The Nucleosome Position-Encoding WW/SS Sequence Pattern is Depleted in Mammalian Genes Relative to Other Eukaryotes

Nucleosomal DNA sequences generally follow a well-known pattern with ∼10-bp periodic WW (where W is A or T) dinucleotides that oscillate in phase with each other and out of phase with SS (where S is G or C) dinucleotides. However, nucleosomes with other DNA patterns have not been systematically analyzed. Here, we focus on an opposite pattern, namely anti-WW/SS pattern, in which WW dinucleotides preferentially occur at DNA sites that bend into major grooves and SS (where S is G or C) dinucleotides are often found at sites that bend into minor grooves. Nucleosomes with the anti-WW/SS pattern are widespread and exhibit a species- and context-specific distribution in eukaryotic genomes. Unlike non-mammals (yeast, nematode and fly), there is a positive correlation between the enrichment of anti-WW/SS nucleosomes and RNA Pol II transcriptional levels in mammals (mouse and human). Interestingly, such enrichment is not due to underlying DNA sequence. In addition, chromatin remodeling complexes have an impact on the abundance but not on the distribution of anti-WW/SS nucleosomes in yeast. Our data reveal distinct roles of cis- and trans-acting factors in the rotational positioning of nucleosomes between non-mammals and mammals. Implications of the anti-WW/SS sequence pattern for RNA Pol II transcription are discussed

Predicting Human Nucleosome Occupancy from Primary Sequence

Nucleosomes are the fundamental repeating unit of chromatin and comprise the structural building blocks of the living eukaryotic genome. Micrococcal nuclease (MNase) has long been used to delineate nucleosomal organization. Microarray-based nucleosome mapping experiments in yeast chromatin have revealed regularly-spaced translational phasing of nucleosomes. These data have been used to train computational models of sequence-directed nuclesosome positioning, which have identified ubiquitous strong intrinsic nucleosome positioning signals. Here, we successfully apply this approach to nucleosome positioning experiments from human chromatin. The predictions made by the human-trained and yeast-trained models are strongly correlated, suggesting a shared mechanism for sequence-based determination of nucleosome occupancy. In addition, we observed striking complementarity between classifiers trained on experimental data from weakly versus heavily digested MNase samples. In the former case, the resulting model accurately identifies nucleosome-forming sequences; in the latter, the classifier excels at identifying nucleosome-free regions. Using this model we are able to identify several characteristics of nucleosome-forming and nucleosome-disfavoring sequences. First, by combining results from each classifier applied de novo across the human ENCODE regions, the classifier reveals distinct sequence composition and periodicity features of nucleosome-forming and nucleosome-disfavoring sequences. Short runs of dinucleotide repeat appear as a hallmark of nucleosome-disfavoring sequences, while nucleosome-forming sequences contain short periodic runs of GC base pairs. Second, we show that nucleosome phasing is most frequently predicted flanking nucleosome-free regions. The results suggest that the major mechanism of nucleosome positioning in vivo is boundary-event-driven and affirm the classical statistical positioning theory of nucleosome organization

Nucleosomes in gene regulation: theoretical approaches

This work reviews current theoretical approaches of biophysics and bioinformatics for the description of nucleosome arrangements in chromatin and transcription factor binding to nucleosomal organized DNA. The role of nucleosomes in gene regulation is discussed from molecular-mechanistic and biological point of view. In addition to classical problems of this field, actual questions of epigenetic regulation are discussed. The authors selected for discussion what seem to be the most interesting concepts and hypotheses. Mathematical approaches are described in a simplified language to attract attention to the most important directions of this field

Analysis of nucleosomal DNA patterns around transcription factor binding sites

Nucleosomes are ~147bp DNA wrapped around the histone octamer which are involved in regulating gene transcription. They have the ability to disassemble depending on the process they are involved in and the nucleosome positioning controls the output of the genome. Therefore, it is important to understand the nucleosome positioning and how its positioning affects the binding of transcription factors (TFs) and gene expression thereby regulating the transcription outcome of the genome. Many studies suggest that TFs and nucleosomes compete with each other for genome accessibility. However, the majority of the studies focus on the nucleosome organization rather than underlying DNA sequences and its patterns which might actually be playing an important role in understanding the regulatory role of nucleosomes in gene transcription. This research study focuses on identifying the specific sequence patterns at or around TF binding sites. The study specifically focuses on identifying the fraction of nucleosomes with WW/SS and anti - WW/SS sequence patterns as they might be responsible for maintaining the stability of the nucleosomes. This will provide a new molecular mechanism underlying NDR formation around TF binding sites and pioneer TF-induced chromatin opening

Absolute nucleosome occupancy and reconstitution of nucleosome positioning mechanisms for Saccharomyces cerevisiae

Nucleosomes, the basic unit of chromatin, package the genome in a repetitive and non-random way. Genome-wide nucleosome maps revealed that nucleosomes form a stereotypical pattern at actively transcribed genes. This pattern is characterized by a nucleosome depleted region (NDR) upstream of the transcription start site followed by an array of regularly spaced nucleosomes. This stereotypical NDR-array pattern is pivotal for proper transcription initiation and therefore a major regulatory element for gene expression. Additionally, nucleosome positioning plays an important role in DNA replication and DNA repair. The NDR-array pattern is to some extent encoded in the DNA sequence, which is mainly read out by a combination of non-histone DNA binding proteins (general regulatory factors, GRFs) and ATP dependent chromatin remodeling enzymes (remodelers). Deletion experiments in Saccharomyces cerevisiae revealed that remodelers usually have redundant functions, whereas GRFs are essential for viability, which both complicates the detailed mechanistical dissection of these proteins in vivo. Therefore, the Korber group established a genome-wide remodeling assay with in vitro-assembled chromatin, which reconstitutes the individual contribution of each remodeling enzyme/GRF to the stereotypical NDR-array pattern. This approach revealed that some remodelers, like INO80, position in vivo-like nucleosomes on their own, whereas other remodelers, like ISW1a and ISW2, need an alignment point provided by GRFs. However, it remained unclear how remodelers generate nucleosome regularity in arrays and how arrays are aligned at GRFs. In particular, it was unclear to which extent remodelers generate the array-defining distances between nucleosomes, and between nucleosomes and GRFs by themselves, or if rather the nucleosome density and the underlying DNA sequence dominate these distances. Here, we showed that not just ISWI-type remodelers, but also INO80 as well as Chd1 align nucleosomes at GRFs and that all remodelers with spacing activity contain a ruler element as they generate remodeler-specific regular spacing in arrays and array alignment (phasing). This ruler most likely resides in the DNA-binding domain/subunit of each remodeler and can in some cases respond to nucleosome density. The extent of the nucleosomal arrays depends on the nucleosome density and mildly on the underlying DNA-sequence. Based on structural information of the INO80 remodeling complex, we generated INO80 mutants, which generated altered spacing and phasing distances in our reconstitution assay. This tuned for the first-time array generation by a remodeler and revealed the location of the ruler element in INO80. Not only the information where a nucleosome is positioned, but also how often this position is occupied, is fundamental for all nucleosome-related processes. However, all available genome-wide nucleosome mapping techniques are not able to provide nucleosome occupancy in absolute terms but rather measure nucleosome densities relative to the maximal nucleosome peak height in a single sample. To overcome this limitation, we established two orthogonal approaches to map absolute nucleosome occupancy. The first genome-wide high-resolution occupancy map of the Saccharomyces cerevisiae genome reveals that nucleosomal arrays exhibit uniformly high nucleosome occupancy. This contrasts other nucleosome maps, which often suggested drastic changes in nucleosome occupancy within single genes. Furthermore, we did not find any correlation between high transcription rates and low nucleosome occupancy as indicated by other studies, but we revealed a correlation between low nucleosome occupancy and high RSC occupancy, a nucleosome-evicting remodeling enzyme.Nukleosomen sind die grundlegenden Strukturelemente in Chromatin und verpacken das Genom auf eine repetitive und nicht zufällige Art und Weise. Genomweite Nukleosom-Karten zeigten, dass Nukleosomen eine stereotypische Verteilung an aktiv transkribierten Genen aufweisen. Dieses Muster ist charakterisiert durch eine nukleosomendepletierte Region (NDR) vor der Transkriptionsstartstelle gefolgt von einer Abfolge äquidistanter Nukleosomen. Dieses stereotypische NDR-Reihenmuster ist zentral für richtige Transkriptionsinitation und deshalb ein wichtiges regulatorisches Element für Genexpression. Zusätzlich spielt Nukleosomenpositionierung eine wichtige Rolle in DNA-Replikation und DNA-Reparatur. Das NDR-Reihen-muster ist teilweise in der DNA-Sequenz kodiert, welche v.a. von einer Kombination aus nicht-Histon DNA-Bindeproteinen (generelle Regulationsfaktoren, GRFs) und ATP-abhängigen Chromatinumbauenzymen (‚Remodeler‘) gelesen wird. Deletions-experimente in Saccharomyces cerevisiae zeigten, dass Remodeler normalerweise redundant arbeiten, wohingegen GRFs essenziell für die Überlebensfähigkeit der Zelle sind. Beides verkompliziert die detaillierte mechanistische Analyse dieser Proteine in vivo. Deshalb etablierte das Korber-Labor einen genomweiten Remodeler-Assay mit In vitro-Chromatin. Dieser Assay rekonstituiert die individuellen Beiträge von jedem Remodeler oder GRF zu dem stereotypischen NDR-Reihenmuster. Dieser Ansatz zeigte, dass einige Remodeler, wie ISW1a und ISW2 aus Hefe, einen Bezugspunkt brauchen in Form von GRFs. Trotzdem blieb es unklar, wie genau Remodeler Regularität in Nukleosomenabfolgen erzeugen und wie diese Nukleosomenabfolgen an den GRFs ausgerichtet werden. Speziell war unklar, bis zu welchem Grad Remodeler die reihendefinierenden Abstände zwischen Nukleosomen und zwischen GRFs und Nukleosomen selbst einstellen oder ob eher die Nukleosomendichte und die zugrundeliegende DNA-Sequenz diese Abstände dominieren. Hier zeigen wir, dass nicht nur Remodeler vom ISWI-Typ, sondern auch INO80 und Chd1 aus Hefe Nukleosomen an GRFs ausrichten können und dass alle Remodeler, die reguläre Nukleosomenabstände erzeugen, ein strukturelles Element ähnlich einem Lineal besitzen, da sie Remodeler-typische reguläre Abstände innerhalb Nukleosomreihen oder zwischen Nukleosomreihen und Ausrichtungspunkt erzeugen. Dieses Lineal-Element liegt wahrscheinlich in der DNA-binde-Domäne/Untereinheit des einzelnen Remodelers und in manchen Fällen reagiert dieses Lineal auf die Nuklesomendichte. Das Ausmaß der Nukleosomreihung hängt hauptsächlich von der Nukleosomendichte ab und teilweise von der DNA-Sequenz. Basierend auf strukturellen Daten des INO80-Komplexes konnten wir INO80-Mutanten erzeugen, welche veränderte Abstände in unserem Rekonstitutionssystem einstellten. So gelang zum ersten Mal die Manipulation der Bildung von Nukleosomreihen durch einen Remodeler. Zudem identifizierte es die Lage des Lineal-Elementes in INO80. Nicht nur die Information, wo ein Nukleosom positioniert ist, sondern auch wie oft es diese Position besetzt, ist fundamental für alle nukleosomen-abhängigen Prozesse. Nichtsdestotrotz sind alle verfügbaren genomweiten Methoden zur Kartierung von Nukleosomen nicht in der Lage, den Nukleosomenbesetzungsgrad vollständig zu messen. Stattdessen messen diese Methoden eher eine Nukleosomendichte, die relativ zur maximalen Nukleosomenbesetzung ein jeder Probe berechnet wird. Um dieses Problem zu überwinden, haben wir zwei orthogonale Methoden entwickelt, die absolute Nukleosomenbesetzung messen. Die daraus resultierende erste genomweite, hochauflösende Nukleosomen-Besetzungskarte für das Saccharomyces cerevisiae Genom zeigt Nukleosomreihen mit gleichmäßig hoher Nukleosomenbesetzung. Das steht im Gegensatz zu anderen Nukleosomkarten, die oft einen zweifachen Unterschied zwischen Nukleosomenbesetzung in demselben Gen suggerieren. Des Weiteren konnten wir keine Korrelation zwischen hohen Transkriptionsraten und niedriger Nukleosomenbesetzung feststellen, obwohl andere Studien darauf hinweisen. Jedoch konnten wir eine Korrelation zwischen niedriger Nukleosomenbesetzung und dem vermehrten Vorkommen des Remodelers RSC ableiten, welcher Nukleosomen entfernt

Centromere Identity and the Nature of the Cenp-A-Containing Nucleosome

The centromere is an essential chromosomal locus that serves as the site of kinetochore formation, ensuring accurate chromosome segregation during mitosis and meiosis. While most centromeres form on repetitive DNA, the underlying DNA sequence is neither necessary nor sufficient to support centromere function, suggesting that this locus is epigenetically defined. The histone H3 variant centromere protein A (CENP-A) replaces H3 in nucleosomes at the centromere and is the best candidate to provide this epigenetic mark. This thesis aims to understand the features of the CENP-A nucleosome that impart its ability to mark and stabilize functional centromeres. In the first part of the thesis, our work provides an in-depth study on the structure of CENP-A-containing nucleosomes and shows that CENP-A nucleosomes adopt an unconventional conformation in solution that results in both an altered histone core and wrap of DNA. Upon binding of the nonhistone protein CENP-C, the histone core and path of DNA wrapping it revert back to a canonical shape, but DNA termini flexibility becomes enhanced. These structural transitions imparted by CENP-C have important functional consequences, as endogenous centromeres lacking CENP-C lose CENP-A and have increased mitotic defects. In the second part of the thesis, I discuss the role that DNA sequence plays in centromere function. While seemingly indispensable, both the quality of DNA sequence and the quantity of DNA repeats are important for maintaining centromeres, and I outline our work that aims to understand both of these phenomena. Taken together, these works greatly increase our understanding of the intrinsic and extrinsic components of the CENP-A nucleosome and its role in centromere identity

Functional characterization of the Saccharomyces cerevisiae chromatin remodeler INO80

Knowing the explicit locations of nucleosomes in a genome is a pre-requisite for understanding the regulation of genes. Predominantly at regulatory active promoter sites, regular spaced arrays phased at reference points shape the chromatin landscape. In eukaryotic cells ATP-dependent chromatin remodeler align nucleosomes at reference points and are pivotal in the formation of the stereotyped promoter pattern. Chromatin remodeler of the ISWI, CHD, SWI/SNF and INO80 family convert energy derived from ATP hydrolysis to operate on their nucleosomal substrates to accomplish nucleosome spacing, eviction and editing reactions. Recent structural elucidations provided mechanistic insights into how chromatin remodelers engage their nucleosomal substrates (Eustermann et al., 2018, Aramayo et al., 2018, Willhoft et al., 2018, Ayala et al., 2018, Farnung et al., 2017, Wagner et al., 2020, Yan et al., 2019, He et al., 2020, Han et al., 2020) and brought about a unifying DNA wave mechanism underpinning ATP-dependent DNA translocation by chromatin remodeling complexes (Yan and Chen, 2020). Understanding how phased arrays of equally spaced nucleosomes are generated by chromatin remodelers represents an ultimate long-term goal in chromatin biology. What remains unclear is the underlying mechanism that directs nucleosome positioning by chromatin remodelers in absolute terms. How do ATP-dependent chromatin remodelers generate phased arrays of regularly spaced nucleosomes? How are the distances between nucleosomes and phasing sites and between adjacent nucleosomes set? Is DNA shape read-out part of nucleosome positioning driven by chromatin remodelers? Do remodelers have intrinsic ruler-like elements that set spacing and phasing distances? The aim of this thesis was to delineate whether, and if so, what type of genomic information is read by a remodeler in the stereotypic placement of nucleosomes at physiological sites, and how the remodeler activities fit into the unifying framework of ATP-dependent DNA translocation mechanism of chromatin remodelers. To gain an insight into nucleosome positioning driven by Saccharomyces cerevisiae (S.c.) ATP-dependent chromatin remodelers, a combination of a minimalistic genome-wide in vitro reconstitution system, biochemical analysis, high-resolution structures and structure-guided mutagenesis of the S.c. INO80 model system was applied. Findings of this work would have an impact on the mechanistic understanding of nucleosome positioning driven by ATP dependent chromatin remodelers based on the ruler concept that has been described earlier for the ISW1a chromatin remodeler (Yamada et al., 2011). The ISW1a, Chd1 and ISW2 remodelers demonstrated “clamping” activity and used ruler elements to set 1 Abstract distances with a defined linker length (21-26 bp at all densities, 12-13bp at all densities, 54-58 bp at low/medium densities, respectively). Mutagenesis of the INO80 model system identified and tuned the INO80 ruler element, which is comprised of the Ino80_HSA domain of the ARP module, the NHP10 module and Ino80 N-terminal residues. Regularly spaced symmetrical arrays were generated at the Reb1 reference point sites as well as at BamHI-introduced dsDNA break sites. Nucleosome positioning on the genomic sequences of S. c., Schizosaccharomyces pombe (S.p.) as well as Escherichia coli (E.coli) showed no significant differences. Mutagenesis of residues located within the Ino80_HSA domain established a causal link between nucleosome positioning by INO80 and DNA shape read-out by the INO80_HSA domain. The spacing and phasing distances generated by ATP-dependent chromatin remodelers point towards a remodeler-intrinsic ruler activity that is independent of underlying DNA sequences and can be sensitive to nucleosome density. This study measured linker lengths set by remodeler-intrinsic ruler-like functionalities in absolute terms, which will be instrumental to dissect contributions from individual remodelers in nucleosome positioning in vivo. This provides the starting point to understand how remodeler-driven nucleosome dynamics direct stable steady-state nucleosome positions relative to DNA bound factors, DNA ends and DNA sequence elements. Sequence-dependent DNA shape features have been mainly associated with binding of transcription factors as well as general regulatory factors and more static DNA binding events. This study augments the general description of nucleosome positioning sequences for chromatin remodelers by establishing nucleosome positioning motifs based on DNA shape analysis. This study provides an intriguing framework to implement DNA shape read-out in the tracking mechanism of DNA-translocating machineries

Transcription factor DNA binding- and nucleosome formation energies determined by high performance fluorescence anisotropy

Protein DNA binding is the core of transcriptional regulation, the process which controls the flow of information stored in an organism’s genome to react to its environment and to maintain its functionality. The initial event of gene expression is the binding of a transcription factor (TF) to its target site. These binding events are integrated over several binding sites and TFs by which a fine tuned regulation can be achieved. The number, combination and strengths of the different binding sites encode the desired gene expression level and the plasticity of the regulated gene. Efforts have been devoted with the goal of identifying the specific DNA sequences bound by different TFs. For more than two decades, it was thought that mutations at each position in this sequence independently contribute to the binding probability of a TF. This binding preference has therefore been described through position weight matrices (PWMs). PWMs describe the binding preference of a TF towards its target sites by assuming that each nucleotide position contributes independently to the total specificity (linearity assumption). However, current research has shown that this simplified view lacks a significant part of the information needed to precisely describe the binding preference of a TF. It was also shown that the most information missing in the PWM is encoded in dinucleotide mutations. Two questions are important in this regard: (1) Which information about TF-DNA interaction are we missing and are currently employed methods able to provide them? and (2) What is a comprehensive description of non-linearity that is based on biophysical properties rather then on abstract probabilities? One important aspect is the three dimensional configuration of the DNA strand (DNA shape) which is known to affect TF binding to a varying degree. Through recent work by the group of Remo Rohs it is possible to predict shape parameters (features) from a DNA sequence and investigate to which degree they influence binding for any given set of measurements. The first aim of this thesis is therefore to determine non-linearity in TF-DNA interaction and investigate the influence of DNA shape on them. Protein-DNA interactions were studied with a variety of methods using structural biology (NMR, crystallography, cryo EM) or quantitative Methods (EMSA, DNA binding arrays, ChIPSeq, B1H, SELEX, MITOMI, Simile-Seq). Most of these quantitative methods to measure TF-DNA interactions, however, are not very sensitive to weak binders due to stringent washing steps or cutoffs they employ. Especially sequences with two positions differing from the consensus can be very weakly bound - therefore a sensitive method is needed to investigate non-linearity. The method called High Performance Fluorescence Anisotropy (HiP-FA, recently developed in our lab) provides the necessary sensitivity. Using HiP-FA, I determined the affinities of 13 TFs from the Drosophila melanogaster segmentation network and found most of them to contain a significant non-linearity in their specificity. The binding energies of the TFs correlated significantly with certain DNA shape features suggesting shape readout by the TFs. These results could be confirmed in existing structural biology data. Besides the influence of information directly encoded in the DNA sequence, the binding of a TF in the genome is most influenced by the DNA accessibility. This property is a result of the genomic DNA being wrapped around histone octamers forming nucleosomes. Since the underlying sequence can also influence the binding of the histone complex to the DNA, a natural question to ask is which features of the DNA sequence are the major determinant of histone-DNA interaction. Attempts to address this question used existing methods which were either MNase based and are therefore prone to the enzymes intrinsic cutting bias or based on dialysis and/or EMSA readout and have in consequence a low throughput and can only be automated to a small degree. This leads to a limited set of measurements which are usually only based on a single measurement point instead of a complete titration curve. The second aim of my thesis is therefore to develop an in vitro assay to determine free energies of nucleosome formation which improves on the limitations of existing methods. Using the sensitive FA-microscopy setup, I developed an automated assay to determine the free energy of nucleosome formation in a competitive titration. In contrast to existing methods, the throughput of the assays allows for full competitor titration curves. By measuring the free binding energies of 42 sequences, I showed that GC-content is the factor most contributing to the free energy. The relationship between these quantities is non-monotonous with an optimal GC-content of 49 percent. The results provided in this thesis give insight into the nature of non-linearity in TF-DNA interactions and highlight the DNA shape readout therein. Methodical advancements developed in this work can be used as a foundation to investigate other kinds of molecular interactions making use of the high sensitivity of FA-based microscopy

Transcription factor DNA binding- and nucleosome formation energies determined by high performance fluorescence anisotropy

Protein DNA binding is the core of transcriptional regulation, the process which controls the flow of information stored in an organism’s genome to react to its environment and to maintain its functionality. The initial event of gene expression is the binding of a transcription factor (TF) to its target site. These binding events are integrated over several binding sites and TFs by which a fine tuned regulation can be achieved. The number, combination and strengths of the different binding sites encode the desired gene expression level and the plasticity of the regulated gene. Efforts have been devoted with the goal of identifying the specific DNA sequences bound by different TFs. For more than two decades, it was thought that mutations at each position in this sequence independently contribute to the binding probability of a TF. This binding preference has therefore been described through position weight matrices (PWMs). PWMs describe the binding preference of a TF towards its target sites by assuming that each nucleotide position contributes independently to the total specificity (linearity assumption). However, current research has shown that this simplified view lacks a significant part of the information needed to precisely describe the binding preference of a TF. It was also shown that the most information missing in the PWM is encoded in dinucleotide mutations. Two questions are important in this regard: (1) Which information about TF-DNA interaction are we missing and are currently employed methods able to provide them? and (2) What is a comprehensive description of non-linearity that is based on biophysical properties rather then on abstract probabilities? One important aspect is the three dimensional configuration of the DNA strand (DNA shape) which is known to affect TF binding to a varying degree. Through recent work by the group of Remo Rohs it is possible to predict shape parameters (features) from a DNA sequence and investigate to which degree they influence binding for any given set of measurements. The first aim of this thesis is therefore to determine non-linearity in TF-DNA interaction and investigate the influence of DNA shape on them. Protein-DNA interactions were studied with a variety of methods using structural biology (NMR, crystallography, cryo EM) or quantitative Methods (EMSA, DNA binding arrays, ChIPSeq, B1H, SELEX, MITOMI, Simile-Seq). Most of these quantitative methods to measure TF-DNA interactions, however, are not very sensitive to weak binders due to stringent washing steps or cutoffs they employ. Especially sequences with two positions differing from the consensus can be very weakly bound - therefore a sensitive method is needed to investigate non-linearity. The method called High Performance Fluorescence Anisotropy (HiP-FA, recently developed in our lab) provides the necessary sensitivity. Using HiP-FA, I determined the affinities of 13 TFs from the Drosophila melanogaster segmentation network and found most of them to contain a significant non-linearity in their specificity. The binding energies of the TFs correlated significantly with certain DNA shape features suggesting shape readout by the TFs. These results could be confirmed in existing structural biology data. Besides the influence of information directly encoded in the DNA sequence, the binding of a TF in the genome is most influenced by the DNA accessibility. This property is a result of the genomic DNA being wrapped around histone octamers forming nucleosomes. Since the underlying sequence can also influence the binding of the histone complex to the DNA, a natural question to ask is which features of the DNA sequence are the major determinant of histone-DNA interaction. Attempts to address this question used existing methods which were either MNase based and are therefore prone to the enzymes intrinsic cutting bias or based on dialysis and/or EMSA readout and have in consequence a low throughput and can only be automated to a small degree. This leads to a limited set of measurements which are usually only based on a single measurement point instead of a complete titration curve. The second aim of my thesis is therefore to develop an in vitro assay to determine free energies of nucleosome formation which improves on the limitations of existing methods. Using the sensitive FA-microscopy setup, I developed an automated assay to determine the free energy of nucleosome formation in a competitive titration. In contrast to existing methods, the throughput of the assays allows for full competitor titration curves. By measuring the free binding energies of 42 sequences, I showed that GC-content is the factor most contributing to the free energy. The relationship between these quantities is non-monotonous with an optimal GC-content of 49 percent. The results provided in this thesis give insight into the nature of non-linearity in TF-DNA interactions and highlight the DNA shape readout therein. Methodical advancements developed in this work can be used as a foundation to investigate other kinds of molecular interactions making use of the high sensitivity of FA-based microscopy

Functional characterization of the Saccharomyces cerevisiae chromatin remodeler INO80

Knowing the explicit locations of nucleosomes in a genome is a pre-requisite for understanding the regulation of genes. Predominantly at regulatory active promoter sites, regular spaced arrays phased at reference points shape the chromatin landscape. In eukaryotic cells ATP-dependent chromatin remodeler align nucleosomes at reference points and are pivotal in the formation of the stereotyped promoter pattern. Chromatin remodeler of the ISWI, CHD, SWI/SNF and INO80 family convert energy derived from ATP hydrolysis to operate on their nucleosomal substrates to accomplish nucleosome spacing, eviction and editing reactions. Recent structural elucidations provided mechanistic insights into how chromatin remodelers engage their nucleosomal substrates (Eustermann et al., 2018, Aramayo et al., 2018, Willhoft et al., 2018, Ayala et al., 2018, Farnung et al., 2017, Wagner et al., 2020, Yan et al., 2019, He et al., 2020, Han et al., 2020) and brought about a unifying DNA wave mechanism underpinning ATP-dependent DNA translocation by chromatin remodeling complexes (Yan and Chen, 2020). Understanding how phased arrays of equally spaced nucleosomes are generated by chromatin remodelers represents an ultimate long-term goal in chromatin biology. What remains unclear is the underlying mechanism that directs nucleosome positioning by chromatin remodelers in absolute terms. How do ATP-dependent chromatin remodelers generate phased arrays of regularly spaced nucleosomes? How are the distances between nucleosomes and phasing sites and between adjacent nucleosomes set? Is DNA shape read-out part of nucleosome positioning driven by chromatin remodelers? Do remodelers have intrinsic ruler-like elements that set spacing and phasing distances? The aim of this thesis was to delineate whether, and if so, what type of genomic information is read by a remodeler in the stereotypic placement of nucleosomes at physiological sites, and how the remodeler activities fit into the unifying framework of ATP-dependent DNA translocation mechanism of chromatin remodelers. To gain an insight into nucleosome positioning driven by Saccharomyces cerevisiae (S.c.) ATP-dependent chromatin remodelers, a combination of a minimalistic genome-wide in vitro reconstitution system, biochemical analysis, high-resolution structures and structure-guided mutagenesis of the S.c. INO80 model system was applied. Findings of this work would have an impact on the mechanistic understanding of nucleosome positioning driven by ATP dependent chromatin remodelers based on the ruler concept that has been described earlier for the ISW1a chromatin remodeler (Yamada et al., 2011). The ISW1a, Chd1 and ISW2 remodelers demonstrated “clamping” activity and used ruler elements to set 1 Abstract distances with a defined linker length (21-26 bp at all densities, 12-13bp at all densities, 54-58 bp at low/medium densities, respectively). Mutagenesis of the INO80 model system identified and tuned the INO80 ruler element, which is comprised of the Ino80_HSA domain of the ARP module, the NHP10 module and Ino80 N-terminal residues. Regularly spaced symmetrical arrays were generated at the Reb1 reference point sites as well as at BamHI-introduced dsDNA break sites. Nucleosome positioning on the genomic sequences of S. c., Schizosaccharomyces pombe (S.p.) as well as Escherichia coli (E.coli) showed no significant differences. Mutagenesis of residues located within the Ino80_HSA domain established a causal link between nucleosome positioning by INO80 and DNA shape read-out by the INO80_HSA domain. The spacing and phasing distances generated by ATP-dependent chromatin remodelers point towards a remodeler-intrinsic ruler activity that is independent of underlying DNA sequences and can be sensitive to nucleosome density. This study measured linker lengths set by remodeler-intrinsic ruler-like functionalities in absolute terms, which will be instrumental to dissect contributions from individual remodelers in nucleosome positioning in vivo. This provides the starting point to understand how remodeler-driven nucleosome dynamics direct stable steady-state nucleosome positions relative to DNA bound factors, DNA ends and DNA sequence elements. Sequence-dependent DNA shape features have been mainly associated with binding of transcription factors as well as general regulatory factors and more static DNA binding events. This study augments the general description of nucleosome positioning sequences for chromatin remodelers by establishing nucleosome positioning motifs based on DNA shape analysis. This study provides an intriguing framework to implement DNA shape read-out in the tracking mechanism of DNA-translocating machineries