Biology subjects

Oberbeckmann, E.

Publications and source records attributed to Oberbeckmann, E..

4 recordsLinked to original sources

bioRxiv2023

Three-step mechanism of promoter escape by RNA polymerase II

The transition from transcription initiation to elongation is highly regulated in human cells but remains incompletely understood at the structural level. In particular, it is unclear how interactions between RNA polymerase (Pol) II and initiation factors are broken to enable promoter escape. Here we reconstitute Pol II promoter escape in vitro, determine high-resolution structures of five transition intermediates, and show that promoter escape occurs in three major steps. First, the growing RNA transcript displaces the B-reader element of the initiation factor TFIIB without evicting TFIIB. Second, rewinding of the upstream edge of the growing DNA bubble evicts TFIIA, TFIIB and TBP and repositions parts of TFIIE and TFIIF. Third, binding of DSIF and NELF evicts TFIIE and TFIIH, establishing the paused elongation complex. This three-step model for promoter escape fills a gap in our understanding of the initiation-elongation transition of Pol II transcription.

biochemistry↗

bioRxiv2023

In vitro reconstitution of chromatin domains

The spatial organization of the genome modulates nuclear processes, including transcription, replication, and DNA repair1,2. Eukaryotic genomes are organized into distinct 3D chromatin domains3. However, the molecular mechanisms that drive the formation of these domains are difficult to dissect in vivo and remain poorly understood. Here, we reconstitute S. cerevisiae chromatin in vitro and determine its 3D organization at sub-nucleosome resolution by MNase-based chromosome conformation capture and molecular dynamics simulations. We show that regularly spaced and phased nucleosome arrays form chromatin domains in vitro that resemble domains in vivo. This demonstrates that neither loop extrusion nor transcription are required for domain formation. In addition, we find that the boundaries of reconstituted domains correspond to nucleosome-free regions and that insulation strength scales with their width. Finally, we show that domain compaction depends on nucleosome linker length, with longer linkers forming more compact structures. Together, our results demonstrate that nucleosome positioning is sufficient to reconstitute chromatin domains and provide a proof-of-principle for bottom-up 3D genome studies.

genomics↗

bioRxiv2020

Genome information processing by the INO80 chromatin remodeler positions nucleosomes

The fundamental molecular determinants by which ATP-dependent chromatin remodelers organize nucleosomes across eukaryotic genomes remain largely elusive. Here, chromatin reconstitutions on physiological, whole-genome templates reveal how remodelers read and translate genomic information into nucleosome positions. Using the yeast genome and the multi-subunit INO80 remodeler as a paradigm, we identify DNA shape/mechanics encoded signature motifs as sufficient for nucleosome positioning and distinct from known DNA sequence preferences of histones. INO80 processes such information through an allosteric interplay between its core- and Arp8-modules that probes mechanical properties of nucleosomal and linker DNA. At promoters, INO80 integrates this readout of DNA shape/mechanics with a readout of co-evolved sequence motifs via interaction with general regulatory factors bound to these motifs. Our findings establish a molecular mechanism for robust and yet adjustable +1 nucleosome positioning and, more generally, remodelers as information processing hubs that enable active organization and allosteric regulation of the first level of chromatin.

biochemistry↗

bioRxiv2020

Ruler elements in chromatin remodelers set nucleosome array spacing and phasing

Arrays of regularly spaced nucleosomes dominate chromatin and are often phased by alignment to reference sites like active promoters. How the distances between nucleosomes (spacing), and between phasing sites and nucleosomes are determined remains unclear, and specifically, how ATP dependent chromatin remodelers impact these features. Here, we used genome-wide reconstitution to probe how Saccharomyces cerevisiae ATP dependent remodelers generate phased arrays of regularly spaced nucleosomes. We find that remodelers bear a functional element named the ruler that determines spacing and phasing in a remodeler-specific way. We use structure-based mutagenesis to identify and tune the ruler element residing in the Nhp10 and Arp8 modules of the INO80 remodeler complex. Generally, we propose that a remodeler ruler regulates nucleosome sliding direction bias in response to (epi)genetic information. This finally conceptualizes how remodeler-mediated nucleosome dynamics determine stable steady-state nucleosome positioning relative to other nucleosomes, DNA bound factors, DNA ends and DNA sequence elements.

biochemistry↗