Single-molecule studies on the accessibility of chromatin : post-translational modiﬁcations and nucleosome remodeler CHD1
Gebhardt, J. Christof M.
FacultiesFakultät für Naturwissenschaften
InstitutionsInstitut für Biophysik
The organization of large amounts of DNA within eukaryotic nuclei and especially its accessibility for transcription and replication has been a large research interest. Cells use nucleosomes arranged in chromatin ﬁbers to systematically pack large amounts of DNA and still be able to access certain areas when needed. This thesis focuses on two methods used by cells to address the processing of chromatin - Post-translational modiﬁcations and chromatin remodeling. Post-translational modiﬁcations are small chemical groups attached to speciﬁc histone positions and thereby aﬀecting the stability of those. Chromatin remodelers on the other hand are proteins that allow for nucleosomes not only to be moved along DNA, but also for histones to be excluded or exchanged from nucleosomes as well as whole histone octamers to be excluded. We addressed both projects using recombinantly expressed human histones assembled into histone octamers. Nucleosomes are constructed using ﬂuorescent labeled 200bp ’601’ Widom positioning sequence. Our study focuses on using single-molecule Förster Resonance Energy Transfer (smFRET) to image changes in conformation of the nucleosomal constructs. smFRET allows for unique insights in ambient conditions and varying buﬀer conditions. This advantage is used to observe the salt dependent stability of post-translational modiﬁed nucleosomes. smFRET allows also to draw distance information from the measurements and thereby gives the opportunity to build structural models. By single-labeling the chromatin remodeler CHD1 and side-speciﬁcally labeling nucleosomes we could measure a network consisting 47 measurements. The Fast-Nano-Positioning System (FastNPS). gives the unique opportunity to use previously existing partial structural data to build full models. Investigating the interaction of CHD1 with nucleosomes in presence of diﬀerent nucleotides gave us the opportunity to gain new insights into the remodeling mechanisms. Comparison of our ﬁndings with other known remodelers allows to put these results in context and ﬁnd similarities between remodeler families. It also gives inspirations for further experiments that would help to better understand chromatin remodeling.
Subject HeadingsMolekulare Biophysik [GND]
Konfokale Mikroskopie [GND]
Fluorescence resonance energy transfer [MeSH]
Fluorescent dyes [MeSH]
Microscopy, Confocal [MeSH]