Fig 1.
FISH damages chromatin domain structure across the nucleus.
A) Representative image of a nucleus labeled with a chromosome 3 probe (magenta) and a DAPI nuclear counterstain (blue). The two chromosome 3 foci are denoted by a circle and an arrow. The image is a max projection of ~15 planes within a z-stack. Scale bar: 5 μm. B) The average D for each nucleus undergoing the cumulative 3D FISH protocol (following the full protocol by performing each step consecutively; light blue) and treated with the individual 3D FISH reagents (fixing cells first, then performing the step listed; dark blue) compared to a live cell control and a 4% PFA fixed cell control (gray). The 3D FISH protocol has seven main steps: (1) cell fixation with 4% PFA, (2) permeabilization with Triton X-100, (3) deproteinization with hydrochloric acid (HCl), (4) degradation of RNA using RNase A, (5) lowering DNA melting point through formamide treatment, (6) heat denaturation of DNA, and (7) hybridization of the probe to DNA during overnight incubation. There are no protective benefits to performing the protocol in order. After completing the whole protocol, the average D of nuclei is far below control cells. All data is an average of between 240–800 nuclei, across three biological replicates. Dashed lines within violins denote the 75th percentile, median, and 25th percentile from top to bottom. C) A comparison of brightfield and PWS microscopy images (brighter red indicates higher D) after treatment with each reagent. Some treatments of the 3D FISH protocol cause nuclei to appear vastly different, even on brightfield images. Since the nuclear membrane is visible after heat treatment and overnight incubation, it is unlikely that these treatments are completely degrading the nuclear membrane. However, it is impossible to visually detect the location of the nucleus after formamide treatment without using a nuclear counterstain. Scale bar: 5 μm. D) The probability distribution function for D of each pixel within all analyzed nuclei in the live cell control, the fixed cell control, and the formamide-treated cells. Formamide causes the PDF to shift towards a random distribution, centered around D ≈ 2.1. Formamide lowers the most probable D to ~1.8, much lower than the values observed in healthy cells.
Table 1.
Statistics of the change in D resultant from each 3D FISH reagent.
Fig 2.
Optimization of the 3D FISH protocol does not prevent or reverse the formamide-induced chromatin structural alterations.
A) Formamide causes changes to chromatin structure regardless of the mechanism or length of fixation. The most common fixative for FISH, 4% paraformaldehyde (PFA), is normally varied in the length of incubation with in vitro cell cultures to ensure that the cell remains stable over time and allows proper labeling of DNA with the desired probe. Increasing the incubation time indeed better preserves the original chromatin structure by reducing the change in D due to fixation. Other fixation reagents such as glutaraldehyde (GA) mixed with PFA, ethanol, and methanol seem to perform as well or better than increasing the incubation time. However, it is notable that ethanol fixation increases D, leading to formamide appearing to have less of an impact. Methanol, while having negligible effect during fixation, experiences the largest change because of formamide treatment. The 2% PFA with 2.5% GA solution has the least impact on D during fixation and does not experience as great a change from formamide. All data is an average of between 100–1000 nuclei, across three biological replicates. Dashed lines within violins denote the 75th percentile, median, and 25th percentile from top to bottom. B) Representative PWS microscopy images for live cell controls and fixed cells before and after formamide incubation (brighter red indicates higher D). Although the 10-minute 4% PFA fixation causes the largest change in average nuclear D values, qualitatively, the nuclei look the most like the chromatin structures visible in live cells. Most other stronger fixatives contain smaller high D clumps (chromatin packing domains) as compared to the live cells. After formamide treatment, however, none of the cells bear any resemblance to the original chromatin organization seen in live cells. Scale bar: 5 μm. C) The average nuclear D for two steps in the 3D FISH protocol that are commonly optimized to mitigate nuclear changes: heat denaturation and overnight incubation. Regardless of the temperature chosen for the heating step, D is close to 2, indicating that the temperature used for denaturing chromatin neither causes different chromatin structures nor reorganizes chromatin more than formamide treatment alone. Although the overnight incubation step shows a slight increase in D as the incubation time is increased, even a 48-hour incubation is not able to recover the original chromatin structure. Plotted data are from between 210 and 430 nuclei across three biological replicates. Dashed lines within violins denote the 75th percentile, median, and 25th percentile from top to bottom. D) PWS microscopy images were collected from nuclei treated with different temperatures or incubation times for the heat denaturation step and the overnight incubation step, respectively (brighter red indicates higher D). Scale bar: 5 μm.
Fig 3.
Alternate DNA sequence labeling methods have minimal impact on global nuclear chromatin organization.
A) The average D for nuclei labeled with either 3D FISH (light blue), RASER-FISH (light blue), or CRISPR-Sirius (dark blue) compared to a live cell control (gray) and a 4% PFA fixed cell control (gray). All data is an average of between 70 and 170 nuclei, across three biological replicates. Dashed lines within violins denote the 75th percentile, median, and 25th percentile from top to bottom. B) A comparison of brightfield and PWS microscopy images (brighter red indicates a higher D) for each DNA labeling method. RASER-FISH labeled cells are visually different in both Brightfield and PWS, potentially due to the two fixation steps and the presence of formamide in the hybridization buffer. CRISPR-Sirius labeled cells look identical to the unstained live cells. Scale bar: 5 μm. C) The probability distribution function for D of each pixel within the analyzed nuclei. 3D FISH dramatically shifts the PDF to lower D values, while RASER-FISH and CRISPR-Sirius have almost no impact. D) Representative images of nuclei labeled with each of the three tested DNA labeling protocols. A chromosome 3 probe was used in the 3D FISH-labeled cell and a chromosome 19 probe was used for the RASER-FISH-labeled cell. The CRISPR-Sirius cell was labeled using a sgRNA targeting the XXYLT1 gene with the MS2 aptamer and stained using Janelia Fluor 646 (JF646). All cells were counterstained with DAPI. The magenta foci are denoted by a circle and an arrow while the nucleus is pseudocolored in blue. Each image is a max projection of ~15 planes within a z-stack. Scale bar: 5 μm. E) Analysis of the labeling efficiency of each tested protocol. Between 15 to 20 confocal images containing nuclei were used to determine the number of foci within each nucleus. The 3D FISH protocol (N = 76 nuclei) had the most visible foci, followed by CRISPR-Sirius (N = 81 nuclei) had the fewest visible foci, and RASER-FISH (N = 99 nuclei). The control sample (N = 61 nuclei) had foci detected in one nucleus.
Table 2.
Comparison of DNA sequence labeling protocol effects on chromatin.