Single-molecule regulatory architectures captured by chromatin fiber sequencing

Abstract

Gene regulation is chiefly determined at the level of individual linear chromatin molecules, yet our current understanding of cis-regulatory architectures derives from fragmented sampling of large numbers of disparate molecules. We developed an approach for precisely stenciling the structure of individual chromatin fibers onto their composite DNA templates using nonspecific DNA N6-adenine methyltransferases. Single-molecule long-read sequencing of chromatin stencils enabled nucleotide-resolution readout of the primary architecture of multikilobase chromatin fibers (Fiber-seq). Fiber-seq exposed widespread plasticity in the linear organization of individual chromatin fibers and illuminated principles guiding regulatory DNA actuation, the coordinated actuation of neighboring regulatory elements, single-molecule nucleosome positioning, and single-molecule transcription factor occupancy. Our approach and results open new vistas on the primary architecture of gene regulation.

Massively multiplex single-molecule oligonucleosome footprinting

“…MNase-Southern) for studying constitutive heterochromatin. This is strongly supported by orthogonal analysis of heterochromatin-spanning K562 reads generated using the recently published, conceptually similar Fiber-seq method (Stergachis et al, 2020), which also reveal that H3K9me3 domains are enriched for irregular chromatin fibres ( Figure 6 -Figure Supplement 2). Given the robustness of this finding, it is tempting to speculate that this irregularity may be linked to the dynamic restructuring of heterochromatic nucleosomes by factors like HP1 (Sanulli et al, 2019), which may promote phaseseparation of heterochromatin.…”

“…SAMOSA adds to the growing list of technologies that use high-throughput single-molecule sequencing to explore the epigenome (Baldi et al, 2018;Lee et al, 2019;Shipony et al, 2020;Wang et al, 2019;Stergachis et al, 2020). We foresee the broad applicability of this and similar approaches to dissect gene regulatory processes at previously intractable length-scales.…”

“…The workflow for genomic samples will be presented first in each sections, and the deviations for homogeneous samples detailed at the end. 500U hia5 K562 Fiber-seq data from Stergachis et al (2020) were downloaded using Google Cloud Services via SRA accession SRP252718 and processed as below.…”

Identification of determinants of differential chromatin accessibility through a massively parallel genome-integrated reporter assay

“…Although SLOT was able to determine the relative accessibility of classes of DNA sequences, it had poor resolution to measure accessibility of individual DNA sequences, because of the low cleavage probability of DNase I at enzyme concentrations capable of discriminating levels of chromatin accessibility. We hypothesized that we could measure changes in DNA accessibility with higher sensitivity by observing the chromatin accessibility-dependent methylation of Escherichia coli adenine DNA adenine methylase (Dam) to the locus, given the high efficiency and stability of Dam methylation in cells (van Steensel and Henikoff 2000) and the known propensity of Dam to methylate more frequently in accessible chromatin (van Steensel and Henikoff 2000;Szczesnik et al 2019;Abdulhay et al 2020;Stergachis et al 2020). We further hypothesized that fusing Dam to retinoic acid receptor-gamma (RAR) would enhance the differential methylation of this RAR-Dam fusion protein at genomic loci with RAR binding motifs, and we make use of a mutant version of Dam methyltransferase shown to display increased signal-to-noise over wild-type Dam (van Steensel and Henikoff 2000;Szczesnik et al 2019).…”

“…However, bisulfite sequencing requires constrained library design to ensure sufficient CpG sites that act as a substrate for bisulfite conversion, and MNase-seq requires measurement over multiple MNase concentrations to fully measure accessibility (Schwartz et al 2019). Restriction enzyme strategies have been used to measure nucleosome occupancy and accessibility in yeast (Oberbeckmann et al 2019) and mouse hepatocyte (Chereji et al 2019) and stem cells (Soenmezer et al 2020), and recently, adenine methyltransferase has been used to map nucleosome positioning in human cell lines (Abdulhay et al 2020;Stergachis et al 2020). Here, we aim to develop an assay that takes advantage of adenine methyltransferase and restriction enzyme digestion for measuring the local DNA accessibility of genomically integrated large-scale reporter libraries, and probe the regulatory sequence determinants driving differential chromatin accessibility between stem cells and definitive endoderm.…”