A Quantitative Telomeric Chromatin Isolation Protocol (Q-TIP) to Characterize Telomere Composition Changes

Telomeres protect linear ends of eukaryotic chromosomes by preventing chromosomal end-to-end fusions and telomere attrition. They consist of repetitive DNA sequences, telomeric repeat containing RNAs (TERRAs) and proteins. Telomeres play a crucial role in chromosome stability, cancer biology, and ageing. With each cell division, telomeres shorten, as the replication machinery of the cell is not capable to fully copy the very terminal part of chromosomes. Therefore, after a certain number of cell divisions, telomeres get critically short and elicit signals in order to stop the proliferation. A cell can circumvent the signal for critically short telomeres by activating mechanisms that elongate telomeres. In most of the cases, telomere elongation is achieved by the expression of the telomerase enzyme. In this case, cells gain the ability to divide indefinitely, which can, in combination with other events, eventually lead to cancer. The telomeric protein composition changes during tumorigenesis, aging and in telomere syndromes are poorly defined. Here, we develop a quantitative telomeric chromatin isolation protocol (q-TIP) for human cells in which chromatin is crosslinked, immunopurified and analyzed by mass spectrometry. Q-TIP involves stable isotope labeling by amino acids in cell culture (SILAC) in order to compare and identify quantitative differences in telomere protein composition of cells from various states. With q-TIP, we specifically enrich telomeric DNA and all shelterin components. We identify and validate known and novel telomere associated polypeptides including all THO subunits, SMCHD1 and LRIF1. We apply q-TIP to long and short telomeres and detect increased density of SMCHD1 and LRIF1 and increased association of the shelterin components TRF1, TIN2, TPP1 and POT1 with long telomeres. Our results validate q-TIP to study telomeric states during normal development and in disease.