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Super-Resolution Imaging of Individual Human Sub-Chromosomal Regions In Situ Reveals Nanoscopic Building Blocks of Higher-Order Structure Ke Fang, Xuecheng Chen, Xiaowei Li, Yi Shen, Jielin Sun, Daniel M. Czajkowsky, and Zhifeng Shao ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b01963 • Publication Date (Web): 01 May 2018 Downloaded from http://pubs.acs.org on May 1, 2018

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Super-Resolution Imaging of Individual Human Sub-Chromosomal Regions In Situ Reveals Nanoscopic Building Blocks of Higher-Order Structure

Ke Fang, † Xuecheng Chen, † Xiaowei Li, ‡ Yi Shen, ‡ Jielin Sun, † Daniel M. Czajkowsky,*, ‡ and Zhifeng Shao*, ‡ †

Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems

Biomedicine, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China ‡

State Key Laboratory for Oncogenes and Bio-ID Center, School of Biomedical Engineering,

Shanghai Jiao Tong University, Shanghai 200240, China

*To whom correspondence may be addressed: [email protected] or [email protected]

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ABSTRACT

It is widely recognized that the higher-order spatial organization of the genome, beyond the nucleosome, plays an important role in many biological processes. However, to date, direct information of even such fundamental structural details as the typical sizes and DNA content of these higher-order structures in situ is poorly characterized. Here, we examine the nanoscopic DNA organization within human nuclei using super-resolution direct stochastic optical reconstruction microscopy (dSTORM) imaging and 5-ethynyl-2’-deoxyuridine (EdU) click chemistry, studying single fully-labeled chromosomes within an otherwise unlabeled nuclei to improve the attainable resolution. We find that, regardless of nuclear position, individual subchromosomal regions consist of three different levels of DNA compaction: (i) dispersed chromatin; (ii) nanodomains of sizes ranging tens of nanometers containing a few kilobases (kb) of DNA; and (iii) clusters of nanodomains. Interestingly, the sizes and DNA content of the nanodomains are approximately the same at the nuclear periphery, nucleolar proximity, and nuclear interior, suggesting that these nanodomains share a roughly common higher-order architecture. Overall, these results suggest that DNA compaction within the eukaryote nucleus occurs via the condensation of DNA into few-kb nanodomains of approximately similar structure, with further compaction occurring via the clustering of nanodomains.

Keywords: click chemistry, DNA density, genome structure, dSTORM, super-resolution, cluster analysis


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One of the hallmark features of the spatial organization of chromatin in the eukaryotic nucleus is a pronounced heterogeneity in DNA density across the nucleus, most notably between peripheral and interior regions of the nucleus.1-4 In particular, in almost all cells that have been studied to date, the DNA density in the former has been found to be 3 to 4 times higher than that in the latter.5, 6 A difference in DNA density has also been observed between nucleolar-proximal and nucleolar-distal (but still nuclear interior) chromatin, with the former exhibiting a similar density as in the nuclear periphery.7 These differences in local DNA density clearly reflect differences in the local folding of the chromatin, and thus are expected to play important roles in many fundamental nuclear processes.8-14 Yet, despite their importance, many details of the physical attributes of the chromatin condensates in the nucleus, including such basic information as their size range and local DNA content, are still not well known. Such information is critical for an understanding of the overall architecture of these higher-order structures as well as the mechanisms by which they assemble and disassemble in response to biological cues.15-17 Resolving this information ideally requires experimental techniques that can directly visualize the DNA with nanoscopic resolution. A recent electron microscopy-based method, ChromEMT, provided spectacular nanometer-scale images of the eukaryotic interphase chromatin structure, revealing differences in local chromatin density nucleus-wide.18 However, it is presently not known how to quantitatively relate the observed contrast differences in this method to differences in the DNA density.19 As an alternative, optical super-resolution microscopy has recently emerged as a highly effective methodology to resolve biological complexes down to ~20 to 30 nm resolution.20-24 As such, it has been applied to determine the structure of genomic regions in the nucleus,25-28 including the entire nuclear genome using DNA-binding dyes.25, 28


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However, these studies did not resolve finer details in the nucleus, perhaps owing to significant background from out-of-focus fluorophores. Also, quantification of the DNA content of localized regions using such DNA-binding dyes can be challenging, owing to the dynamic nature of the binding process that is sensitive to the local environment28,

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Still, the use of super-

resolution optical methods to characterize the distribution of nuclear histones has shown the power of these techniques to reveal higher resolution details of chromatin structure,30-32 although similar, high-resolution data of the DNA organization itself is ultimately necessary to provide the most direct measure of the local DNA density distribution within the nucleus. We recognized that click chemistry coupled with the incorporation of EdU,33, 34 which had previously been used to image replication foci and whole genomes with super-resolution microscopy,28, 35, 36 could be a useful strategy to effectively quantify nuclear DNA density in super-resolution imaging. Click chemistry is a highly specific reaction that occurs between azide and alkyne-bearing moieties that can occur under biologically relevant conditions.34 However, imaging the fully labeled genome by this method to the highest possible resolution is complicated by the significant aforementioned out-of-focus fluorescence. Here we use super-resolution, direct stochastic optical reconstruction microscopy (dSTORM)21,

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combined with click chemistry of EdU-incorporated DNA to investigate the

higher order organization of sub-chromosomal regions of single fully-labeled chromosomes within an otherwise unlabeled nucleus. In this way, we show a substantial decrease in background noise and an increase in attainable resolution, from >80 nm with the fully-labeled nucleus down to ~20 nm with the single labeled chromosome per nucleus. Surprisingly, we find that, regardless of their location within the nucleus, the sub-chromosomal regions consist of three different types of structure: dispersed chromatin, well-defined nanodomains containing several


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kilobases (kb) of DNA whose spatial extent ranges over tens of nanometers, and clusters of these nanodomains, with only the proportion of each type differing between the different nuclear locations. Interestingly, we find that there is a single dominant DNA density for the nanodomains within each region. We speculate that these nanodomains are the building blocks of higher-order chromosome structure in the mammalian nucleus, in where DNA, regardless of nuclear location, is compacted by the aggregation of DNA into nanodomains, with further condensation occurring via the assembly of nanodomains into clusters.

Results and Discussion High resolution dSTORM imaging of the nuclear genome To determine the spatial distribution of genomic DNA within the eukaryotic nucleus, we employed super-resolution dSTORM optical microscopy37 to image Alexa Fluor 647-labeled EdU within chromosomes of human fibroblasts. To ensure continuous and full incorporation of EdU into chromosomes, EdU was added to G1 phase cells (two hours before the start of S phase) that were synchronized using serum starvation (Figure S1 and Figure S2).38 Following progression through S phase, we collected cells in the following G1 phase, and, in all experiments, examined the genomic structure of these G1-phase nuclei. We also optimized the click-chemistry labeling reaction (see Materials and Methods), finally achieving an Alexa Fluor 647 density of 1 fluorophore every 315±79 bp (Figure S3A,B, Table 1).

Figure 1A shows a typical dSTORM image of the full genome-labeled nuclei. Such images generally agree with previous super-resolution studies that employed DNA-incorporated dyes.4,


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However, while these images reveal finer features than conventional optical microscopy (see

inset to Figure 1A), they nonetheless exhibit a fuzziness that is particularly apparent in zoomedin regions (Figure 1B). This fuzziness is in stark contrast to the sharply defined features previously observed of other biological molecules within cells using STORM.39,

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Indeed,

Fourier Ring Correlation (FRC) analysis revealed that the characteristic spatial frequency across these images, defined conventionally as the frequency associated with a FRC value of 1/7 (see Materials and Methods), is only 0.0086 ± 0.0022 nm-1 (n=10) (Figure 1C,F), which is four to five-fold lower than is typically possible with STORM imaging.41 We reasoned that, with such a highly labeled whole nucleus, a high background noise resulting from out-of-focus fluorescence could have significantly limited the localization accuracy and thus lower the final resolution.42 Consistent with this, we found that the background noise determined from the fitting of the single localizations in these nuclei (see Materials and Methods) is significantly greater than that from single fluorophores adsorbed on glass (Figure S4). As a result, we sought to prepare a different sample that would significantly reduce this effect. In particular, we developed a procedure to prepare samples in which there is only a single labeled chromosome within an otherwise unlabeled nucleus (Figure S1). Namely, following the labeling of cells for one complete S phase, we washed the cells with EdU-free media, and allowed them to grow for seven additional generations in EdU-free media, which ensured a very low probability (0.05) of there being more than one labeled chromosome in the same nucleus. dSTORM imaging of single labeled chromosomes in such nuclei revealed much more sharply defined fine features than in the fully labeled nuclei (Figure 1D,E). FRC analysis demonstrated a


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nearly four-fold improvement in the characteristic frequency across these images (0.035 ± 0.016 nm-1) compared with that of the fully labeled genome (Figure 1C,F). Further, examination of the background noise in these images revealed roughly similar values to that of single fluorophores on glass (Figure S4B). An analysis of the full width at half maximum (FWHM) of the localizations of several regions revealed a resolution of ~20 nm in these images (Figure S5).26, 43 These higher resolution images enabled identification of otherwise undetectable details in the original fully labeled genome images.

Structural hierarchy within individual chromosomes To identify chromosomal regions at each nuclear position, we used Hoechst 33342 to label the whole nucleus and an anti-Nucleolin antibody to label the nucleolus.44 Further, for all measurements, we limited our study to the central mid-plane of the nucleus and to the most intense region of the chromosomes located therein. Examination of the relationship between the localization standard deviation and focal depth from single Alexa Fluor 647 fluorophores on glass (Figure S6) provided an estimate of the imaging focal depth of these single fully-labeled chromosomes of ~400 nm. Thus, these images reflect the spatial distribution of the DNA density of a sub-chromosomal ~400 nm-thick slice through single labeled chromosomes. In total, we analyzed 20 STORM images for each sub-chromosomal region near the nuclear periphery (NuP), proximal to the nucleolus (NoP), and within the nuclear interior (INT) (Figure 2 and Figure S7). Overall, we found that the DNA within each region consists of several regions of discrete, spatially resolved higher density DNA that span tens of nanometers as well as extended regions containing substantially less dense DNA (Figure 2). To obtain a quantitative


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understanding of these nanodomains, we employed a clustering algorithm that groups the localizations in these images based on their spatial proximity (see Materials and Methods), similar to that used in an earlier STORM study31 (Figure 2). In this way, we found that all of the sub-chromosomal regions consist of three levels of structure: dispersed chromatin with lower DNA density, nanodomains of more condensed DNA whose spatial extent is generally tens of nanometers, and clusters of these nanodomains. Notably, we find regions in the nuclear periphery and the nucleolar proximity, which are generally thought to be associated only with highly condensed DNA,1-4 that clearly contain significant amounts of less compact DNA (Figure 2). Likewise, within the nuclear interior, where the chromatin is generally believed to be open, there are clearly condensed DNA nanodomains and clusters of nanodomains (Figure 2).

Quantitative comparison of the sub-chromosomal regions located at different nuclear locations Surprisingly, we found that the areal extent of the nanodomains from each nuclear location spans across a similar range, and in fact exhibits a similar size distribution, including the magnitude of the most common size (Figure 3A). Thus, while the local protein composition associated with these specific nuclear regions is expected to be different,1, 12, 45, 46 the sizes of the condensed DNA nanodomains are nonetheless roughly the same. Interestingly, the number of localizations per nanodomain (roughly 20 to 1200) is similar at each nuclear location. To express this in terms of DNA content, we sought a means to convert


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the number of localizations per nanodomain to the amount of DNA.47, 48 To this end, we purified 1 kb DNA fragments from EdU-incorporated genomic DNA and examined well-isolated fragments under identical imaging conditions as our in vivo experiments. In this way, we found 6.77 ± 1.58 localizations per kb DNA (Figure S2B,C). We obtained a similar value when we examined 2 kb DNA fragments (Figure S2B,C), as well as from an analysis of the number of localizations of single Alexa Fluor 647 molecules and the measured density of Alexa Fluor 647 in our genomic sample (Figure S2A). With this, we found that the amount of DNA per nanodomain ranges mainly from 1 to 10 kb at each nuclear position (Figure 3B), and in fact also exhibits a similar size distribution (Figure 3A) as does the DNA density (Figure 3C). Thus, these observations show that the nanodomains at each of these nuclear positions are generally of a similar size and DNA content/density. This is also the case when considering the sizes and DNA content/densities of isolated single nanodomains or just those nanodomains within clusters that contain two or more nanodomains (Figure S8). However, we found a significant difference in the areal extent of the dispersed chromatin or nanodomain/clusters within a given sub-chromosomal region at different nuclear positions (Figure 4). In particular, while the total areal fraction of the nanodomain/clusters within the nuclear periphery and nucleolar proximity are similar, they are both roughly 3-fold larger than that observed in the nuclear interior. Likewise, the areal fraction of the dispersed, lower DNA density region is 1.6-fold greater for chromosomal regions within the nuclear interior than that at the nuclear periphery/nucleolar proximity (Figure 4). We note that, combining the differences in DNA density between the dispersed regions and the nanodomains together with the differences in areal extent between the disordered and


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nanodomain/clusters, we find a 2.6-fold difference in total DNA density between the nuclear periphery and interior regions (Table 2). This difference in DNA density is similar to values obtained with previous, lower-resolution techniques.28 Thus our findings provide a more detailed view of the higher-order structures underlying this long-observed difference in DNA density across the eukaryotic nucleus.

Implication for the mechanisms of DNA compaction in the eukaryotic nucleus The data presented here provide the highest resolution images of the higher-order organization of the DNA density within the eukaryotic nuclei to date. We observe well-resolved DNA nanodomains generally containing a few kb DNA whose size spans tens of nanometers, as well as clusters of these nanodomains, regardless of nuclear location. Importantly, though, neither the nanodomains nor clusters exhibit a spatial extent predominantly of 30 nm or 120 nm (Figure 3 and Figure S9), sizes which have been previously suggested to underlie higher-order folding of chromatin in eukaryotes49-54 but which have also been discounted in other recent studies.18, 32, 55 Moreover, the DNA density distribution of these nanodomains and clusters is also not consistent with a predominance of 30 nm or 120 nm structures (Figure 3 and Figure S10). In fact, our results also discredit other potential models of chromatin condensation in the nucleus (Figure S10). Interestingly though, the single-peak distribution in both size and DNA density of these nanodomains (Figure 3 and Figure S8) is consistent with a recently proposed “liquid phase transition” model for heterochromatin that predicts a single dominant DNA density that is a consequence of the threshold protein density required for protein condensation.56 In this scenario, the nanodomains that we observe may then be associated with the smallest droplet size that can


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form and the clusters of nanodomains would be associated with the fusion of these minimal droplets. Further work is needed to test this intriguing possibility. However, it is also possible that the nanodomains form by different mechanisms than just those associated with heterochromatin formation. This is suggested by the observation of extensive nanodomains within the nuclear interior, which are generally enriched for transcriptionally active chromatin. The presence of clusters of nanodomains indicates that there is no intrinsic property of the nucleus that would limit the prospective size of a DNA condensate to that observed of the nanodomains. Thus, we speculate that the common size and DNA density of the nanodomains in all nuclear locations indicates that the structures of the nanodomains are roughly the same. This could occur, for example, if the condensing agents are, in general, centrally located within the nanodomains and the associated nucleosomal fibers are localized on the periphery, as such an arrangement would produce structures of a limited size and of a limited (and similar) DNA density (Figure S11). In this light, we note that such a structural arrangement is remarkably similar to a recent proposal of transcriptional control by enhances and super-enhancers resulting from the phaseseparation (akin to liquid droplet condensation) of enhancer binding proteins.57 Thus, it may be that the nanodomains we observe here reflect a general architecture of DNA-binding proteins, whether heterochromatic or active regulators of transcription, that undergo phase-separation into localized condensates as a means of performing their functions. Our data also suggest that further condensation of the DNA occurs via the subsequent assembly of the nanodomains into clusters. In support of this, we find that the size and DNA densities of the isolated single nanodomains are the same as of those nanodomains within


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clusters (Figure S8). Thus, our results point to the possibility of a two-stage hierarchical process of maximal compaction of DNA: first, the condensation into nanodomains and then second, the assembly of nanodomains into clusters. This second level of assembly (unlike the first) might occur with a condensing agent that associates on the periphery of each nanodomain, which could thus occur indefinitely (unlike the formation of nanodomains), without significant change in DNA density from the nanodomains (Figure S11). Likewise, we speculate that there are processes that would involve the disassembly of nanodomains from clusters, while maintaining the nanodomain architecture, as well as those that involve the complete dissolution of the nanodomains into the dispersed chromatin. These nanodomains, of only a few kb in size, are much smaller than the well-studied topologically associated domains (TADs) that have been characterized in mammalian cells (median size, 185 kb).58 Interestingly, though, many of the clusters of nanodomains are roughly of this TAD size. Thus, we speculate that some of the nanodomain clusters correspond to these TADs, while the nanodomains constitute sub-structures that can self-associate with other nanodomains to form a particular TAD. Consistent with this, a recent study using structured illumination microscopy (SIM) of specifically designed probes in Drosophila cells revealed that TADs (~100 to 200 kb) adopt physically-connected “nanocompartments” that are similar in size to the clusters of nanodomains described in the present work.59 Finally, we note that our results generally agree with previous super-resolution work that described the nuclear distribution of labeled nucleosomes.18, 32, 53 In particular, a recent live-cell study using photoactivated localization microscopy identified clustered domains of approximately 160 nm in diameter.32 Although this size was inferred from a less direct method than that described in our work, the size of many of the nanodomain clusters described here are


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of this magnitude (Figure S9). Thus, our results may provide a higher-resolution image of the underlying structure described in this previous work. In addition, an earlier work using STORM identified nanodomains of nucleosomes of a similar size to the DNA nanodomains we observe.31 Future work that would show whether these two nanodomain structures are indeed precisely the same structure would enable a greater understanding of the underlying structure of these building blocks of higher-order genomic structure.

Conclusion In conclusion, we have identified a simple experimental strategy to significantly improve the resolution of dSTORM images of EdU-incorporated chromosomes, enabling the highest resolution view to date of the DNA density across the eukaryotic nucleus. We find that all subchromosomal regions studied consist of three levels of DNA compaction, from low-density dispersed chromatin to few-kb nanodomains to clusters of nanodomains. This work sets the stage for further studies with DNA-binding proteins, including those nucleosomal as well as those nonnucleosomal, and studies of different cell states that can be directly correlated with the changes in the higher-order structures described herein. Only with such a quantified, integrated view of the entire genome architecture can we begin to fully understand its role in the myriad biological processes in which it has heretofore been implicated.

Materials and Methods Cell culture, synchronization, and EdU incorporation


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Human skin fibroblasts (BJ, ATCC CRL-2522) were cultured in DMEM (10-013-CVR, Corning) supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco) at 37 °C in 5% CO2. For serum starvation, cells were cultured in low FBS medium (DMEM with 0.5% FBS and 1% penicillin/streptomycin) for 48 h. The medium was then replaced with normal growth medium, in which the cells were allowed to grow for another 8 h at which point most of the cells were in G1 phase, based on flow cytometry (Figure S2). Flow cytometry (LSRFortessa, BD Biosciences) was performed with cells harvested at 4 to 22 h (at 2h intervals) after release from serum starvation conditions. The harvested cells were fixed with 70% methanol at -20 °C for 6 h, washed twice with 1× PBS, and stained with 0.1 mg/mL Propidium Iodide (P4864, SigmaAldrich) for 30 min before flow cytometry. The G1 phase cells were then cultured in normal growth medium with 20 µM F-ara-EdU (Sigma-Aldrich, and a kind gift from Prof. Dr. Nathan W. Luedtke, that exhibits minimal cytotoxicity60) for 12 h to ensure that all of the cells proceed through S phase. The cells were then washed twice with 1× PBS and cultured in normal growth medium without F-ara-EdU. We note that we refer to this EdU derivative in this manuscript as “EdU”. Measurement of EdU-labeling efficiency in situ by the click reaction After EdU incorporation, the cells were divided into two groups. Cells from group A were harvested using Trypsin-EDTA, and then fixed and permeabilized for 10 minutes by a mixture of 4% paraformaldehyde (15812-7, Sigma-Aldrich) and 0.5% Triton X-100 in PBS. The click reaction kit (C10340, Thermo Fisher Scientific) was used to label the nuclei with Alexa Fluor 647. The labeled DNA was then purified using QIAamp DNA Mini Kit (51304, QIAGEN). For group B, the cells were harvested, the DNA was purified with the QIAamp DNA Mini Kit, and


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then the sample was treated with the click reaction kit for 2h. Free fluorophores were removed from the sample by phenol chloroform extraction and ethanol precipitation. Finally, to normalize the DNA concentration of the two groups, DNA was stained with YOYO-1 (Y3601, Thermo Fisher Scientific) at the ratio of 0.3 nmol dyes to 1 µg DNA. All reactions were performed at room temperature. The relative fluorescent units (RFU) of Alexa Fluor 647 and YOYO-1 in the DNA solution were measured using spectrophotometry (NanoDrop 3300, Thermo Scientific). We assumed that the click reaction could achieve full labeling on purified DNA, and so the EdU-labeling efficiency in situ by click reaction was calculated by comparing the ratio of RFUAlexa Fluor 647 to RFUYOYO-1 from the two groups. Preparation of samples for STORM Cells were cultured on 29 mm glass bottom dishes (D29-10-1.5-N, In Vitro Scientific) before STORM imaging. For the preparation of fully labeled nuclei, the cells were used directly after the EdU incorporation. For the single chromosome-labeled samples, the cells after EdU incorporation were grown for an additional seven generations (~5 days) and synchronized at G1 phase using serum starvation as described above. For DNA labeling, the cells were fixed and permeabilized as described above and the free aldehyde was quenched with 100 mM glycine for 30 min. This protocol has been shown to preserve the chromatin structure in previous superresolution studies.26, 31 We then added 10% BSA (BAH 68, Equitech-Bio) in PBS to block the samples for 30 min, followed by incubation in click reaction buffer for 12 h. For immunostaining the nucleoli, samples were incubated for 2 h with the primary antibody anti-Nucleolin Rabbit mAb (14574, Cell Signaling Technology) at 1:800 dilution in PBST (PBS and 0.05% Tween 20)


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and for 30 min with the secondary antibody Alexa Fluor 488 goat anti-rabbit Ig G (A11034, Thermo Fisher Scientific). Finally, the samples were counterstained with 5 µg/mL Hoechst 33342 (H3570, Thermo Fisher Scientific) and 100 nm TetraSpeck Microspheres (T7279, Thermo Fisher Scientific) were added for drift correction. Repeated washing with PBST was done at every step. For single fluorophore imaging, we attached Alexa Fluor 647-conjugated streptavidin (S21374, Thermo Fisher Scientific) to biotin-coated coverslips (CS-BN-5, Nanocs). On average each streptavidin has 3 covalently-attached fluorophores according to the manufacturer. The streptavidin solution (200 ng/ml) was added to the coverslips, allowed to incubate for 15 min, and then washed with PBS. STORM imaging To ensure accurate quantification of the DNA density, precisely the same imaging conditions were followed for all samples. The imaging buffer was a fresh-prepared solution of 10% w/v Glucose (49159, Sigma-Aldrich), 0.5 mg /mL glucose oxidase (G2133, Sigma-Aldrich), 53 µg /mL catalase (C3556, Sigma-Aldrich), 10 mM Cysteamine (M9768, Sigma-Aldrich) and 2 mM cyclooctatetraene (COT) (C0505, Tokyo Chemical Industry)61 in TN buffer (50 mM Tris and 10 mM NaCl, pH 8.0). A commercial microscope system from Nikon Instruments (N-STORM) was used for all imaging experiments. The samples were imaged under the highly inclined illumination of a 50 mW 647 nm laser through a 100x objective lens (PlanApo TIRF, NA 1.49; Nikon). All images were recorded onto a 128 × 128 pixel region of an electron-multiplying charge coupled device (EMCCD) camera (iXon 3; Andor). The exposure time was set at 20 ms,


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while the EM gain and conversion gain were set at 200 × and 1 ×, respectively. The temperature of the imaging environment was controlled at 23 °C. For the estimation of the imaging focal depth, Alexa Fluor 647- conjugated streptavidin bound to the biotinylated coverslips were imaged on a Piezo stage (Mad City Labs) at 10 nm/frame (z direction). The standard deviation (σ) of the fitted Gaussian PSF relative to the z position of fluorophores was measured. Based on an analysis of the number of false-positives and falsenegatives from this curve, localizations in the experiments with the chromosomes were used if their σ value was within 150 to 160 nm to constrain the focal depth to ~400 nm (Figure S6). STORM data analysis All STORM images were analyzed using the ImageJ plugin ThunderSTORM.62 Quantitative information for each localization, including the x- and y-position, standard deviation (σ), background noise, and localization precision, is automatically determined by this procedure. The final images are rendered by representing each x-y localization position as a Gaussian with a width that corresponds to the localization precision (11 nm and 15 nm for the images of the single labeled chromosomes and fully labeled nuclei, respectively (Figure S4)). Sample drift during image acquisition was corrected by tracking fluorescent microspheres (T7279, Thermo Fisher Scientific). The sub-chromosomal regions at the nuclear periphery and at the nucleolar boundary were identified based on distances determined from fully labeled nuclei. Cross-sectional intensity profiles in ImageJ were obtained from these images, which revealed a mean full width at half maximum of 260 ± 45 nm and 289 ± 45 nm (from 10 cells, making 20 measurements per cell) for the nuclear periphery and nucleolar boundary, respectively.


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To determine the FRC curves from the STORM images, we used the FIRE ImageJ plugin.41 Following convention, we associated the characteristic spatial frequency across these images as the frequency associated with a FRC value of 1/7. We note though that FRC provides a global estimate of resolution within an image that is significantly dependent on the image features.63 Since these features are expected to be the same in the fully labeled nucleus and the single labeled chromosome, this FRC calculation indeed provides a useful measure to compare the difference in resolution between these two cases. However, as a measure of the absolute resolution in the images, we, like others,26,

43

prefer the calculation of the FWHM of

representative features in the image. To this end, local regions were binned at 10 nm x 10 nm, and the region was fitted to a Gaussian curve using SigmaPlot (Systat Software Inc). DNA Calibration DNA was purified from the group B cells described above. The DNA was sheared by sonication (Digital Sonifier, Branson) at 10% of full power for 10s. DNA fragments of 1 kb and 2 kb were isolated using agarose gel electrophoresis followed by gel extraction using the MinElute Gel Extraction Kit (28604, QIAGEN). The purified DNA was stored in TE buffer (10 nM Tris-HCl, 0.5 mM EDTA, pH 8.0) at a concentration of 200 ng/µL. Before imaging, the click reaction buffer was added into the sample (as described above) and after 2 h, the free fluorophores were removed as described above. The labeled DNA solution was diluted 10,000fold and incubated at 50 °C for 10 min to promote the disaggregation of DNA. Afterwards, DNA fragments were adsorbed to coverslips that were modified with poly-lysine (P8920, SigmaAldrich). For the latter, the poly-lysine (0.1% w/v in water) was added to the coverslip, allowed to incubate for 30 min, and then washed with water. All imaging was performed with Nikon NSTORM.


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We used the localization number observed during the “equilibrium stage” (400 to 600 s) of the imaging process to quantify the DNA content.47, 48 To correlate the localization number with the DNA content, we analyzed STORM images of the 1 kb and 2 kb DNA fragments, choosing 70 spots in each of 3 independent assays and measured the overall localization number (for these 70 spots) every 100s from 0 s to 900 s using ThunderSTORM. In this way, we obtained 6.8 ± 1.6 and 12.5 ± 1.4 localizations per spot for the 1 kb and 2 kb DNA fragments, respectively. To determine the labeling density of fluorophores in DNA, we examined Alexa Fluor 647conjugated streptavidin (S21374, Thermo Fisher Scientific) which contains, on average, three fluorophores (according to the manufacturer). The streptavidin solution (200 ng/mL in PBS) was added to biotin-coated coverslips (CS-BN-5, Nanocs), allowed to incubate for 15 min, and then the sample was washed with PBS. From the final set of STORM images, we chose 70 spots (~210 fluorophores in total) and recorded the overall localization number (for these 70 spots) every 100 s from 0 to 1900 s. This experiment was performed in three independent assays. In this way, we measured 6 ± 0.6 localizations per spot of streptavidin during the equilibrium stage (400 to 600 s). From the aforementioned number of localizations from the 1 kb and 2 kb DNA fragments, we obtained 3.3±0.9 fluorophores per 1 kb DNA, or 1 Alexa Fluor 647 molecule per 315±79 bp.

Quantitative analysis of the dispersed region, nanodomains, and clusters The clustering of the localizations was performed following a previously described method.31 Briefly, a histogram image with 10 nm pixel size was produced from the list of localizations generated from ThunderSTORM, with the value of each pixel equal to the number of localizations within that region. A density map was then determined using a 5 × 5 convolution of


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the histogram image. The density map was then transformed into a binary image. The threshold for this binary image was determined from a similar analysis of nuclei without incorporated EdU but that were nonetheless treated with click-it reaction chemicals and Alexa Fluor 647. We generated a binary image from the density map of the unlabeled sample using different values of threshold and determined the threshold value such that less than 0.2% of the pixels had a value of 1. This threshold value was then subsequently used in the analysis of the labeled chromosomes. The connected components, consisting of adjacent non-zero pixels with 4 connected neighbors in the binary images, were then determined and used to identify nanodomains and clusters. To determine the number of nanodomains in a cluster, we measured the number of local intensity maximums within the connected component region using Matlab, and equated the number of local intensity maximums to the number of nanodomains in the cluster. A script in Matlab was used to sort the localizations within the connected component to the nearest nanodomain, and then calculate the localization number and the area of each nanodomain, corrected for background (0.001/nm2 determined from the unlabeled samples). The dispersed chromatin of the sub-chromosomal regions was identified by those regions in the Gaussianrendered image that exhibit a greater intensity per pixel than this background value.


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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: zz.zzzz/acsnano.zzzzzzz. Overview of the experiment, flow cytometry images following the release of cells from serum starvation, temporal analysis of the localization counts for DNA calibration, examination of the parameters used to fit the localizations enables a determination of the background noise and localization precision, FWHM analysis to calculate the image resolution, standard deviation of the fitted Gaussian PSF from single fluorophores to determine the z-position of the fluorophores within the nucleus, additional images of single chromosomes used in the analysis, comparison of the physical features of single nanodomains and individual nanodomains within clusters, the size distribution of the clusters of nanodomains at different nuclear locations, schematic depiction of the expected distributions of the DNA condensate sizes and DNA densities according to three models presently proposed to explain chromatin condensation within the eukaryotic nucleus, and schematic

model

of

the

nanodomain

and

cluster

of

nanodomains.

(PDF)

The authors declare that they have no conflict of interest.

AUTHOR INFORMATION Corresponding Author *Email: [email protected] or [email protected]. Author Contributions


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D.M.C. and Z.S. conceived the project. K.F., D.M.C. and Z.F. designed the experiments. K.F. conducted all experiments. K.F., X.C., X.L., Y.S and J.S. performed the data analysis. K.F., D.M.C., and Z.S. wrote the manuscript.

ACKNOWLEDGMENT We thank N. Luedtke for the kind gift of F-ara-EdU. We also thank C. Hu for help with data analysis. The authors are grateful for the generous support from Nikon Instruments Co, Ltd. This work was supported by grants from the National Natural Science Foundation of China (nos. 11374207, 31370750, 31670722, 81627801, 91129000, and 31501054), Shanghai Jiao Tong University (no. 16x120030015), and the K.C. Wong Education Foundation (H.K.) (to Z.S.).

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Human Genome at Kilobase Resolution Reveals Principles of Chromatin Looping. Cell 2014, 159, 1665-1680. 59. Szabo, Q.; Jost, D.; Chang, J. M.; Cattoni, D. I.; Papadopoulos, G. L.; Bonev, B.; Sexton, T.; Gurgo, J.; Jacquier, C.; Nollmann, M. et al. TADs Are 3D Structural Units of Higher-Order Chromosome Organization in Drosophila. Sci. Adv. 2018, 4, 13. 60. Neef, A. B.; Luedtke, N. W. Dynamic Metabolic Labeling of DNA in Vivo with Arabinosyl Nucleosides. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 20404-20409. 61. Olivier, N.; Keller, D.; Gonczy, P.; Manley, S. Resolution Doubling in 3D-STORM Imaging through Improved Buffers. PLoS One 2013, 8, 9. 62. Ovesny, M.; Krizek, P.; Borkovec, J.; Svindrych, Z. K.; Hagen, G. M. ThunderSTORM: A Comprehensive ImageJ Plug-in for PALM and STORM Data Analysis and Super-Resolution Imaging. Bioinformatics 2014, 30, 2389-2390. 63. Legant, W. R.; Shao, L.; Grimm, J. B.; Brown, T. A.; Milkie, D. E.; Avants, B. B.; Lavis, L. D.; Betzig, E. High-Density Three-Dimensional Localization Microscopy across Large Volumes. Nat. Methods 2016, 13, 359-365.

FIGURE LEGENDS Figure 1. Improved resolution of sub-chromosomal regions by imaging single labeled chromosomes within an otherwise unlabeled nucleus. (A) Typical STORM image of a fully labeled nucleus. Shown in the inset is an image obtained with conventional microscopy. Scale bar = 2 µm. (B) Enlarged view of boxed region in (A). Scale bar = 0.5 µm. (C) FRC analysis of STORM images of the fully labeled nucleus (A) and single labeled chromosome within an otherwise unlabeled nuclei (E). (D) Merged image of bright field and conventional fluorescent microscopy images of a single labeled chromosome within the nucleus. The chromosome is white in the image. Scale bar = 2 µm. (E) STORM image of the chromosome shown in (D). Scale bar = 0.5 µm. (F) Characteristic spatial frequency of the fully labeled nuclei or single labeled chromosome STORM images, defined conventionally as the frequency associated with a FRC value of 1/7.


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Figure 2. High-resolution images of individual chromosomes within the nucleus reveal a common three-level higher order organization of DNA condensation. Typical images are shown for sub-chromosomal regions within the nuclear interior (“INT”), nuclear periphery (“NuP”), and nucleolar proximity (“NoP”) for the upper, middle, and bottom row panels, respectively. Within each row, the images reflect (from left to right): a conventional fluorescence microscopy image (blue – DNA, green – nucleolus, red – labeled chromosome), scale bar = 2 µm; STORM image of the boxed region in the conventional image, scale bar = 0.5µm; the density map of the STORM image; the binary image of the connected components determined from the density map, scale bar = 0.5 µm; and a schematic image of the sub-chromosomal region with the dispersed chromatin (“D”) colored grey and the individual nanodomains (“N”) in a range of different colors. The areal fraction of the dispersed chromatin and nanodomain is also shown. Figure 3. The nanodomains from different nuclear locations exhibit a similar area, DNA content and density distribution. Shown are the distributions for the sub-chromosomal regions within the nuclear interior (“INT”), nuclear periphery (“NuP”), and nucleolar proximity (“NoP”). (A) The distribution of nanodomain area. (B) The distribution of the DNA content per nanodomain. (C) The distribution of the DNA density per nanodomain. Figure 4. Difference in the areal fraction of the dispersed and nanodomain in the subchromosomal regions across the nucleus. Measurements from sub-chromosomal regions within the nuclear interior, nuclear periphery, and nucleolar proximity are indicated as “INT”, “NuP”, and “NoP”, respectively.


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RFU of Alexa Fluor 647 /YOYO (10-2)

Labeling efficiency

Group A (in vivo, click reaction for 2 h)

2.69 ± 0.03

82%

Group A (in vivo, click reaction for 12 h)

3.06 ± 0.05

93%

Group B (in vitro, click reaction for 2 h)

3.29 ± 0.05

Table 1. Labeling efficiency of the click reaction in situ.

Density: nanodomain

Areal fraction:

Density: dispersed

Areal fraction:

Total density

(Mb /µm2)

nanodomain

(Mb /µm2)

dispersed

(Mb /µm2)

Interior

4.86 ± 2.82

0.15 ± 0.05

0.81 ± 0.06

0.85 ± 0.05

1.41 ± 0.75

Nuclear periphery

6.45 ± 4.27

0.46 ± 0.06

1.08 ± 0.05

0.54 ± 0.06

3.55 ± 2.44

Nucleolar proximity

5.10 ± 3.03

0.51 ± 0.09

0.91 ± 0.05

0.49 ± 0.09

3.04 ± 2.11

Table 2. Source of the different DNA densities at the different nuclear locations.


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Figure 1. Improved resolution of sub-chromosomal regions by imaging single labeled chromosomes within an otherwise unlabeled nucleus. (A) Typical STORM image of a fully labeled nucleus. Shown in the inset is an image obtained with conventional microscopy. Scale bar = 2 µm. (B) Enlarged view of boxed region in (A). Scale bar = 0.5 µm. (C) FRC analysis of STORM images of the fully labeled nucleus (A) and single labeled chromosome within an otherwise unlabeled nuclei (E). (D) Merged image of bright field and conventional fluorescent microscopy images of a single labeled chromosome within the nucleus. The chromosome is white in the image. Scale bar = 2 µm. (E) STORM image of the chromosome shown in (D). Scale bar = 0.5 µm. (F) Characteristic spatial frequency of the fully labeled nuclei or single labeled chromosome STORM images, defined conventionally as the frequency associated with a FRC value of 1/7. 177x116mm (300 x 300 DPI)


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Figure 2. High-resolution images of individual chromosomes within the nucleus reveal a common three-level higher order organization of DNA condensation. Typical images are shown for sub-chromosomal regions within the nuclear interior (“INT”), nuclear periphery (“NuP”), and nucleolar proximity (“NoP”) for the upper, middle, and bottom row panels, respectively. Within each row, the images reflect (from left to right): a conventional fluorescence microscopy image (blue – DNA, green – nucleolus, red – labeled chromosome), scale bar = 2 µm; STORM image of the boxed region in the conventional image, scale bar = 0.5µm; the density map of the STORM image; the binary image of the connected components determined from the density map, scale bar = 0.5 µm; and a schematic image of the sub-chromosomal region with the dispersed chromatin (“D”) colored grey and the individual nanodomains (“N”) in a range of different colors. The areal fraction of the dispersed chromatin and nanodomain is also shown. 177x116mm (300 x 300 DPI)


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Figure 3. The nanodomains from different nuclear locations exhibit a similar area, DNA content and density distribution. Shown are the distributions for the sub-chromosomal regions within the nuclear interior (“INT”), nuclear periphery (“NuP”), and nucleolar proximity (“NoP”). (A) The distribution of nanodomain area. (B) The distribution of the DNA content per nanodomain. (C) The distribution of the DNA density per nanodomain. 177x102mm (300 x 300 DPI)


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ACS Nano

Figure 4. Difference in the areal fraction of the dispersed and nanodomain in the sub-chromosomal regions across the nucleus. Measurements from sub-chromosomal regions within the nuclear interior, nuclear periphery, and nucleolar proximity are indicated as “INT”, “NuP”, and “NoP”, respectively. 82x60mm (300 x 300 DPI)


Super-Resolution Imaging of Individual Human ... - ACS Publications

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