In vitro reconstitution of chromatin domains shows a role for nucleosome positioning in 3D genome organization.

Eukaryotic genomes are organized into chromatin domains. The molecular mechanisms driving the formation of these domains are difficult to dissect in vivo and remain poorly understood. Here we reconstitute Saccharomyces cerevisiae chromatin in vitro and determine its 3D organization at subnucleosome resolution by micrococcal nuclease-based chromosome conformation capture and molecular dynamics simulations.

scTAM-seq enables targeted high-confidence analysis of DNA methylation in single cells.

Single-cell DNA methylation profiling currently suffers from excessive noise and/or limited cellular throughput. We developed scTAM-seq, a targeted bisulfite-free method for profiling up to 650 CpGs in up to 10,000 cells per experiment, with a dropout rate as low as 7%. We demonstrate that scTAM-seq can resolve DNA methylation dynamics across B-cell differentiation in blood and bone marrow, identifying intermediate differentiation states that were previously masked.

Analysis of sub-kilobase chromatin topology reveals nano-scale regulatory interactions with variable dependence on cohesin and CTCF.

Enhancers and promoters predominantly interact within large-scale topologically associating domains (TADs), which are formed by loop extrusion mediated by cohesin and CTCF. However, it is unclear whether complex chromatin structures exist at sub-kilobase-scale and to what extent fine-scale regulatory interactions depend on loop extrusion. To address these questions, we present an MNase-based chromosome conformation capture (3C) approach, which has enabled us to generate the most detailed local interaction data to date (20 bp resolution) and precisely investigate the effects of cohesin and CTCF depletion on chromatin architecture.