Multiplexing phasor FLIM of histone FRET with IF. Visualisation of a histone PTM’s spatial distribution throughout intact nuclear architecture by IF requires: (1) cell fixation and (2) incubation with secondary IF antibodies conjugated to fluorescent dyes. Both these sample preparation steps have the potential to disrupt phasor FLIM of histone FRET. Thus, here we first aimed to demonstrate that histone FRET remains a quantitative readout of nanoscale chromatin compaction in fixed cells (Fig. 1a-c), and then identify IF labels that do not interfere with this photophysical phenomenon (Fig. 1d-f). To do so we first transiently transfected HeLa cells with H2B-eGFP (donor) in the absence (donor control) versus presence of H2B-mCH (acceptor) and treated samples containing both donor and acceptor (i.e., histone FRET experiment) with drugs known to loosen (Trichostatin A, TSA) or condense (Actinomycin D, Act D) chromatin (i.e., reduce or increase histone FRET) (Baum et al. 2014; Bensaude 2011) (Fig. 1a). Then in fixed and PBS washed HeLa nuclei expressing the donor control versus histone FRET experiments, we acquired FLIM data in the H2B-eGFP channel where quenching of the donor lifetime in the presence of H2B-mCh reports histone FRET. Quantification of these donor control and histone FRET experiments by the phasor approach to FLIM analysis (Methods) enabled definition of a phasor-based palette that extended from no histone FRET (\(\sim\) 2.5 ns, unquenched H2B-eGFP) to 16% histone FRET (\(\sim\) 2.1 ns, quenched H2B-eGFP) (Fig. 1b), which spatially maps and reports the expected fractional distribution of open (blue pixels) versus compact (orange pixels) chromatin throughout HeLa nuclei treated with TSA or Act D prior to fixation (Fig. 1c).

Phasor FLIM of histone FRET is compatible with cell fixation and IF. a. Fixed HeLa nuclei expressing H2B-eGFP in the absence versus presence of H2B-mCh and chromatin opening (Trichostatin A) versus compacting (Actinoymcin D) drug treatments. b-c. FLIM data recorded in the H2B-eGFP channel of the experiments presented in (a) transformed into a phasor plot (b) alongside maps of histone FRET (c). A theoretical FRET trajectory is superimposed (black curve) over the phasor plot (b) that extends from the unquenched donor lifetime (blue cursor) (2.5 ns). This FRET trajectory enables characterisation of the histone FRET efficiency as 16% (orange cursor) (2.1 ns) and definition of phasor cursors that spatially map no FRET (blue pixel) versus histone FRET (orange pixels) (c). d. Fixed HeLa nuclei expressing H2B-eGFP in the absence versus presence of H2B-mCH and IF against H3K9Me3 labelled with Alexa AF405 or AF647. e. Histone FRET maps derived from phasor analysis of FLIM data recorded in the H2B-eGFP channel of the experiments presented in (d). f. Quantification of the fraction of pixels in the no FRET (open chromatin) versus histone FRET (compact chromatin) state in the maps presented in (e). g. Quantification of the fraction of pixels in the histone FRET state (compact chromatin) across multiple cells in donor only, donor plus H3K9Me3-AF405 (donor + AF405), donor plus H3K9Me3-AF647 (donor + AF647), donor/acceptor plus H3K9Me3-AF405 (donor + Acc. + AF405), and donor/acceptor plus H3K9Me3-AF647 (donor + Acc. + AF647). Scale bars, 5 µm. N ≥ 4 cells, 1 biological replicate. The box and whisker plot in panel g shows the minimum, maximum, and sample median, with ns P > 0.05, ****P < 0.0001 (unpaired t-test)

Upon establishing phasor FLIM of histone FRET as being compatible with cell fixation, we next explored the impact of fluorescent secondary antibodies that are a requirement for IF of the epigenetic landscape, on this assay’s capacity to report chromatin compaction. To do so we first performed IF against H3K9me3 labelled with no secondary antibody versus AF405 (blue, H3K9me3-AF405) or AF647 (far-red, H3K9me3-AF647), in fixed and PBS washed HeLa nuclei expressing H2B-eGFP (green, donor), in the absence versus presence of H2B-mCh (red, acceptor) (Fig. 1d). Then after an additional PBS wash, we acquired FLIM data in the H2B-eGFP channel, and from construction of pseudo-coloured maps of histone FRET (Fig. 1e) that report the fraction of open (no FRET) versus compact (FRET) chromatin (Fig. 1f), we investigated whether H3K9me3-AF405 or H3K9me3-AF647 quench this histone FRET donor’s fluorescence lifetime in the absence versus presence of H2B-mCh via an unwanted FRET interaction. Importantly, from quantitation of the fraction of pixels exhibiting FRET throughout multiple HeLa nuclei, we find neither H3K9Me3-AF405 or H3K9Me3-AF647 significantly quench the fluorescence lifetime of H2B-eGFP in the absence versus presence of H2B-mCh compared to histone FRET (Fig. 1g). Therefore, phasor FLIM of histone FRET can report nucleosome proximity in the presence of IF labelled histone PTMs.

Phasor FLIM of histone FRET faithfully reports the chromatin nanostructure underpinning histone PTMs. In the absence of intact nucleus architecture, advanced methods in genomic sequencing have identified several histone post-translational modifications as being strongly associated with either a compact (e.g., H3K9Me3 and H3K27me3) or open (e.g., H3K4me3 and H3K9Ac) chromatin structure that represses or activates transcription, respectively (Martire and Banaszynski 2020). Thus, here to investigate whether these key histone mark associations are preserved within intact nucleus architecture at the level of nucleosome proximity, we performed phasor FLIM analysis of histone FRET between H2B-eGFP and H2B-mCh co-expressed in fixed HeLa nuclei that have been labelled with IF against either H3K9Me3-AF647, H3K27Me3-AF647, H3K4Me3-AF647 or H3K9Ac-AF647 (Fig. 2a-b). This resulted in construction of pseudo-coloured maps of histone FRET (Fig. 2c), which upon subjection to a region of interest (ROI) analysis derived from an IF intensity mask (Fig. 2d), enabled the fraction of compact chromatin (i.e., histone FRET) associated with the high intensity regions of each histone mark (foci), versus the surrounding nucleoplasm, to be quantified (Fig. 2e). Strikingly, from application of this IF masked analysis of histone FRET to multiple HeLa nuclei we found that indeed the fraction of pixels exhibiting histone FRET inside the markers of ‘compact’ chromatin (H3K9Me3 and H3K27Me3) was statistically higher than the surrounding chromatin environment (Fig. 2f), while the fraction of pixels exhibiting histone FRET inside the markers of ‘open’ chromatin (H3K4Me3 and H3K9Ac) was statistically lower than the surrounding chromatin environment (Fig. 2g). This result can be summarised by normalising the fraction of pixels exhibiting histone FRET inside each epigenetic mark with respect to the surrounding nucleoplasm (i.e., chromatin compactors are > 1 while chromatin openers are < 1) (Fig. 2h). And although this result demonstrates phasor FLIM of histone FRET does faithfully report the expected shift in chromatin nanostructure underpinning key histone PTMs characterised by genome sequencing, the median value and range of the normalised histone FRET fractions calculated (Fig. 2h), also suggest a surprising level of heterogeneity in the chromatin compaction status defined by a histone PTM, at not only the single cell level, but also spatially throughout a single nucleus.

Phasor FLIM of histone FRET reports the nuclear wide chromatin nanostructure imparted by ‘open’ and ‘compact’ histone PTMs labelled by IF. a-b. HeLa nuclei fixed with IF (AF647) against H3K9Me3, H3K27Me3, H3K4Me3, and H3K9Ac (a) that are co-expressing the histone FRET pair H2B-eGFP and H2B-mCH (b). c. FLIM of the cells presented in panels (a)-(b) pseudo-coloured to report histone FRET (orange pixels) versus no-FRET (blue pixels). d. Mask based on H3K9Me3 IF signal presented in (a) (left panel) that selects pixels in the corresponding histone FRET map (c) that are inside (middle panel) versus outside (right panel) of high H3K9Me3 intensity regions. e. Fraction of pixels reporting FRET (compact chromatin) versus no FRET (open chromatin) within masked FLIM maps presented in panel d. f-g. Quantification of the fraction of histone FRET (compact chromatin) inside versus outside of high H3K9Me3, H3K27Me3, H3K4Me3, or H3K9Ac intensity regions. h. The ratio of histone FRET fraction (compact chromatin) inside versus outside of high intensity histone marker regions. Scale bars, 5 µm. N ≥ 15 cells, 3 biological replicates. In f-g **P < 0.01, ***P < 0.001, ****P < 0.0001 (paired t-test). The box and whisker plot in panel h shows the minimum, maximum, and sample median, with ns P > 0.05, *P < 0.05, **P < 0.01 (unpaired t-test).

Phasor FLIM of histone FRET enables spatial heterogeneity in the chromatin nanostructure imparted by a histone PTM to be explored. Phasor FLIM analysis of histone FRET coupled with IF has so far enabled the nuclear wide chromatin nanostructure associated with histone PTMs to be quantified at the single cell level. Next to visualise and investigate the degree of spatial heterogeneity in this chromatin nanostructure at the level of single histone PTM foci, we applied a line profile and particle analysis (Fig. 3) to the phasor FLIM of histone FRET experiments performed in HeLa, which were labelled with IF against H3K9Me3, H3K27Me3, H3K4Me3 and H3K9Ac (Fig. 2). To do this, we overlaid each IF intensity mask of a histone PTM with its corresponding map of histone FRET only (compact chromatin) (Fig. 3a), and then compared histone PTM accumulation with compact chromatin localisation along a line across a selected ROI (Fig. 3b-c). This analysis revealed that H3K9Me3 foci predominantly correlated with compact chromatin and H3K4Me3 foci predominantly anti-correlated with compact chromatin, while H3K27Me3 and H3K9Ac foci co-localised with both compact and open chromatin to an equivalent degree (Fig. 3c). In order to statistically quantify this heterogeneity in histone PTM chromatin nanostructure, we next assigned a unique particle number to each histone PTM focus identified (Fig. 3d, left) and then calculated the normalised fraction of histone FRET within each particle’s area, with respect to the fraction of pixels exhibiting histone FRET throughout the cell nucleoplasm (Fig. 3d, right). When applied to multiple histone PTM foci (Fig. 3e), this analysis confirmed that there is significant heterogeneity in the chromatin nanostructure imparted by histone PTMs classified as chromatin ‘compactors’ (i.e., H3K9Me3 and H3K27Me3) versus chromatin ‘openers’ (i.e., H3K4Me3 and H3K9Ac). For example, although H3K9Ac foci statistically co-localise with chromatin that is more open than the surrounding nucleoplasm, there are H3K9Ac foci that exhibit up to ~ fivefold more histone FRET (compaction) than the nucleoplasm. And in contrast, while H3K27Me3 foci statistically co-localise with chromatin that is more compact than the surrounding nucleoplasm, there are H3K27Me3 foci that exhibit no FRET (open chromatin). Thus, phasor FLIM of histone FRET coupled with IF has the potential to enable study of chromatin foci sub-populations defined by a specific histone PTM.

Phasor FLIM of histone FRET enables visualisation and quantification of the chromatin nanostructure imparted by a histone PTM at the single foci level. a. Intensity images of H3K27Me3, H3K9Me3, H3K4Me3, and H3K9Ac IF (magenta) merged with their corresponding binary chromatin compaction maps (yellow), which were generated via the workflow presented in Fig. 2. b. Zoomed in images of ROIs selected from the merged images presented in (a) (white square). c. Intensity of H3K27Me3, H3K9Me3, H3K4Me3, and H3K9Ac accumulation with respect to compact chromatin localisation along the central line within the selected ROIs in (b). d. An example ROI with individual histone PTM foci indexed (left) and the corresponding binary FRET map indexed based on foci detection (right). e. Quantification of the FRET pixel fraction in individual epigenetic foci normalized against FRET pixel fraction in the whole nucleus across multiple nuclei. Scale bars, 5 µm. N > 1000 foci from ≥ 10 cells, 3 biological replicates). The violin plot in panel e shows the sample median, 25% percentile and 75% percentile, with ns P > 0.05, *P < 0.05, **P < 0.01 (one way ANOVA)

Phasor FLIM of histone FRET enables the interplay between the epigenetic landscape, chromatin nanostructure and transcription to be visualised within an intact nucleus. RNAP2 transcribes all protein-coding genes in eukaryotic genomes and therefore is a good biomarker of where inside an intact nucleus transcription is occurring. Thus, to demonstrate how phasor FLIM of histone FRET multiplexed with IF can directly probe the interplay between histone PTMs known to associate with compact versus open chromatin structure and transcriptional activity, here in fixed HeLa co-expressing H2B-eGFP and H2B-mCh, we performed IF against RNAP2-AF647 alongside H3K9Me3-AF405 (Fig. 4a-f) versus H3K4me3-AF405 (Fig. 4g-l). As expected, H3K9Me3-AF405 that is a marker of gene repression, anti-colocalises with RNAP2-AF647 (Fig. 4c) and is associated with a significantly higher fraction of histone FRET than this biomarker of transcription, as well as the surrounding nucleoplasm (Fig. 4d-f and Fig. 4m). While in direct contrast, H3K4me3-AF405 that is a marker of gene activation, co-localises with RNAP2-AF647 (Fig. 4i) and is associated with a significantly lower fraction of histone FRET than this biomarker of transcription as well as the surrounding nucleoplasm (Fig. 4j-l and Fig. 4n). Thus collectively, this result (Fig. 4m-n) demonstrates that transcription does indeed prefer an open chromatin environment underpinned by an increase in nucleosome spacing, and this chromatin nanostructure is defined by an epigenetic landscape within an intact nucleus that agrees with genomic sequencing data. Thus, phasor FLIM of histone FRET multiplexed with IF against RNAP2 and histone PTMs offers an opportunity for cell biologists to investigate the chromatin nanostructure of more ambiguous histone post-translational modifications, with the added advantage of sensitivity toward spatial as well as cell to cell heterogeneity.

RNAP2 is associated with open chromatin. a-b. HeLa nucleus co-expressing the histone FRET pair H2B-eGFP and H2B-mCH that has been fixed with IF against H3K9Me3 and RNAP2 (a) and a ROI (b) selected for line and particle analysis. c. Line profile from the ROI in (b) showing H3K9me3 and RNAP2 anti-colocalise. d-e. FLIM of the cell presented in panel (a)-(b) pseudo-coloured to report histone FRET (orange pixels) versus non-FRET (blue pixels) (d) with a mask applied to the ROI (e) that associates with H3K9me3 versus RNAP2 intensity. f. Fraction of pixels reporting FRET (compact chromatin) in high H3K9Me3 intensity region, versus whole nucleus and high RNAP2 intensity region. g-h. HeLa nucleus co-expressing the histone FRET pair H2B-eGFP and H2B-mCH that has been fixed with IF against H3K4Me3 and RNAP2 (g) and a ROI (h) selected for line and particle analysis. i. Line profile from the ROI in (h) showing H3K4me3 and RNAP2 colocalise. j-k. FLIM of the cell presented in panel (g)-(h) pseudo-coloured to report histone FRET (orange pixels) versus non-FRET (blue pixels) (j) with a mask applied to the ROI (k) that associates with H3K4me3 versus RNAP2 intensity. l. Fraction of pixels reporting FRET (compact chromatin) in high H3K4Me3 intensity region, versus whole nucleus and high RNAP2 intensity region. m–n. Quantification of the fraction of histone FRET (compact chromatin) inside high H3K9Me3, total nucleus, and high RNAP2 intensity regions (m), versus inside high H3K4Me3, total nucleus, and high RNAP2 intensity regions (n) across multiple nuclei. Scale bars, 5 µm. N ≥ 10 cells, 2 biological replicates. **P < 0.01, ****P < 0.0001 (paired t-test)