DNA torsion as a feedback mediator of transcription and chromatin dynamics

Abstract

The double helical structure of DNA lends itself to topological constraints. Many DNA-based processes alter the topological state of DNA, generating torsional stress, which is efficiently relieved by topoisomerases. Maintaining this topological balance is crucial to cell survival, as excessive torsional strain risks DNA damage. Here, we review the mechanisms that generate and modulate DNA torsion within the cell. In particular, we discuss how transcription-generated torsional stress affects Pol II kinetics and chromatin dynamics, highlighting an emerging role of DNA torsion as a feedback mediator of torsion-generating processes.

Keywords: RNA Polymerase II; nascent RNA; nucleosome turnover; topoisomerase; torsional stress.

Figure 1. Effects of torsion on DNA and chromatin structure. (A) The relaxed state of the DNA double helix consists of ~10 bp per helical turn (center). When DNA-based processes exert torsional force on the DNA, it manifests as a change in twist or formation of writhe. (B) The nucleosome is composed of a left-handed wrapped DNA around an octameric core. H3-H4 tetramer is colored blue. H2A-H2B dimers are colored green. (C) Potential writhe in the context of a nucleosomal template. (D) Molecular structure of psoralen (top), tri-methyl psoralen (TMP) (middle), and biotin-TMP (bottom) (E) TMP intercalates into the DNA double-strand and forms monoadducts and interstrand crosslinks under UV light.

Figure 2. Transcribed gene regions experience torsional stress (A) Method for mapping DNA supercoiling genome-wide using next-generation sequencing. (I) Following Exo I-enrichment of crosslinked strands, Illumina paired-end (PE) adapters were ligated. (II) Lambda Exonuclease (Exo λ) was used to resect the 5′ strand until the crosslinking site. (III) Using a primer complementary to PE adapters, several rounds of primer extension were performed. (IV) The single-stranded products were appended with ribo-Gs at the 3′ ends using terminal transferase, (V) followed by ligation with a double-stranded PE adaptor with a CCC overhang. (VI) After a single round of primer extension to generate a double-stranded fragment, the products were amplified using PE sequencing primers. Sequencing from the CCC overhang allowed for the precise mapping of TMP-crosslinked sites throughout the whole genome. (B) Genes in Drosophila genome were split as expressed (top) or silent (bottom). High-resolution genome-wide maps of TMP crosslinking surrounding the transcription start site (TSS) and transcription end site (TES) are shown under normal conditions (black), Topo I inhibition (orange), and Topo II inhibition (teal). Expressed genes experience higher torsional stress, consistent with the twin-supercoil domain model.

Figure 3. Torsional stress affects nucleosome dynamics and Pol II kinetics. Genes were grouped based on change in torsional stress as measured by change in TMP crosslinking before and after Topo I or II inhibition. The highest (High TD) and lowest (Low TD) 20% of genes are shown. For these two groups, the change in nucleosome turnover (A), the change in Pol II pausing (B), and the change in Pol II elongation as measured by nascent RNA production (C) are plotted for regions surrounding the TSS.

Figure 4. Torsional stress affects the affinity of DNA-binding proteins at the TSS. (A) Micrococcal nuclease digested chromatin is extracted using low salt and sequenced using the paired-end Illumina platform. Reads were parsed computationally by size, with short fragments representing sites for DNA binding protein. The global pattern of short fragments from low-salt extraction of chromatin is analyzed for all genes surrounding the TSS before and after Topo I or II inhibition. (B) The change in short fragments after Topo I (left) or II (right) inhibition relative to control is displayed as a heat map for all genes arranged by expression level in untreated cells. (C) Unbiased k-means clustering of (B) with k = 2. (D) Venn diagram of group 2 genes in Topo I (orange) and Topo II (teal) inhibited samples. (E) Gene ontology analysis of group 2 genes performed as described previously.^,