mapping-mammalian-3d-genome-interactions-with-micro-c-xl

Mapping Mammalian 3D Genome Interactions with Micro-C-XL

Three-dimensional (3D) chromosome organization is a major factor in genome regulation and cell-type specification. For example, cis-regulatory elements, ...

University of Copenhagen

Chromosome Conformation Capture (3C) methods are a family of sequencing-based assays to measure the three-dimensional structure of genomes, with Hi-C as the most prominent method in widespread use. The Micro-C-XL protocol is technical variant that improves the resolution and signal-to-noise ratio of the Hi-C protocol and therefore offers enhanced detection of chromatin features such as chromosome loops and fine-grained resolution of topologically associated domains. Here we describe a detailed step-by-step protocol for Micro-C-XL in mammalian cells.

Mammalian Micro-C-XL.

Digestion with restriction enzymes is a classical approach for probing DNA accessibility in chromatin. It allows to monitor both the cut and the uncut fraction and thereby the determination of accessibility or occupancy (= 1 - accessibility) in absolute terms as the percentage of cut or uncut molecules, respectively, out of all molecules. The protocol presented here takes this classical approach to the genome-wide level. After exhaustive restriction enzyme digestion of chromatin, DNA is purified, sheared, and converted into libraries for high-throughput sequencing. Bioinformatic analysis counts uncut DNA fragments as well as DNA ends generated by restriction enzyme digest and derives thereof the fraction of accessible DNA. This straightforward principle is technically challenged as preparation and sequencing of the libraries leads to biased scoring of DNA fragments. Our protocol includes two orthogonal approaches to correct for this bias, the "corrected cut-uncut" and the "cut-all cut" method, so that accurate measurements of absolute accessibility or occupancy at restriction sites throughout a genome are possible. The protocol is presented for the example of S. cerevisiae chromatin but may be adapted for any other species.

ORE-Seq: Genome-Wide Absolute Occupancy Measurement by Restriction Enzyme Accessibilities.

How enhancers control target gene expression over long genomic distances remains an important unsolved problem. Here we investigated enhancer-promoter communication by integrating data from nucleosome-resolution genomic contact maps, nascent transcription and perturbations affecting either RNA polymerase II (Pol II) dynamics or the activity of thousands of candidate enhancers. Integration of new Micro-C experiments with published CRISPRi data demonstrated that enhancers spend more time in close proximity to their target promoters in functional enhancer-promoter pairs compared to nonfunctional pairs, which can be attributed in part to factors unrelated to genomic position. Manipulation of the transcription cycle demonstrated a key role for Pol II in enhancer-promoter interactions. Notably, promoter-proximal paused Pol II itself partially stabilized interactions. We propose an updated model in which elements of transcriptional dynamics shape the duration or frequency of interactions to facilitate enhancer-promoter communication.

Article	Year
Mammalian Micro-C-XL. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 35103975	2022
ORE-Seq: Genome-Wide Absolute Occupancy Measurement by Restriction Enzyme Accessibilities. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 36807068	2023
RNA polymerase II dynamics shape enhancer-promoter interactions. Nature genetics\| PubMed ID: 37430091	2023

Nils Krietenstein

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