Cytology of DNA Replication Reveals Dynamic Plasticity of Large-Scale Chromatin Fibers

Abstract

Summary In higher eukaryotic interphase nuclei, the 100s-1000s fold linear compaction of chromatin is difficult to reconcile with its function as a template for transcription, replication, and repair. It is challenging to imagine how DNA and RNA polymerases with their associated molecular machinery would move along the DNA template without transient decondensation of observed large-scale chromatin “chromonema” fibers [1]. Transcription or replication “factory” models [2] in which polymerases remain fixed while DNA is reeled through are similarly difficult to conceptualize without transient decondensation of these chromonema fibers. Here we show how a dynamic plasticity of chromatin folding within large-scale chromatin fibers allows DNA replication to take place without significant changes in the global large-scale chromatin compaction or shape of these large-scale chromatin fibers. Time-lapse imaging of lac-operator tagged chromosome regions shows no major change in the overall compaction of these chromosome regions during their DNA replication. Improved pulse-chase labeling of endogenous interphase chromosomes yields a model in which global compaction and shape of large Mbp chromatin domains remains largely invariant during DNA replication, with DNA within these domains undergoing significant movements and redistribution as they move into and then out of adjacent replication foci. In contrast to hierarchical folding models, this dynamic plasticity of large-scale chromatin organization explains how localized changes in DNA topology allow DNA replication to take place without an accompanying global unfolding of large-scale chromatin fibers while suggesting a possible mechanism for maintaining epigenetic programming of large-scale chromatin domains throughout DNA replication.

CRISPR/Cas9-mediated knock-in of an optimized TetO repeat for live cell imaging of endogenous loci

“…A previous study proposed that TetO or LacO repeats bound by their corresponding repressors can cause replication fork barrier, accompanied by unusual, long-lived PCNA foci overlying the operator repeats, followed by mitotic defects ( 54 ). This previous study is inconsistent with other published studies, including live-cell imaging of LacO and PCNA dynamics during S-phase, which demonstrated that chromosome regions carrying large numbers of LacO arrays replicated at specific times during the cell cycle without any delay in DNA replication ( 55 , 56 ).…”

Genome organization during the cell cycle: unity in division

“…In contrast, a recent study in mammalian cells suggests that large‐scale chromatin remodeling is not needed during replication. This model proposes a mechanism by which only very transient, local decompaction of replicating regions takes place, and that epigenetic marks can be transferred from template DNA to the nearby newly replicated DNA . This study concludes that there is a ‘relatively disordered’ internal structure for chromatin fibers.…”

“…Microscopy has also revealed that chromosomes occupy distinct territories, and that TADs along individual chromosomes are spatially segregated into GC‐rich transcriptionally active chromatin regions in the interior of the nucleus and GC‐poor transcriptionally inactive regions at the nuclear periphery . The gene rich regions display a visually spread out conformation in comparison with gene poor regions, though recent light and electron microscopy results suggest that fairly compact fiber structures exist for both active and inactive genomic regions . To understand the impact of genome structure changes that happen during and after mitosis, it is important to note the stability of the 3D genome structures during interphase.…”

“…The fact that proliferating cell types tend to be affected more in Progeria patients than postmitotic cells provides further evidence that cell division is a dangerous chance for cells with defective epigenetics to lose important chromatin architecture. Similarly, unfaithful inheritance of architectural proteins after cell division has been linked with disease …”

Confocal Laser Scanning Microscopy of Living Cells

“…Instead of these algorithms, conventional microscopy uses deconvolution to improve the quality of images as in confocal microscopy [12]. For example, deconvolution was applied to wide-field microscopy images to resolve DNA replication units that are studied only by electron and structured illumination microscopy [13]. Unlike these later methods, confocal and classical light microscopy possesses the ability to study these structures in the living cells.…”