Optical tweezers force spectroscopy of chromatin structure

Malte Bussiek (BPE group)

Background

The basic structural unit of chromatin is the nucleosome, consisting of 147 bp of DNA wrapped around an octamer of 4 different core histones (H2A, H2B, H3 and H4) in 1.65 left-handed windings. Nucleosomes are repeated along the DNA at an interval of 180 to 220 bp, yielding an array of particles connected by short pieces of linker DNA. This array is, under physiological conditions, condensed to a fiber with a diameter of 30 nm and a mass density of about 6 nucleosomes per 11 nm [20]. Although the exact structure of the 30 nm-fiber is still a matter of research [21], the determinants of chromatin condensation have been established [22, 23]: These are (i) internucleosome interactions mainly mediated by the N-terminal histone tails, (ii) the geometry of the DNA entering and exiting the nucleosome and (iii) the spacing of the nucleosomes along DNA. In the cell, chromatin is subject to extensive structural modifications which are associated with localized functions and which are mediated by a number of factors. The nucleosome is no longer considered as a static structure, since many works in recent years have demonstrated its extensive conformational flexibility [24].

Objective

In this project we plan to investigate the elastic response of reconstituted nucleosome arrays towards external stretching forces using optical tweezers (OT). The force versus extension curves reveals that nucleosomal disruption lengths do not always correspond to the full expected length of 147 bp. The current work aims to gain insight into this discrepancy by stretching defined nucleosomal under varying conditions. In a next step the influence of post-translational modifications on the level of the histone proteins or the additional of chromatin remodeling proteins will be studied. Comparative AFM measurements are carried out to determine the conformation of the nucleosome arrays, that complement the interpretation.