Schwenk, S. | Search bioRxiv

bioRxiv2019

Nanopore sequencing of DNA concatemers reveals higher-order features of chromatin structure

Higher-order chromatin structure arises from the combinatorial physical interactions of many genomic loci. To investigate this aspect of genome architecture we developed Pore-C, which couples chromatin conformation capture with Oxford Nanopore Technologies (ONT) long reads to directly sequence multi-way chromatin contacts without amplification. In GM12878, we demonstrate that the pairwise interaction patterns implicit in Pore-C multi-way contacts are consistent with gold standard Hi-C pairwise contact maps at the compartment, TAD, and loop scales. In addition, Pore-C also detects higher-order chromatin structure at 18.5-fold higher efficiency and greater fidelity than SPRITE, a previously published higher-order chromatin profiling technology. We demonstrate Pore-Cs ability to detect and visualize multi-locus hubs associated with histone locus bodies and active / inactive nuclear compartments in GM12878. In the breast cancer cell line HCC1954, Pore-C contacts enable the reconstruction of complex and aneuploid rearranged alleles spanning multiple megabases and chromosomes. Finally, we apply Pore-C to generate a chromosome scale de novo assembly of the HG002 genome. Our results establish Pore-C as the most simple and scalable assay for the genome-wide assessment of combinatorial chromatin interactions, with additional applications for cancer rearrangement reconstruction and de novo genome assembly.

genomics↗

bioRxiv2017

Cell-wall synthesis and ribosome maturation are co-regulated by an RNA switch in Mycobacterium tuberculosis

The success of Mycobacterium tuberculosis as a pathogen relies on the ability to switch between active growth and non-replicating persistence, associated with latent TB infection. Resuscitation promoting factors (Rpfs) are essential for the transition of M. tuberculosis to dormancy and for emergence from the non-replicating persistent state. But these enzymes are double-edged swords, as their ability to degrade the cell wall, is potentially lethal to the bacterium itself. Hence, Rpf expression is tightly regulated. We have identified a novel regulatory element in the 5 untranslated region (UTR) of rpfB. We demonstrate that this element is a transcriptionally regulated RNA switch/riboswitch candidate, which is restricted to pathogenic mycobacteria, suggesting a role in virulence. Moreover, we have used translation start site mapping to re-annotate the RpfB start codon and identified and validated a ribosome binding site that is likely to be targeted by an RpfB antisense RNA. Finally, we show that rpfB is co-transcribed with downstream genes, ksgA and ispE. ksgA encodes a universally conserved methyl transferase involved in ribosome maturation and ispE encodes an essential ATP-dependent kinase involved in cell wall synthesis. This arrangement implies co-regulation of resuscitation, cell wall synthesis and ribosome maturation via the RNA switch. We propose that deregulation of this switch, associated with cell wall synthesis and ribosome function, presents a new target for anti-tuberculosis drug development.\n\nImportanceThis work describes the identification and characterisation of a novel regulatory RNA element/attenuator that controls cell wall synthesis and ribosome function in Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB). By switching between two different conformations, this RNA switch can either enable or inhibit transcription of a tri-cistronic mRNA that encodes a cell-wall remodelling enzyme crucial for activation of latent TB, an RNA methytransferase that is important for ribosome function and a protein kinase essential for early steps in cell wall synthesis. This RNA switch is only present in a subset of pathogenic mycobacteria, and by regulating the expression of three genes associated with classical antimicrobial targets we believe that it offers a novel important target for future anti-tuberculosis drugs.

molecular biology↗