Capture-C: a modular and flexible approach for high-resolution chromosome conformation capture

Downes, Damien J.; Smith, Alastair; Karpinska, Magdalena A; Velychko, Taras; Rue-Albrecht, Kévin; Sims, David; Milne, Thomas A.; Davies, James O. J.; Oudelaar, A. Marieke; Hughes, Jim R.

doi:10.1038/s41596-021-00651-w

Cited by 40 publications

(41 citation statements)

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“…Capture-C was performed as described previously [62,63] for three biological replicates per experimental condition. Briefly, 10 million cells per biological replicate were crosslinked, followed by cell lysis.…”

Section: Capture-cmentioning

confidence: 99%

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The Mediator complex regulates enhancer-promoter interactions

Ramasamy¹,

Aljahani²,

Karpinska³

et al. 2022

Preprint

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Enhancer-mediated gene activation generally requires physical proximity between enhancers and their target gene promoters. However, the molecular mechanisms by which interactions between enhancers and promoters are formed are not well understood. Here, we investigate the function of the Mediator complex in the regulation of enhancer-promoter interactions, by combining rapid protein depletion and high-resolution MNase-based chromosome conformation capture approaches. We show that depletion of Mediator leads to reduced enhancer-promoter interaction frequencies, which are associated with a strong decrease in gene expression. In addition, we find increased interactions between CTCF-binding sites upon Mediator depletion. These changes in chromatin architecture are associated with a re-distribution of the Cohesin complex on chromatin and a reduction in Cohesin occupancy specifically at enhancers. Our results indicate that enhancer-promoter interactions are dependent on an interplay between the Mediator and Cohesin complexes and provide new insights into the molecular mechanisms by which communication between enhancers and promoters is regulated.

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Section: Capture-cmentioning

confidence: 99%

“…The final libraries were assessed on a fragment analyzer using the High Sensitivity NGS Analysis Kit (Agilent, DNS-474-FR) and sequenced using the NextSeq550 Illumina platform (75-bp paired-end reads). Data analysis was performed using the CapCruncher pipeline [62] (https://github.com/sims-lab/CapCruncher).…”

Section: Capture-cmentioning

confidence: 99%

The Mediator complex regulates enhancer-promoter interactions

Ramasamy¹,

Aljahani²,

Karpinska³

et al. 2022

Preprint

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“…We hypothesized that MLL-AF4 and the complex of proteins it assembles at enhancers may play a role in driving these interactions. We used Next Generation Capture-C (Davies et al, 2016; Downes et al, 2022) to test whether physical contact between MLL-AF4 enhancers and promoters was dependent on the presence of the fusion protein. Using promoter-specific capture probes, we observed a striking correlation between MLL-AF4 binding at enhancers and promoter interaction frequency (Figure 5A, S5A, purple shading).…”

Section: Resultsmentioning

confidence: 99%

“…Capture-C was conducted as described previously (Davies et al, 2016; Downes et al, 2022), using 2×10 7 cells per replicate. Briefly, DpnII-generated 3C libraries were sonicated to a fragment size of 200 bp and Illumina paired-end sequencing adaptors (New England BioLabs, E6040, E7335 and E7500) were added using Herculase II (Agilent) for the final PCR.…”

Section: Methodsmentioning

confidence: 99%

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PAF1 and FACT cooperate with MLL-AF4 to drive enhancer activity in leukemia

Crump

Smith

Godfrey

et al. 2022

Preprint

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Aberrant enhancer activation has been identified as a key mechanism driving oncogene expression in many cancers. Here we use TOPmentation (Transcription factor-OPtimized ChIPmentation) to probe enhancer usage in primary MLL-rearranged acute lymphoblastic leukemia. We find that MLL-AF4, commonly held to promote transcription by binding to gene promoters, is also present at many active enhancers, where it assembles a complex of transcriptional co-activators normally found in the gene body. This includes DOT1L, ENL, PAF1, and a newly identified interaction with the histone chaperone FACT. By chemical degradation, we demonstrate that PAF1 and FACT are required for enhancer activity, including maintaining histone H3K27 acetylation, enhancer RNA transcription and enhancer-promoter interactions. This work identifies novel roles for PAF1 and FACT in enhancer function, and reveals an enhancer-targeting mechanism by which MLL-AF4 upregulates transcription, recruiting transcription machinery through a network of multivalent interactions to control enhancer activity and gene expression in acute leukemias.

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