Introduction
Overview of nuclear architecture studies
Thus, it is critical to understand how the 3D nuclear architecture is shaped in many biological contexts. Here, I highlight key advances in sequence-based and image-based technologies to study 3D nuclear architecture.
Sequencing-based technologies to study nuclear architecture
Those TAD boundaries are enriched with insulator-binding protein CTCF and housekeeping genes ( Dixon et al., 2012 ). Using ChIA-PET, Fullwood et al. 2009) revealed long-range chromatin interactions at gene promoters mediated by estrogen receptor α in the human genome.
Imaging-based technologies to study nuclear architecture
In particular, Wang et al. 2016) performed sequential DNA FISH to resolve dozens of genomic loci in one chromosome in single cells. More recently, sequential DNA FISH together with transcriptional measurements can be performed within the same single cells by technologies such as Hi-M (Cardozo Gizzi et al., 2019), opt.
Summary
In particular, Nguyen et al. 2020) developed OligoFISSEQ, which hybridizes barcoded Oligopaint probes to the sample and reads out those barcodes by fluorescence in situ sequencing (FISSEQ) (Lee et al., 2014), to detect chromosome structures in single cells. 2021) developed in situ genome sequencing (IGS), which performs sequencing and imaging of the genome simultaneously from the same single cells, to capture genomic sequences and map obtained sequences back into the nucleus in 3D space.
The spatial organization of chromosomes and TAS in individual cells can be reconstructed from intron seqFISH data (Figure 2A). We integrated our genome-wide analysis (DNA seqFISH+) with transcripts (RNA seqFISH) as well as histone modifications and subnuclear structures (immunofluorescence (IF)) (Figure 1a and Extended Data Figure 1a).
Multiplexed dynamic imaging of genomic loci by combined
Abstract
Visualization of chromosome dynamics allows the investigation of spatiotemporal chromatin organization and its role in gene regulation and other cellular processes. Our approach first marks and sequences chromosomal loci in living cells with the CRISPR-Cas9 system, then sequences those loci by DNA seqFISH in fixed cells and resolves their identities.
Introduction
However, current approaches to labeling multiple genomic loci in living cells have a fundamental limitation in the number of loci that can be labeled and uniquely identified. Recently, a color-barcoding approach using engineered sgRNA scaffolds that recruit different combinations of spectrally distinct fluorescent proteins demonstrated simultaneous imaging of six chromosomal loci in single cells ( 10 ).
Results
A set of dye-labeled adapter probes hybridized to the overhang sequences (Fig. S2 A). We imaged cells again to confirm the probe displacement, and subsequent rounds of hybridizations were performed (Fig. S2 B,C).
Discussion
In situ transcriptional profiling of single cells reveals the spatial organization of cells in the rat hippocampus.
Main figures
Note that DNA seqFISH spots do not perfectly colocalize with CRISPR imaging spots because they target adjacent regions in the genome. Trajectories of three loci in the magnified images (B–E) were also marked as xy projections (inset). I) Cumulative square displacement traces (n = 30) calculated with two adjacent frames as a function of time from the enlarged cell.
Supplemental figures
Number of subtelomeric spots per cell separated by color barcode with three rounds of hybridizations. The trajectories of the three loci per cell were also highlighted as xy projections (inset). B) Traces of cumulative quadratic displacement versus time.
Methods
Note that each time point shown in the figure and movie was the start time of the z-stacks. The microscope's Perfect Focus system was used to automatically correct focus drift during imaging.
Supplemental items
Values correspond to the number of TAS or mRNA detected for each gene in a given cell. Even the Xi region, which appears unstructured in the bulk of the data from both DNA seqFISH+ (Fig. 6F and Fig.
Dynamics and spatial genomics of the nascent transcriptome by
Abstract
Visualization of the transcriptome and nuclear organization in situ in the individual cell is the holy grail of single-cell analysis. Surprisingly, global nascent transcription fluctuated asynchronously in individual cells with a period of 2 h in mouse embryonic stem cells as well as fibroblasts.
Introduction
Pioneering work with single-molecule fluorescence in situ hybridization (smFISH) ( Femino et al., 1998 ; Raj et al., 2006 ) observed that nascent mRNAs are produced in bursts at transcriptional active sites (TAS) in individual nuclei. Furthermore, the relatively short lifetimes of TAS compared to the longer lifetimes of mRNAs (Sharova et al., 2009) mean that intron seqFISH can capture rapid dynamics in the nascent transcriptome that would otherwise be hidden in mRNA measurements.
Results
Surprisingly, there is great variability in the global nascent transcriptional states of cells, as observed from the total number of active transcriptional sites in each nucleus in the 10,421 gene-intron experiments (Figure 5A), even after considering differences in cell cycle phase and cell size (Figure S4E-I). The variability in the 5-EU signal in individual cells (Figure S5B) is similar to the intron variability observed (Figure 5A), confirming emerging transcriptional heterogeneity in single cells.
Discussion
Further investigation of the dynamics can benefit from intron seqFISH together with multiplexed live cell imaging of genomic loci (Takei et al., 2017). Hes1 has been shown to oscillate in many cell lines, including mESCs and fibroblasts ( Kobayashi et al., 2009 ; Yoshiura et al., 2007 ).
For example, cells can use fluctuations in global transcriptional activity to coordinate the stoichiometry of many transcripts in mechanisms similar to the frequency-modulated signaling observed in yeast and mammalian pathways (Cai et al., 2008; Yissachar et al., 2013). Finally, an exciting recent work has shown that intron-to-exon ratios across the transcriptome can be used to determine the direction of cells on the developmental trajectory (La Manno et al., 2017).
Main figures
Barcoding round I image of 10,421 gene intron seqFISH from three z-sections (top left), and zoomed view of the yellow box region through five rounds of barcoding (top right panels). More long-range contacts are observed in the intron seqFISH contact maps compared to ligation-based Hi-C maps.
Supplemental figures
The merged image (right) shows many colocalized spots (white) between these two images (green and magenta), demonstrating the robustness of the intron seqFISH protocol over 20 rounds of hybridizations without significant signal reduction. The panel on the right displays the quantitative fluorescence intensity of intron FISH, DNA FISH, and chromosome dye along the yellow line within a single z section.
Methods
Probe sequences of the genes in the same chromosome were amplified from one primer pair, and those primer binding sequences (5' and 3' end) were targeted with 20-nt dye-conjugated readout probes to stain introns in specific chromosomes. The number of each barcode was then counted in each of the assigned cell volumes and transcript numbers were assigned based on the number of barcodes present on the target in the cell volume.
Supplemental items
We observed similar cell-type-dependent association of chromosomes with the nucleus in the mouse brain cortex ( Fig. 3A ). At the same time, we observed a cell type dependence in the average interchromosomal spatial distances (Fig. 5E, top).
Integrated spatial genomics reveals global architecture of single
Abstract
These loci form "fixed spots" on nuclear arrangements in single cells and often appear on the surfaces of nuclear bodies and areas defined by combinatorial chromatin marks. Furthermore, highly expressed genes appear to be pre-positioned in active nuclear zones, independent of blast dynamics in single cells.
Introduction
Here we report the imaging of 3,660 chromosomal loci in single mouse embryonic stem cells (mESCs) by DNA seqFISH+, along with 17 chromatin marks and subnuclear structures by sequential immunofluorescence (IF) and the expression profile of 70 RNAs. Recent methods using Oligopaint and sequential DNA fluorescence in situ hybridization (DNA FISH) have imaged many DNA loci in single cells.
Results
In addition, 4 repetitive regions related to nuclear organization32,33 were sequenced by DNA FISH (Extended Data, Figure 5a). At the individual cell level, many DNA loci appear to be consistently close to specific IF tags in a large percentage of cells (Figure 2c, Extended Data Figure 6f).
Discussion
Concentric organization of A- and B-type laminae predicts their respective roles in the spatial organization and stability of the nuclear lamina. Normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regression.
Main figures
Magnified views (right) show gene loci such as Pou5f1 in zone 1 or interfaces 1/2 (top) and loci around the nucleolus and heterochromatin zones (bottom). Tfcp2l1 is shown at 1 Mb resolution and Bdnf at 25 kb resolution DNA seqFISH+ data.
Supplemental figures
For the box plots in b and g, the center line in the box marks the median, the upper and lower bounds of the boxes mark the interquartile range, the whiskers extend to the most distant data points within 1.5 times the interquartile range, and the gray dots mark the external. For box plots, the center line in the box marks the median, the upper and lower bounds of the boxes mark the interquartile range, the whiskers extend to the most distant data points within 1.5 times the interquartile range, and the gray dots mark the outliers .
Methods…
Starting from the 5' end of the forward strand, candidate probes were tested for stability, moving one base at a time. In total, it took about 80 hours to complete the 80 rounds of the seqFISH+ DNA hybridization and imaging routine.
Supplemental items…
Nuclear dots appeared to be more distributed in cells with preferential localization in the nuclear interior (Fig. 5A and 5C). Additional scaling relationships are shown in fig. H) Spatial distance between pairs of intra-chromosomal loci within cells by DNA seqFISH+ in different cell types at 1-Mb resolution (top row, quarter spatial distance within cells) and at 25-kb resolution (bottom row, spatial distance mean within alleles ).
Integrated spatial genomics in tissues reveals invariant and cell
Abstract
Nuclear architecture in tissues can result from the specific organization of nuclear bodies, chromatin states and chromosome structures that are specific to the cell type. We have revealed chromatin fixed points in combination with the cell-specific organization of nuclear bodies that govern the interchromosomal organization and radial positioning of chromosomes in different cell types.
Introduction
On the other hand, imaging-based approaches allow direct integration of multimodal measurements, including chromosome structures. We recently developed an imaging-based integrated spatial genomics approach that enables the analysis of nuclear organization beyond chromosome structures ( 32 ).
Results
For example, individual cells showed colocalization of nuclear speckle components (Malat1 and SF3a66), X chromosome inactive (Xi) components (Xist, mH2A1, H3K27me3, LINE1) and heterochromatin components (DAPI, MajSat, H3K9me3, H4K20) S1K and S1L ). The overall intensity of the IF markers was highly variable across single cells (Fig. 2A and fig. S3A).
Discussion
We observed that Xa and Xi have distinct mean distances between pairs of loci at different scales of genomic length and in different cell types (fig. S8G and S8H). Interestingly, both Xa and Xi have heterogeneous domain structures in individual cells ( Figs. 6F and 6G and fig. S9).
Sims, SCOPE-Seq: a scalable technology for coupling live cell imaging and single-cell RNA sequencing. Satija, Normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regression.
Main figures
Cells are colored according to their transcriptional cell types in (A). F) Hierarchical clustering of DAPI meta features compared to the RNA seqFISH clusters shown above. Gene density (top panel) and 15% variable loci in terms of radial positioning among the three cell types (middle panel) are shown.
Supplemental figures
Good colocalization (white in right panel) of H4K20me3 immunofluorescence signals before and after DNA-seqFISH+ preparation including a heating denaturation step. J) Quantification of the H4K20me3 immunofluorescence signal retention in the nuclei before and after DNA-seqFISH+. F and G) Spatial distance between pairs of intra-chromosomal loci in cells by DNA seqFISH+ in different cell types at 1-Mb resolution (quartile spatial distance in cells for chromosome 15) in (F) and at 25-kb resolution (median spatial distance within homologous chromosomes for chromosome 5) in (G).
Methods…
Supplemental items…
Conclusion
Future directions
