Sven Sewitz

Lineage-Specific Genome Architecture Links Enhancers and Non-coding Disease Variants to Target Gene Promoters

Summary Long-range interactions between regulatory elements and gene promoters play key roles in transcriptional regulation. The vast majority of interactions are uncharted, constituting a major missing link in understanding genome control. Here, we use promoter capture Hi-C to identify interacting regions of 31,253 promoters in 17 human primary hematopoietic cell types. We show that promoter interactions are highly cell type specific and enriched for links between active promoters and epigenetically marked enhancers. Promoter interactomes reflect lineage relationships of the hematopoietic tree, consistent with dynamic remodeling of nuclear architecture during differentiation. Interacting regions are enriched in genetic variants linked with altered expression of genes they contact, highlighting their functional role. We exploit this rich resource to connect non-coding disease variants to putative target promoters, prioritizing thousands of disease-candidate genes and implicating disease pathways. Our results demonstrate the power of primary cell promoter interactomes to reveal insights into genomic regulatory mechanisms underlying common diseases.

A Small Molecule That Disrupts G-Quadruplex DNA Structure and Enhances Gene Expression

It has been hypothesized that the formation of G-quadruplex structures in the DNA of gene promoters may be functionally linked to transcription and consequently that small molecules that interact with such G-quadruplexes may modulate transcription. We previously reported that triarylpyridines are a class of small molecules that selectively interact with G-quadruplex DNA. Here we describe an unexpected property of one such ligand that was found to disrupt the structure of two different DNA G-quadruplex structures, each derived from sequence motifs in the promoter of the proto-oncogene c-kit. Furthermore, cell-based experiments in a cell line that expresses c-kit (HGC-27) showed that the same ligand increased the expression of c-kit. This contrasts with G-quadruplex-inducing ligands that have been previously found to inhibit gene expression. It would thus appear that the functional consequence of small molecule ligands interacting with G-quadruplex structures may depend on the specific mode of interaction. These observations provide further evidence to suggest that G-quadruplex forming sequence motifs play a role that relates to transcription.

Genome-wide analysis of a G-quadruplex-specific single-chain antibody that regulates gene expression

G-quadruplex nucleic acids have been proposed to play a role in a number of fundamental biological processes that include transcription and translation. We have developed a single-chain antibody that is selective for G-quadruplex DNA over double-stranded DNA, and here show that when it is expressed in human cells, it significantly affects the expression of a wide variety of genes, in a manner that correlates with the presence of predicted G-quadruplexes. We observe cases where gene expression is increased or decreased, and that there are apparent interactions with G-quadruplex motifs at the beginning and end of the genes, and on either strand. The outcomes of this genome-wide study demonstrate that G-quadruplex recognition by the antibody has physiological consequences, and provides insights into some of the complexity associated with G-quadruplex-based regulation.

Targeting the c-Kit Promoter G-quadruplexes with 6-Substituted Indenoisoquinolines

Herein, we demonstrate the design, synthesis, biophysical properties, and preliminary biological evaluation of 6-substituted indenoisoquinolines as a new class of G-quadruplex stabilizing small molecule ligands. We have synthesized 6-substituted indenoisoquinolines 1a-e in two steps from commercially available starting materials with excellent yields. The G-quadruplex stabilization potential of indenoisoquinolines 1a-e was evaluated by fluorescence resonance energy transfer-melting analysis, which showed that indenoisoquinolines show a high level of stabilization of various G-quadruplex DNA structures. Indenoisoquinolines demonstrated potent inhibition of cell growth in the GIST882 patient-derived gastrointestinal stromal tumor cell line, accompanied by inhibition of both c-Kit transcription and KIT oncoprotein levels.

DNA looping and catalysis; the IHF-folded arm of Tn10 promotes conformational changes and hairpin resolution.

DNA loops and bends are common features of DNA processing machines. The bacterial transposon Tn10 has recruited integration host factor (IHF), a site-specific DNA-bending protein, as an architectural component for assembly of the higher-order nucleoprotein complex within which the transposition reaction takes place. Here, we demonstrate additional roles for the IHF loop during the catalytic steps of the reaction. We show that metal ion-dependent unfolding of the IHF-bent transposon arm is communicated to the catalytic center, inducing a substantial conformational change in the DNA. Partial disruption of the IHF loop shows that this step promotes resolution of the hairpin intermediate on one transposon end and initiation of catalysis at the other. Further evidence suggests that the molecular mechanism responsible may be mechanical stress in the IHF loop, related to a change in the relative position of the transposase contacts that anchor the loop on either side.

An Integrated Model of Transcription Factor Diffusion Shows the Importance of Intersegmental Transfer and Quaternary Protein Structure for Target Site Finding

We present a computational model of transcription factor motion that explains both the observed rapid target finding of transcription factors, and how this motion influences protein and genome structure. Using the Smoldyn software, we modelled transcription factor motion arising from a combination of unrestricted 3D diffusion in the nucleoplasm, sliding along the DNA filament, and transferring directly between filament sections by intersegmental transfer. This presents a fine-grain picture of the way in which transcription factors find their targets two orders of magnitude faster than 3D diffusion alone allows. Eukaryotic genomes contain sections of nucleosome free regions (NFRs) around the promoters; our model shows that the presence and size of these NFRs can be explained as their acting as antennas on which transcription factors slide to reach their targets. Additionally, our model shows that intersegmental transfer may have shaped the quaternary structure of transcription factors: sequence specific DNA binding proteins are unusually enriched in dimers and tetramers, perhaps because these allow intersegmental transfer, which accelerates target site finding. Finally, our model shows that a ‘hopping’ motion can emerge from 3D diffusion on small scales. This explains the apparently long sliding lengths that have been observed for some DNA binding proteins observed in vitro. Together, these results suggest that transcription factor diffusion dynamics help drive the evolution of protein and genome structure.

Higher order assembly: folding the chromosome

The linear molecules of DNA that constitute a eukaryotic genome have to be carefully organised within the nucleus to be able to correctly direct gene expression. Microscopy and chromosome capture methods have revealed a hierarchical organisation into territories, domains and subdomains that ensure the accessibility of expressed genes and eventually chromatin loops that serve to bring gene enhancers into proximity of their target promoters. A rapidly growing number of genome-wide datasets and their analyses have given detailed information into the conformation of the entire genome, allowing evolutionary insights, observations of genome rearrangements during development and the identification of new gene-to-disease associations. The field is now progressing into using computational models of genome dynamics to investigate the mechanisms that shape genome structure, placing increasing importance on the role of chromatin associated proteins for this process.

Functional coupling between the two active sites during Tn10 transposition buffers the mutation of sequences critical for DNA hairpin processing

DNA processing reactions often involve multiple components acting in concert to achieve the desired outcome. However, it is usually difficult to know how the components communicate and cooperate to orchestrate an ordered series of events. We address this question in the context of the Tn10 transposition reaction, in which the DNA cleavage and joining events occur within a higher‐order complex containing a transposase dimer, two transposon ends and the DNA‐bending host‐factor IHF (Integration Host Factor). Previously it was shown that the complex is asymmetric. The α side consists of an IHF protomer initially immobilized by a DNA‐loop, but subsequently used to promote conformational changes required for the cleavage steps. The β side of the complex was considered to fulfil a more passive role. Here we show that the α side of the complex promotes coupled conformational changes at both transposon ends, while the α and β sides communicate and cooperate to dominate different phases of the transposition reaction. Together, these effects provide for a robust response to critical changes in the transposon end. These findings also explain the intriguing genetic phenotypes of a series of previously reported Tn10 mutants and have consequences for the evolution of new elements.

Heterogeneous chromatin mobility derived from chromatin states is a determinant of genome organisation in S. cerevisiae

Spatial organisation of the genome is essential for the activation of correct gene expression profiles, but the mechanisms that shape three-dimensional genome organisation in eukaryotes are still far from understood. Here, we develop a new approach, combining bioinformatic determination of chromatin states, dynamic polymer modelling of genome structure, quantitative microscopy and Hi-C to demonstrate that differential mobility of yeast chromosome segments leads to self-organisation of the genome in three dimensions. More than forty percent of chromatin-associated proteins display a poised distribution and coordinated relocations. They are distributed heterogeneously along the chromosome, and by simulating the dynamics of this heteropolymer, we observe structural features that match our experimental results. Further, we show that this mechanism directly contributes to the directed relocalisation of active genes to the nuclear periphery. One Sentence Summary Unequal protein occupancy and chromosome segment mobility drive 3D organisation of the genome.

Systems Biology Approaches for Understanding Genome Architecture

The linear and three-dimensional arrangement and composition of chromatin in eukaryotic genomes underlies the mechanisms directing gene regulation. Understanding this organization requires the integration of many data types and experimental results. Here we describe the approach of integrating genome-wide protein-DNA binding data to determine chromatin states. To investigate spatial aspects of genome organization, we present a detailed description of how to run stochastic simulations of protein movements within a simulated nucleus in 3D. This systems level approach enables the development of novel questions aimed at understanding the basic mechanisms that regulate genome dynamics.