Pairwise Proximity-Differentiated Visualization of Single-Cell DNA Epigenetic Marks

Article abstract of DOI:10.1002/anie.202011172

Spatial positioning and proximity of relevant biomolecules such as DNA epigenetic marks are fundamental to a deeper understanding of life. However, it remains poorly explored and technically challenging. Here we report the pairwise proximity-differentiate

Full text of DOI:10.1002/anie.202011172

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: Pairwise Proximity-differentiated Visualization of Single-cell DNA
Epigenetic Marks
Authors: Jing Xue, Feng Chen, Li Su, Xiaowen Cao, Min Bai, Yue
Zhao, Chunhai Fan, and Yongxi Zhao
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202011172
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Pairwise Proximity-differentiated Visualization of Single-cell DNA
Epigenetic Marks
Jing Xue,+ Feng Chen,+ Li Su,+ Xiaowen Cao, Min Bai, Yue Zhao, Chunhai Fan, and Yongxi Zhao*
Abstract: Spatial positioning and proximity of relevant biomolecules
such as DNA epigenetic marks are fundamental to a deeper
understanding of life process. However, it remains poorly explored
and technically challenging. Here we report pairwise proximity-
differentiated visualization of single-cell 5-formylcytosine (5fC) and
5-hydroxymethylcytosine (5hmC). These two marks on chromatin in
fixed cells are successively labeled and crosslinked with their DNA
primer probes via click chemistry. Based on a pairwise proximity-
differentiated mechanism, proximal 5fC/5hmC sites firstly and
residual 5fC or 5hmC sites then are encoded with respective
circularized barcodes. These barcodes are simultaneously amplified
for multiplexed single-molecule imaging. Thus we demonstrate
differentiated visualization of 5fC or 5hmC spatial positioning and
their pairwise proximity in single cells. Such multi-level subcellular
information may provide insights into regulation functions and
mechanisms of chromatin modifications, and the spatial proximity
can expose the potential crosstalk or interaction between their
reader proteins.
their potential crosstalk or interaction of their reader proteins.
Unfortunately, single-cell visualization of both spatial positioning
and proximity of epigenetic marks has not been explored.
Traditional fluorescence in situ hybridization-based cell
imaging is limited to detect sequences of interest and
inadequate for the assessment of base modifications.[6]
Immunoassays use antibodies to bind to base modifications
such as 5hmC only in naked DNA.[7] The base sites covered by
DNA-binding proteins or in condensed chromatins are sterically
excluded from antibodies with relatively large sizes. Such large
sizes also disturbed the exploration of spatial proximity of
different marks. Chemoenzymatic- or chemical-based methods
have been well developed for the covalent labeling of 5hmC or
3a, 3d]
5fC coupled with bioorthogonal click tags.[2a,
These
methods provide considerable improvement over antibody-
based ones as they ensure more accurate and comprehensive
labeling of DNA epigenetic sites. But the recognition and
visualization of proximity between these epigenetic marks
remains limited. DNA nanotechnology may be a promising
solution ascribed to predictable and programmable assembly
with nanoscale precision.[8] We and others have developed
various nanostructured DNA probes for molecule positioning,
proximity recognition, assembly or disassembly.[9] In particular,
DNA walkers or motors are designed based on assembly-
disassembly cycles to continuously recognize adjacent substrate
molecules.[10] And proximity DNA ligation assay can detect the
pairwise proximity of two molecules such as proteins to
investigate their spatial distances and interactions.[11] In a typical
proximity ligation assay, both pairwise proximity target sites and
residual target sites were labeled with the DNA recognition
probes. These probes then participated in hybridization and
ligation reaction. However, this method only detected the
pairwise proximity sites and can’t record the residual target sites.
Moreover, the probes labeled at residual target sites have been
still occupied by ligation probes for proximity assay, thus they
were difficult to be detected differently from pairwise proximity
target sites. Therefore, differentiated visualization of spatial
positioning and proximity of two different DNA epigenetic marks
remains challenging.
Intracellular positioning of relevant biomolecules and their
nanoscale colocalization or proximity are important to the fate
and function of both single cells and living organisms. For
example, a serial of DNA epigenetic marks exist in mammalian
genomes. They have profound influence in gene expression and
chromatin architecture, and are implicated in pathological
associations including cancers.[1] The best-known ones are 5-
methylcytosine (5mC) and its oxidized derivatives 5-
hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC).[2]
Several deep sequencing strategies have been well developed
for genome-wide analysis of these modifications, revealing their
important functions in normal biological processes and
diseases.[3] Yet these methods rely on cell population averaging,
and are disabled to visualize the subcellular distribution. The
spatial positioning of epigenetic marks in single cells is
fundamental to understanding their functions.[4] Furthermore,
accumulated evidence has verified the co-occurrence of two
epigenetic marks at the same gene regions and suggested their
5]
spatial proximity on the basis of chromatin interactions.[2a,
Exploring the proximity of different epigenetic marks may reveal
Herein,
we
report
pairwise
proximity-differentiated
visualization of single-cell 5fC and 5hmC (Scheme 1 and S1).
These two oxidized cytosines are selected as representative
epigenetic marks due to their well-performed studies. We require
to synthesize an specific 5fC labeling probe azido derivative of
1,3-indandione (AI)[2a]. And we report its practicability in the
complex intracellular environment against the interferences from
various analogous such as 5-formyluracil (5fU). In addition,
5hmC is routinely labeled with click tags by T4 phage β-
glucosyltransferase (β-GT). Both 5fC and 5hmC on chromatins
in fixed cells are sequentially labeled and crosslinked with their
DNA primer probes via click chemistry. Based on a pairwise
[a]
J. Xue, Prof. F. Chen, L. Su, X. W. Cao, M. Bai, Dr. Y. Zhao, Prof. Y.
X. Zhao
Institute of Analytical Chemistry and Instrument for Life Science,
The Key Laboratory of Biomedical Information Engineering of
Ministry of Education
School of Life Science and Technology, Xi’an Jiaotong University
Xianning West Road, Xi’an, Shaanxi 710049, China
E-mail: yxzhao@mail.xjtu.edu.cn
Prof. C. H. Fan
Institute of Molecular Medicine, Renji Hospital, School of Medicine
and School of Chemistry and Chemical Engineering, Shanghai Jiao
Tong University, Shanghai 200127, China
[+]
These authors contributed equally to this work.
proximity-differentiated
mechanism,
our
method
can
successively encode proximal 5fC/5hmC sites and residual 5fC
or 5hmC sites with respective circularized DNA barcodes. In
brief, a pair of splint ligation probes captured at 5fC/5hmC
Supporting information for this article is given via a link at the end of
the document.
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molecule sensitivity. Thus we realize the differentiated
visualization of 5fC or 5hmC spatial positioning and 5fC/5hmC
proximity in single cells. Such multi-level spatial information may
promote deeper investigations of their regulation functions and
mechanisms. Notably, our method is designed to detect hetero-
proximity (5fC/5hmC) instead of homo-proximity (5fC/5fC or
5hmC/5hmC). And it can be easily applied to the differentiated
visualization of monovalent or bivalent histone modifications and
to the detection of protein-protein interactions.
To demonstrate the proposed method generally, we designed
DNA origami target substrates containing two kinds of target
sites (A and B) with various distances (Figure 1A and Table S1).
The sets of DNA probes used here mainly include primer probes
for target stie labeling, a pair of splint ligation probes for pairwise
proximity (A1/B1, ~10 nm) RCA, two pre-circularized DNA
barcodes for respective targrt site (A2 or B2) barcoding RCA,
two displacement probes and three fluorescence probes. The
displacement probes are designed to be complementary to splint
ligation probes for toehold-mediated strand displacement. Their
squence lengths can significantly affect the thermodynamics and
performance of pairwise proximity-differentiated recognition and
visualization. Thus we firstly predicted the standard free
energies of different displacement probes hybridizing to the
ligated splint ligation probes (Figure S1). And as shown in the
bottom panel of Figure 1A, we demonstrated that only the
displacement probes with an appropriate squence length can
Scheme 1. Workflow of pairwise proximity-differentiated visualization of
single-cell DNA epigenetic marks. The cells are fixed before the recognition
and differentiation of target sites.
proximity sites form two nicks, and then they are ligated as one
circularized DNA barcode. In contrast, molecules of these
probes at residual, unpaired modification sites are released by
DNA strand displacement. The restored DNA primer probes can
hybridize with the circularized DNA barcodes for 5fC or 5hmC.
These three barcodes will work as templates for rolling circle
amplification (RCA). The long single-stranded RCA products
(RCPs) contain periodically repeated sequences to capture
fluorescent DNA probes for signal amplification with single-
Figure 1. Pairwise proximity-differentiated visualization on DNA origami. (A) Principle of the method. Influence of strand displacement reaction on differentiated
recognition and visualization was investigated. Disp, Disp-11 and Disp+14 indicate the displacement probes (a1*a2*) with different sequence lengths. The
process of b2*b1* is similar to that of a1*a2*. Cy5, Cy3 and FAM indicate the fluorescence channels. (B) Distance-dependent proximity visualization. (C) The
effect of primer probe length on proximity visualization. The scale bars in the images are 15 μm. 10 images are randomly selected for each statistical analysis.
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distinguish A1/B1 pairwise proximity sites versus two kinds of
residual sites (A2 or B2), which is in agreement with the energy
prediction. In contrast, overlong displacement probes caused the
loss of fluorescence signals of A1/B1 pairwise proximity sites,
and too short displacement probes were disabled to trigger the
fluorescence signals of A2 or B2 sites. Furthermore, we
investigated the effect of different separation or distances (~10,
~20 and ~30 nm) between two target sites on the pairwise
proximity analysis (Figure 1B). We observed very low
fluorescence signals when the distance of two sites reached ~30
nm. And we also demonstrated that the detection distance or
spatial resolution of pairwise proximity sites was mainly
determined by the length of two kinds of DNA primer probes
(Figure 1C). Notably, the distances of about 10-20 nm match the
study of two protein interactions or protein complexes. Overall,
these results verified the pairwise proximity-differentiated
visualization analysis by our proposed method.
For the analysis of 5hmC and 5fC, we first confirmed the
synthesis of AI, 5fC labeling by AI and 5hmC labeling by β-GT
using UDP-N3-glucose (Figure S2-S4). We also demonstrated
the pairwise proximity visualization of 5hmC/5fC at the same
dsDNA substrates in vitro (Figure S5), which is similar to above
experiments of DNA origami substrates. Notably, the
quantitative assessment of labeling these marks with DNA
primer is of great significance but difficulty, and it may be
realized by ultrasensitive analytical mass spectrometry and DNA
quantitative techniques such as qPCR or unique molecular
identifier-used next generation sequencing. Then the spatial
positioning of intracellular 5hmC and 5fC was investigated. We
demonstrated that no potential uncrosslinked 5fC-AI adduct was
detected by the set of DNA probes for 5hmC analysis (Figure
S6-S8). Considering the diversity of chromatin modifications
including histone modifications,[12] we explored the colocalization
relationship between 5hmC and 5fC, and between 5hmC or 5fC
and one of five model histone modifications by routine
fluorescence colocalization analysis (Figure 2A and S9). These
colocalization values presented remarkable single-cell
heterogeneity (Figure 2B and 2C).[13] Especially,
a low
colocalization level of 5hmC with γH2AX is observed. Previous
studies also reported their low colocalization level in untreated
cells but a high value after DNA damage treatment.[7] However,
the spatial resolution for colocalization analysis by traditional
fluorescence microscopes is only about 250 nm in the lateral
dimension.[14] It can’t explore the nanoscale proximity (e.g., ~10
nm) between 5fC and 5hmC, which may be associated with their
protein interaction, gene regulation and other functions.
Finally, we demonstrated differentiated visualization of 5fC or
5hmC spatial positioning and their nanoscale proximity in single
cells by our proposed method. As described above, barcoding
amplification was performed to generate three kinds of RCPs
after pairwise proximity- differentiated recognition. These RCPs
can be observed as bright spots (Figure 3 and S10). And the
spot count of individual cells were routinely extracted using
Image J. Both fluorescence intensity and spot count information
are obtained to evaluate the distribution features. Fluorescence
intensity of individual cells was used to roughly depict the global
or total abundance of labeled sites, and spot count was roughly
hypothesized as the number of representative detection sites.
Notably, one bright spot with big sizes may cover several RCP
Figure 2. Spatial positioning of single-cell 5fC and 5hmC. (A) Cell images of
5fC, 5hmC and H3K4me1 in different cell lines. Scale bar, 10 µm. (B)
Colocalization analysis of 5fC+5hmC, 5fC+H3K4me1 and 5hmC+H3K4me1 in
different cell lines (N=50). (C) Colocalization analysis of 5fC or 5hmC with five
histone modifications in MCF-10A cells (N=50). N: the number of cells.
molecules or detection sites which are localized closely.
Negative controls were also performed to verify the assay
specificity (Figure S10). We confirmed the spatial proximity
between 5fC and 5hmC in cells for the first time.
In summary, we demonstrate differentiated visualization of 5fC
or 5hmC spatial positioning and their proximity in single cells. A
pairwise proximity-differentiated mechanism is proposed for
successive recognition and amplification barcoding of proximal
5fC/5hmC sites and residual, unpaired 5fC or 5hmC sites. More
comprehensive subcellular distribution information is provided
for deeper epigenetics studies. Especially, the spatial proximity
can expose the potential crosstalk or interaction between their
reader proteins and may suggest new function in related
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Support Plan” of Xi’an Jiaotong University. We thank Miss Hao
and Miss Lu at Instrument Analysis Center of Xi'an Jiaotong
University for the assistance with confocal fluorescence imaging
and LCMS/MS analysis.
Keywords: DNA nanotechnology • epigenetic marks• pairwise
proximity • differentiated recognition • single-cell imaging
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This research was funded by the National Natural Science
Foundation of China (grant number 31671013, 21705124 and
21874105), the China Postdoctoral Science Foundation [grant
number 2017M613102, 2018T111032 and 2019M663658], the
Natural Science Foundation of Shaanxi Province (grant number
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Jing Xue, Feng Chen, Li Su, Xiaowen
Cao, Min Bai, Yue Zhao, Chunhai Fan,
and Yongxi Zhao*
Page No. – Page No.
Pairwise Proximity-differentiated
Visualization of Single-cell DNA
Epigenetic Marks
Pairwise proximity-differentiated visualization in single cells has been developed.
This method achieves the differentiated recognition and barcoding amplification
visualization of both 5fC/5hmC proximity sites and residual sites of 5fC or 5hmC
on chromatins. It can be used to probe the valence of histone modifications and
even protein interactions in single cells.
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