Advancing Epigenetics Towards Systems Biology

Proteomics of isolated chromatin segments (PICh) on human telomeres (Prot 52)

Paulina Marzec, Jérôme Déjardin

Introduction

Proteomics of isolated chromatin segments (PICh) is a powerful method of repetitive chromatin isolation that allows identifying proteins associated to specific genomic loci. Purification is based on nucleic acid probe hybridization to the DNA moieties present in formaldehyde-crosslinked chromatin. Hybridized chromatin is captured on magnetic beads, eluted and bound proteins can be identified by mass spectrometry. In order to increase the stability of the probe-chromatin interaction, Locked Nucleid Acids (LNA) containing oligos are used. LNA probes have higher melting temperature than DNA probes of the same sequence. This facilitates strong interactions with DNA and stabilizes probe invasion. Between the LNA probe and the immobilization tag there is a long spacer that reduces steric hindrance effects. Desthiobiotin is used as the immobilization tag. Desthiobiotin has weaker affinity to streptavidin (Kd~10-12 M) which allows a competitive gentle elution from streptavidin beads with regular biotin (Kd~10-14 M).

The following protocol is optimized for 109 cells per pull down (which is approximately the number of adherent cells growing on 50 cell culture dishes of 150 mm diameter or 25 dishes of mouse stem cells cultured on gelatin).

Each experiment should consist of at least two pulldowns: one using a scrambled and the other a telomere specific probe. In both conditions, chromatin should be processed in the same way and split just before adding LNA probes.

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Paulina Marzec, Jérôme Déjardin

Institute of Human Genetics. CNRS UPR1142. 141 rue de la Cardonille. 34000 MONTPELLIER, FRANCE.

Corresponding author: Paulina Marzec
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Paulina Marzec, Jérôme Déjardin