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Chromatin in situ proximity (ChrISP): Single-cell analysis of chromatin proximities at a high resolution
 
Xingqi Chen*1, Chengxi Shi*1, Samer Yammine*1, Anita Göndör1, Daniel Rönnlund2, Alejandro Fernandez-Woodbridge1, Noriyuki Sumida1, Jerker Widengren2, and Rolf Ohlsson1
1Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden
2Applied Physics, AlbaNova University Center, Royal Institute of Technology, Sweden


*X.C., C.S., and S.Y. contributed equally to this work
BioTechniques, Vol. 56, No. 3, March 2014, pp. 117–124
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Supplementary Material
Abstract

Current techniques for analyzing chromatin structures are hampered by either poor resolution at the individual cell level or the need for a large number of cells to obtain higher resolution. This is a major problem as it hampers our understanding of chromatin conformation in single cells and how these respond to environmental cues. Here we describe a new method, chromatin in situ proximity (ChrISP), which reproducibly scores for proximities between two different chromatin fibers in 3-D with a resolution of ~170Å in single cells. The technique is based on the in situ proximity ligation assay (ISPLA), but ChrISP omits the rolling circle amplification step (RCA). Instead, the proximities between chromatin fibers are visualized by a fluorescent connector oligonucleotide DNA, here termed splinter, forming a circular DNA with another circle-forming oligonucleotide, here termed backbone, upon ligation. In contrast to the regular ISPLA technique, our modification enables detection of chromatin fiber proximities independent of steric hindrances from nuclear structures. We use this method to identify higher order structures of individual chromosomes in relation to structural hallmarks of interphase nuclei and beyond the resolution of the light microscope.

Higher order chromatin conformations result from packaging of the genome within the confines of the nucleus. This packaging must not only allow for the regulated accessibility of trans-acting factors to generate local chromatin loops but also enable the anchoring of the chromatin to structural hallmarks (1,2) to facilitate the emergence of robust phenotypes.

Our understanding of chromatin conformations and how these relate to hallmarks of nuclear structures is currently based on a combination of confocal DNA fluorescence in situ hybridization (DNA FISH) and the so-called C techniques (3C, 4C, 5C, and Hi-C) (2-4). The latter methods, which are based on the in situ ligation of cross-linked chromatin fibers to assess chromatin fiber proximities in a high-throughput manner, can have a very high resolution defined by the formaldehyde crosslinking agent (4). However, these methods do not robustly address the frequency of such features within a cell population, complicating the interpretation of chromatin conformation during complex biological processes. Conversely, single-cell studies of chromatin proximities using DNA FISH are severely hampered by that technique's low 3-D resolution (5).

Although the lateral resolution of a confocal microscope is limited to ~250 nm by diffraction, this resolution can be considerably increased in a stimulated emission depletion (STED) microscope (6). However, in many realizations of STED microscopy, the axial resolution is comparable to that of confocal microscopy. Even though recent developments in far field optical super resolution microscopy now allows the resolution in all three planes to be improved by about an order of magnitude, i.e. from about 1 μm down to approximately 100 nm (7, 8), this is still is not sufficient to discern different types of chromatin structures.

Method summary

Combining features of the in situ proximity ligation technique with conventional DNA FISH/immunostaining, the method enables the identification of the proximity of two different locations in chromatin, such as different chromatin regions or a chromatin region and a chromatin mark, in relation to structural hallmarks of the nucleus.

Given that complex biological processes, such as those represented by dynamic changes in chromatin structure, cannot be understood unless they are examined in single cells (9), there is a strong need for a new method that has the pros of 3C (or other C techniques) and DNA FISH technologies while minimizing their cons. Although a single-cell Hi-C method has recently been described, the resolution of chromatin proximities was very poor (10).

This study describes the ChrISP method, which not only explores chromatin fibre proximities beyond the current resolution of the microscope, but also allows an analysis of the frequencies of such features in cell populations. The new method is based on the analysis of proximities between chromatin fibres brought together in the context of higher order chromatin structures (Figure 1A) with a resolution that goes far beyond that of a conventional confocal microscope. It exploits features of the in situ proximity ligation assay (ISPLA) (11), but without the rolling circle amplification (RCA) step. This modification neutralizes steric hindrances provided by nuclear substructures and provides a continuum of signals rather than large dots typical of the rolling circle amplification step. DNA probes labeled with either digoxigenin or biotin are hybridized to formaldehyde-fixed cells followed by primary antibody detection. After the primary antibodies are bound by secondary antibodies conjugated to different oligonucleotide DNA sequences, backbone (long connector) and fluorescently labeled splinter (short connector) oligonucleotides are added. If two different secondary antibodies are within 170Å of each other, their respective conjugated oligonucleotides are close enough to allow the backbone and splinter oligonucleotides to simultaneously anneal to them, and these can then be ligated to each other to form a fluorescently labeled circular DNA molecule. The signal derived from the labeled splinter oligonucleotide will only survive subsequent washing if it has been ligated into the circular DNA. ChrISP represents a major improvement over existing techniques in terms of resolution at the light microscopic level and with a versatility that makes it suitable for numerous applications focusing on chromatin structures and associated marks as well as how these relate to nuclear hallmarks.

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