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ATP dependent nucleosome remodelling - Single molecule studies and super-resolution microscopy

Final Report Summary - REMODELLING (ATP dependent nucleosome remodelling - Single molecule studies and super-resolution microscopy)

The DNA in the nucleus of eukaryotic cells is highly compressed allowing for efficient compaction of the genome as well as control of its access. The first step of compaction is the wrapping of DNA arround histone octamers forming so called nucleosomes. Neighbouring nucleosomes are thought to arrange in higher order structures often referred to as 30nm fibers, but the precise architecture as well as the dynamics of such arrangements are currently not well understood.

Most DNA binding proteins are not able to bind DNA protected within a nucleosome. Therefore, two strategies have been developed to access the genetic information. First, the histones and in particular their tales can be post-translationally modified in order to either stabilize or more importantly destabilize the nucleosome. This dynamically imprinted information is oftentimes referred to as the histone code or epigenetic information. Secondly, a class of enzymes has evolved which use the free energy from ATP hydrolysis to dynamically assemble, disassemble or simply move nucleosomes. These enzymes are called nucleosome remodellers and the mechanistic study of these enzymes was the aim of this research project.
We developed single molecule methods to study the effect of ATP dependent nucleosome remodelling at the level of mononucleosomes. To this end we developed strategies for the site specific dye labelling of nucleosomes and immobilised the labelled nucleosomes to the surfaces of micro-fluidic chambers.

In a first step we wanted to determine the conformation of a remodeller bound to a nucleosome. We therefore used single cysteine mutants of the nucleosome remodeller Chd1 from yeast, to site specifically label different domains of the enzyme. For each position we employed several dye labels on the nucleosome to determine the domain arrangement of the remodeller with respect to the nucleosome using smFRET measurements. In order to arrive at a three-dimensional structural model, we developed the nano-positioning system (NPS).

Secondly, we used dual labelled nucleosomes to monitor the dynamics by the remodelling complex due to the hydrolysis of ATP. We performed this analysis for remodellers from three distinct familys, namely Chd1, Acf and Ino80.

We also performed experiments emplyoing time-correlated single photon counting and fluorescence correlation spectroscopy to determine the stability of nucleosomes in salt dependent assays. We compared the behaviour of unmodified nucleosomes to those carrying an acetylation at specific sites and to nucleosomes carrying a different version of histone H2, namely H2AZ.2.2. Both acetylation as well as the histone variant led to a destabilisation of the nucleosome.

Nucleosome remodelling is especially important for determining the transcription start site by creating both a nucleosome free region as well as the well localised +1 nucleosome. Therefore in order to better understand this process we applied our NPS tool to study the architecture of RNA polymerase II (Pol II) transcription initiation complexes.

Lastly, we wanted to study the architecture of higher order nucleosomal arrays using super-resolution optical microscopy. To this end we developed strategies to build nucleosomal arrays using repeats of the 601 localisation sequence and label these arrays site specifically. First super-resolution experiments showed that while the quality of these arrays was well established using biochemical assays, binding of the arrays to surfaces caused a partial dis-assembly of the array.