Single-molecule regulatory architectures — as captured by chromatin-fiber sequencing

Any trait (phenotype) reflects the contribution of genes (i.e. DNA sequence), epigenetic factors (chromosomal events other than DNA sequence: DNA methylation, RNA interference, histone modifications, chromatin remodeling), environmental effects (diet, lifestyle), endogenous influences (e.g. cardiopulmonary disorders, kidney disease), and each individual’s microbiome. Today’s topic discusses chromatin — that portion of a chromosome comprising nucleosome arrays, punctuated by short regulatory regions, containing transcription factors (TFs) and other nonhistone proteins.

Chromatin is fundamental to genome function, yet remains undefined at the level of individual chromatin fibers (i.e. the fundamental units of gene regulation). For example, how is regulatory DNA activated on individual chromatin fibers? To what degree are nearby regulatory regions coordinately stimulated on the same chromatin fiber? How does regulatory DNA triggering affect nucleosome positioning on individual chromatin fibers? How does TF occupancy modulate regulatory DNA actuation and function on single templates? Addressing these questions — requires nucleotide-resolution analysis of individual multikilobase (thousands of DNA base-pairs) chromatin fibers, which is not obtainable using current single-cell or bulk-profiling approaches.

Authors [see attached article] wished to develop a method for measuring the primary architecture of chromatin onto its underlying DNA template at single-nucleotide resolution — thereby enabling simultaneous identification of genetic and epigenetic features along multikilobase segments of the genome. Current approaches to mapping chromatin and regulatory architectures require a researcher to sample large populations of chromatin fibers and rely on dissolution of chromatin, using nucleases such as deoxyribonuclease (DNase I), micrococcal nuclease, restriction enzymes, transposases, or mechanical shearing.

CpG and GpC methyltransferases (enzymes that methylate DNA at GC or GC sites) are capable of marking accessible cytosines in a dinucleotide context without digesting DNA, and approaches using GpC methyltransferases have been extended to single-pass nanopore-sequencing. However, the usefulness of these approaches for gaining insights into the biology of individual chromatin fibers is limited because of: [a] sporadic occurrence and linear clustering of CpG and GpC dinucleotides in animal genomes as a result of mutation and selection; [b] confounding influence of endogenous cytosine methylation machineries; [c] marked DNA degradation induced by bisulfite conversion; and [d] the intrinsically limited ability of nanopore-sequencing to accurately identify modified bases on a per-molecule basis.

Using nonspecific DNA N6-adenine methyltransferases, authors [see attached article] found that single-molecule long-read sequencing of chromatin stencils enabled nucleotide-resolution readout of the primary architecture of multikilobase chromatin fibers (a method abbreviated ‘Fiber-seq’). Fiber-seq is able to expose widespread plasticity in the linear organization of individual chromatin fibers and illuminate factors that guide regulatory DNA activation, to study the coordinated stimulation of neighboring regulatory elements, single-molecule nucleosome positioning, and single-molecule transcription factor occupancy. Authors believe that this approach — and the data presented herein — open up new vistas on our learning about the primary fundamentals of gene regulation.


Science 26 Jun 2020; 368: 1449-1454

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