Biochemical observation of the rapid mobility of nuclear HMGB1

Abstract

Formaldehyde-crosslinked and sonicated chromatin fragments were obtained from 15-day chicken embryo erythrocytes and purified on caesium chloride gradients. Polyclonal antibodies raised against chicken HMGB1 were used to immuno-precipitate fragments carrying HMGB1 in two protocols: (1) affinity purified antibodies covalently coupled to agarose beads and (2) diluted antiserum. The DNA of the antibody-bound chromatin was quantified and its sequence content assessed by quantitative real-time PCR to give values of the absolute enrichments generated. Amplicons were monitored within the active β-globin locus, in the adjacent heterochromatin, in the lysozyme locus (containing an active housekeeping gene and the inactive lysozyme gene) and at the promoter of the inactive ovalbumin gene. For all amplicons the Bound/Input ratio was close to unity, implying no preferential location of HMGB1 on the chromatin. This initially unexpected result can now be understood in the light of the exceptional mobility of HMGB1 revealed by FLIP experiments showing that only 1–2 s are needed for HMGB1 to cross the nucleus: crosslinking times of 1 min were used in the present experiments.