Nucleosomes form the basic structural organization of all eukaryotic genomes. They serve both as barriers that restrict access to the genome and as a medium that accumulates epigenetic marks. The locations of nucleosomes in the genome are precisely controlled by ATP-dependent nucleosome remodeling complexes. Some remodeling complexes can deposit or eject nucleosomes or nucleosomal components in response to epigenetic cues. Other remodeling complexes reposition entire nucleosomes along DNA, set a constant spacing between nucleosomes or align arrays of nucleosomes with regards to specific genomic features. Collectively, these enzymes enable the structural changes of chromatin that underlie a regulated use of the eukaryotic genome. As such, they intimately affect all fundamental nuclear processes such as DNA transcription, replication, repair and recombination. Not surprisingly, a growing list of malignancies is linked to nucleosome remodeling complexes.
Despite intense research, the functions and mechanisms of remodeling have remained elusive. Using multi-disciplinary approaches that span genetic, genomic, biochemical, biophysical and structural techniques we strive to fill this gap. By decoding the design of various remodelers, we hope to obtain insight into the inner workings of these molecular machines. Furthermore, we aim to understand how they sculpt the chromatin landscape and how they thereby affect the cell’s function. What follows is a brief overview about questions currently being addressed in the lab.
The accessibility problem: How do remodelers set up, maintain and dissolve tightly folded chromatin?
How strongly condensed heterochromatin is established, maintained or dissolved is subject of intense research. ATP-dependent nucleosome remodeling complexes facilitate these structural transformations by catalyzing changes to the composition or positioning of nucleosomes. These nucleosome remodeling factors however face one dilemma: How can they access and then remodel tightly folded chromatin structures? We develop innovative in vivo and in vitro tools and approaches to shed light on this question. The results will contribute towards our understanding of how ATP-dependent chromatin remodelers deal with tightly folded chromatin and how they control the packaging and dynamics of heterochromatin.
What are the structural architectures of nucleosome remodeling complexes?
Despite intense efforts, there is a dearth of structural information about remodeling complexes. Reasons lie in their compositional complexity - they often form megadalton complexes - and structural plasticity. Using innovative hybrid approaches, we aim to clarify the general architecture of these remodeling complexes. More specifically, we use an integrated approach of protein crosslinking, high resolution mass spectrometry (MS) and structural modeling. In this technique, we map the amino acids that are involved in any one crosslink by high resolution MS and subsequent bioinformatic analysis of the MS dataset. The structural information provided by these crosslinks is then used to “stitch” together existing structural models of domains and subunits using additional structural data, coming for example from SAXS and computational docking methods.
How does the structural architecture of remodelers relate to their mechanochemical cycle?
The succession of steps throughout the catalytic cycle is tightly regulated in molecular machines. The regulatory framework that allows the enzyme to proceed through catalysis in such a controlled manner must be exposed to understand the overall mechanism. Recent work suggested the existence of several auto-regulatory elements and conformational changes. Using biophysical techniques such as fluorescence spectroscopy and quantitative structural MS, and mechanistic biochemical approaches, we map such conformational changes.
Cellular biochemistry - A mechanistic dissection of remodelers in the living cell
Remodelers mobilize nucleosomes in vivo and thereby sculpt the characteristic nucleosomal organization of the genome. How they are instructed what, where and when to remodel is largely unknown. We test specific mechanistic hypotheses in vivo in yeast by employing a battery of incisively mutated versions of remodelers that lack certain functionalities. By chromatin immunoprecipitation and next generation sequencing we uncover the associated molecular phenotypes. Besides providing critical tests of concrete mechanistic hypotheses, which were derived from in vitro approaches, these experiments tell us which elements of the remodelers are necessary for sliding nucleosomes in vivo, for sufficient nucleosome affinity, for specific recruitment to a subset of nucleosomes, for proper nucleosome positioning, for formation of nucleosomal arrays and for the characteristic spacing of nucleosomes.