Phasor histone FLIM-FRET microscopy quantifies spatiotemporal rearrangement of chromatin architecture during the DNA damage response

Lou, Jieqiong; Scipioni, Lorenzo; Wright, Belinda K.; Bartolec, Tara K.; Zhang, Jessie; Masamsetti, V. Pragathi; Gaus, Katharina; Gratton, Enrico; Cesare, Anthony J.; Hinde, Elizabeth

doi:10.1073/pnas.1814965116

Cited by 62 publications

(68 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Importantly, FLIM-FRET also provides accurate quantification due to the independence of the fluorescence lifetime from the relative concentrations of the interacting proteins, and is independent of their diffusion rates (Llères et al, 2007). In conclusion, our assay measures close contacts in the nuclear space between distant nucleosomes, and thus provides a read-out of nanoscale chromatin compaction (Lou et al, 2019;Sobecki et al, 2016;Baarlink et al, 2017;Wang et al, 2019a).…”

Section: Nanoscale Chromatin Compaction Is Decreased In Set-2 Mutant mentioning

confidence: 73%

Cooperation between Caenorhabditis elegansCOMPASS and condensin in germline chromatin organization

Herbette

Robert

Bailly

et al. 2020

Preprint

View full text Add to dashboard Cite

Deposition of histone H3 lysine 4 (H3K4) methylation at promoters by SET1/COMPASS is associated with context-dependent effects on gene expression and local changes in chromatin organization.Whether SET1/COMPASS also contributes to higher-order chromosome structure has not been investigated. Here, we address this question by quantitative FRET (Förster resonance energy transfer)based fluorescence lifetime imaging microscopy (FLIM) on C. elegans germ cells expressing histones H2B-eGFP and H2B-mCherry. We find that SET1/COMPASS subunits strongly influence meiotic chromosome organization, with marked effects on the close proximity between nucleosomes. We further show that inactivation of SET-2, the C. elegans homologue of SET1, or CFP-1, the chromatin targeting subunit of COMPASS, strongly enhance chromosome organization defects and loss of fertility resulting from depletion of condensin-II. Defects in chromosome morphology resulting from conditional inactivation of topoisomerase II, another structural component of chromosomes, were also aggravated in the absence of SET-2. Combined, our in vivo findings suggest a model in which the SET1/COMPASS histone methyltransferase complex plays a role in shaping meiotic chromosome in cooperation with the non-histone proteins condensin-II and topoisomerase.

show abstract

Section: Nanoscale Chromatin Compaction Is Decreased In Set-2 Mutant mentioning

confidence: 73%

Cooperation between Caenorhabditis elegansCOMPASS and condensin in germline chromatin organization

Herbette

Robert

Bailly

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…Our estimation of the average interaction distance might be inaccurate, as it does not rely on a robust calibration protocol. In this respect, we believe it would be interesting to perform a similar analysis on the histone‐based FRET assays where the use of fluorescent proteins would allow a more robust calibration with constructs of known stoichiometry and a direct estimation of the average nanometer distance between labeled histones.…”

Section: Discussionmentioning

confidence: 99%

“…This FRET assay has been used to reveal distinct domains and quantitatively discriminate different levels of nanoscale chromatin compaction in live HeLa cells and in living Caenorhabditis elegans as a model system . More recently, the same FRET assay has been applied to measure chromatin organization in live cells in combination with the phasor analysis of FLIM . Coupling this technology with laser microirradiation allowed identifying the DDR‐dependent chromatin architectural changes that occur in response to DNA double‐strand breaks (DSBs) .…”

Section: Introductionmentioning

confidence: 99%

“…More recently, the same FRET assay has been applied to measure chromatin organization in live cells in combination with the phasor analysis of FLIM . Coupling this technology with laser microirradiation allowed identifying the DDR‐dependent chromatin architectural changes that occur in response to DNA double‐strand breaks (DSBs) . All these works clearly show that FRET can be a powerful tool to map nanoscale chromatin compaction in vivo.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Chromatin nanoscale compaction in live cells visualized by acceptor‐to‐donor ratio corrected Förster resonance energy transfer between DNA dyes

Pelicci

Diaspro

Lanzanò

2019

Journal of Biophotonics

View full text Add to dashboard Cite

@Chromatin nanoscale architecture in live cells can be studied by Förster resonance energy transfer (FRET) between fluorescently labeled chromatin components, such as histones. A higher degree of nanoscale compaction is detected as a higher FRET level, since this corresponds to a higher degree of proximity between donor and acceptor molecules. However, in such a system, the stoichiometry of the donors and acceptors engaged in the FRET process is not well defined and, in principle, FRET variations could be caused by variations in the acceptor‐to‐donor ratio rather than distance. Here, to get a FRET level independent of the acceptor‐to‐donor ratio, we combine fluorescence lifetime imaging detection of FRET with a normalization of the FRET level to a pixel‐wise estimation of the acceptor‐to‐donor ratio. We use this method to study FRET between two DNA binding dyes staining the nuclei of live cells. We show that this acceptor‐to‐donor ratio corrected FRET imaging reveals variations of nanoscale compaction in different chromatin environments. As an application, we monitor the rearrangement of chromatin in response to laser‐induced microirradiation and reveal that DNA is rapidly decompacted, at the nanoscale, in response to DNA damage induction.

show abstract

“…To interrogate the chromatin network organization within an intact nucleus and define the role this structural framework plays in nuclear factor navigation, two perspectives of investigation have emerged. The first approach is to directly study chromatin organization using electron microscopy [12], super-resolution optical microscopy [13][14][15], histone FRET microscopy [16][17][18][19], or techniques based on high throughput identification of DNA sequences that are in spatial proximity to one another in the nucleus [20][21][22][23][24]. The second perspective is to indirectly probe chromatin organization from the point of view of the molecules exploring this complex environment.…”

Section: Introductionmentioning

confidence: 99%

Fluorescence fluctuation spectroscopy: an invaluable microscopy tool for uncovering the biophysical rules for navigating the nuclear landscape

Priest

Solano

Lou

et al. 2019

Biochemical Society Transactions

Self Cite

View full text Add to dashboard Cite

Nuclear architecture is fundamental to the manner by which molecules traverse the nucleus. The nucleoplasm is a crowded environment where dynamic rearrangements in local chromatin compaction locally redefine the space accessible toward nuclear protein diffusion. Here, we review a suite of methods based on fluorescence fluctuation spectroscopy (FFS) and how they have been employed to interrogate chromatin organization, as well as the impact this structural framework has on nuclear protein target search. From first focusing on a set of studies that apply FFS to an inert fluorescent tracer diffusing inside the nucleus of a living cell, we demonstrate the capacity of this technology to measure the accessibility of the nucleoplasm. Then with a baseline understanding of the exploration volume available to nuclear proteins during target search, we review direct applications of FFS to fluorescently labeled transcription factors (TFs). FFS can detect changes in TF mobility due to DNA binding, as well as the formation of TF complexes via changes in brightness due to oligomerization. Collectively, we find that FFS-based methods can uncover how nuclear proteins in general navigate the nuclear landscape.

show abstract

Phasor histone FLIM-FRET microscopy quantifies spatiotemporal rearrangement of chromatin architecture during the DNA damage response

Cited by 62 publications

References 52 publications

Cooperation between Caenorhabditis elegansCOMPASS and condensin in germline chromatin organization

Cooperation between Caenorhabditis elegansCOMPASS and condensin in germline chromatin organization

Chromatin nanoscale compaction in live cells visualized by acceptor‐to‐donor ratio corrected Förster resonance energy transfer between DNA dyes

Fluorescence fluctuation spectroscopy: an invaluable microscopy tool for uncovering the biophysical rules for navigating the nuclear landscape

Contact Info

Product

Resources

About