kb-long (HeLa S) and 33-kb-long (HeLa L) telomeres were described previously (Grolimund et al 2013). Both cell lines were maintained at 37°C with 5% CO 2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and penicillin/streptomycin.…”

Telomere biology in aging and cancer: early history and perspectives

“…Although many other telomeric accessory factors have since been identified by proteomic analysis (Déjardin and Kingston, 2009;Lee et al, 2011;Grolimund et al, 2013;Bartocci et al, 2014) and the functions of these accessory factors are important in telomere maintenance (Arnoult and Karlseder, 2015), shelterin is still recognized as the only constitutive protein complex with a direct telomerebinding ability (Doksani and de Lange, 2014). Among shelterin components that directly bind to the telomere, TRF2 has a central role in protecting telomeric ends from activating the ATM-dependent DNA damage signaling pathway and from repair through classical NHEJ, while ATR-dependent signaling is repressed by POT1 (Denchi and de Lange, 2007).…”