Abstract
The linear sequence of DNA encodes access to the complete set of proteins that carry out cellular functions. Yet, much of the functionality appropriate for each cell is nested within layers of dynamic regulation and organization, including a hierarchy of chromatin structural states and spatial arrangement within the nucleus. There remain limitations in our understanding of gene expression within the context of nuclear organization from an inability to characterize hierarchical chromatin organization in situ. Here we demonstrate the use of fluorescence lifetime imaging microscopy (FLIM) to quantify and spatially resolve chromatin condensation state using cell-permeable, DNA-binding dyes (Hoechst 33342 and PicoGreen). Through in vitro and in situ experiments we demonstrate the sensitivity of fluorescence lifetime to condensation state through the mechanical effects that accompany the structural changes and are reflected through altered viscosity. The establishment of FLIM for resolving and quantifying chromatin condensation state opens the door for single-measurement mechanical studies of the nucleus and for characterizing the role of genome structure and organization in nuclear processes that accompany physiological and pathological changes.
Highlights
The structural state of DNA in the nucleus, corresponding to varying levels of chromatin condensation, is integral to its function
The fluorescence lifetime is mostly insensitive to properties of the incident light that lead to the initial excitation such as the exposure time, intensity and wavelength as well as the emission artifacts including fluorophore concentration and photobleaching.[29]
The capacity to spatially resolve chromatin condensation state throughout the nucleus has major bearing on understanding both the functional attributes associated with those states and the dynamics of chromatin throughout the nuclear interior
Summary
The structural state of DNA in the nucleus, corresponding to varying levels of chromatin condensation, is integral to its function. This varying of hierarchical condensation is thought to allow or prevent access of transcription factors to the linear sequence[1,2] while serving as a central feature of nuclear organization.[3] Chromatin states are broadly categorized into heterochromatin and euchromatin, owing to their historical association with the density of their appearance with light[4] or electron microscopy.[5] Heterochromatin is generally associated with highly condensed, gene-poor stretches of chromatin consistent with repression.[6] This. PLOS ONE | DOI:10.1371/journal.pone.0146244 January 14, 2016
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