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Live visualization of chromatin dynamics with fluorescent TALEs

Nature Structural & Molecular Biology volume 20, pages 1321–1324 (2013)Cite this article

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Abstract

The spatiotemporal organization of genomes in the nucleus is an emerging key player to regulate genome function. Live imaging of nuclear organization dynamics would be a breakthrough toward uncovering the functional relevance and mechanisms regulating genome architecture. Here, we used transcription activator–like effector (TALE) technology to visualize endogenous repetitive genomic sequences. We established TALE-mediated genome visualization (TGV) to label genomic sequences and follow nuclear positioning and chromatin dynamics in cultured mouse cells and in the living organism. TGV is highly specific, thus allowing differential labeling of parental chromosomes by distinguishing between single-nucleotide polymorphisms (SNPs). Our findings provide a framework to address the function of genome architecture through visualization of nuclear dynamics in vivo.

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Figure 1: Fluorescent TALE allows visualization of major satellites in living cells.
Figure 2: Centromeres and telomeres are specifically labeled by fluorescent TALEs.
Figure 3: Differential labeling of parental chromosomes through distinction of SNPs by fluorescent TALEs.

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Acknowledgements

We thank R. Feil (Institute of Molecular Genetics, University of Montpellier, Montpellier, France) for providing hybrid SF1 ES cells, I. Jackson (Medical Research Council, Edinburgh, UK) and Y. Matsuda (Nagoya University, Nagoya, Japan) for M. spretus genomic DNA, H. Kimura (Osaka University, Osaka, Japan) for anti-H3K9me3 antibody, K. Okamoto (Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan) for anti-TRF1 antibody, A. Sakakibara (Nagoya University School of Medicine, Nagoya, Japan) for the H2B-iRFP construct, K. Yamagata (Osaka University) for pcDNA3.1 H2B-mRFPpA83, C. Ebel (IGBMC, Strasbourg, France) for advice on fluorescence-activated cell sorting, M. Koch (IGBMC) for technical advice on imaging, I. Sumara (IGBMC) and T. Yamamoto (Hiroshima University, Hiroshima, Japan) for helpful discussions, P. Lansdorp (Terry Fox Laboratory, BC Cancer Agency, Vancouver, Canada) for advice on telomere analysis and A. Burton (IGBMC) and A. Boskovic (IGBMC) for critical reading of the manuscript. Y.M. was supported by an European Molecular Biology Organization (EMBO) long-term fellowship (ALTF864-2008, 2009) and a Japan Society for the Promotion of Science postdoctoral fellowship (2010–2011). Work in M.-E.T-P.'s laboratory is funded by Agence Nationale de la Recherche (ANR-09-Blanc-0114), EpiGeneSys Network of Excellence, EMBO Yong Investigator Programme and an European Research Council Starting Grant (ERC-Stg 'NuclearPotency').

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Authors and Affiliations

  1. Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Université de Strasbourg, Illkirch, France

    Yusuke Miyanari, Céline Ziegler-Birling & Maria-Elena Torres-Padilla

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  1. Yusuke Miyanari
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  2. Céline Ziegler-Birling
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  3. Maria-Elena Torres-Padilla
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Contributions

Y.M. designed the project and performed experiments. C.Z.-B. performed experiments. M.-E.T.-P. directed the study. Y.M. and M.-E.T.-P. wrote the manuscript.

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Correspondence to Yusuke Miyanari or Maria-Elena Torres-Padilla.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Validation of the length for target sequence.

(a and b) To validate the binding affinity of TALEs, we designed 4 different TALEs against 13, 15, 18, and 20 nt of MajSat sequence and fused them with the VP64 activation domain (B13, B15, B18, and B20, respectively). The firefly luciferase reporter plasmid contains tandem repeats of the binding sequence upstream of a minimal CMV promoter. (b) The indicated TALE-VP64 fusions and the reporter plasmid were transfected to 293T cells. Relative luciferase activity normalized to Renilla luciferase activity is shown. NC indicates mock transfection control. The mean ± s.d. of three independent biological replicates is shown. We found progressive increase of luciferase activity with the length of the target sequence, in agreement with a previous report 8. (c) The indicated TALE-mClover constructs were transfected in mouse ES cells. Confocal images of TALE-mClover and DAPI are shown. line-profiles (RGB profile) of fluorescence intensities for DAPI (black) and mClover (green) from the A-B line drawn in images are shown in right. Note that B13 displays slightly higher background signal in nucleoplasm contrasting to other TALEs. Scale bar, 2 μm.

Supplementary Figure 2 Characterization of ES cell lines stably expressing TALE against major satellite sequences.

(a) Representative images of cell-line YM9B15 stably expressing TALE-Ty1 against MajSat sequence. The cells were stained with anti-Ty1 antibody (green) and with DAPI (gray). Fluorescent signals for Ty1 were colocalized with DAPI-dense heterochromatin domains, suggesting that the TALE-Ty1 specifically binds to target major satellite sequences. Scale bar, 2 μm. (b) ChIP assay with indicated cell-lines for H3K9me3 and histone H3 on MajSat sequence. The enrichment of both H3K9m3 and histone H3 were indistinguishable between YM9B15 and parental E14. The mean ± s.d. of three independent biological replicates is shown. (c) Representative images of the two clones (E142C-02 and E142C-05) showing fluorescent signals of TALE-mClover_MajSat and Histone H2B fused with tandem iRFP, H2B-tdiRFP. Scale bar, 2 μm. (d) Cell-cycle analysis by FACS with indicated cell-lines stained with propidium Iodide (PI) to measure DNA-content per nucleus. (e) FACS data was analyzed using ModFit to classify cell-cycle phases into S, G2/M, and G1 phase. The proportion of each phase for the indicated cell-lines is shown. (f) Representative images of chromosome spreads from clone E142C-02 and E142C-05 showing 40 acrocentric chromosomes. (g) Percentage of cells displaying normal karyotype consisting of 40 chromosomes. More than 75% of cells for all the clones displayed normal karyotype, indicating that chromosome segregation was not affected by expression of the TALE-mClover_MajSat.

Supplementary Figure 3 Expression of TALE against telomeres had no effect on cell-cycle profile or karyotype.

(a) Two ES cell-lines stably expressing TALE-mClover_telomere (E14Telo1 and E14Telo2), TALE-mRuby_MajSat, and H2B-tdiRFP, and perental E14 were analyzed by FACS. Histograms of PI staining are shown. (b) FACS data was analyzed using ModFit to classify cell-cycle phases into S, G2/M, and G1 phase. The proportion of each phase for the indicated cell-lines is shown. Note that the proportion of cells in each cell-cycle is indistinguishable between parental E14 and the two clones expressing TALE-mClover_telomere. Shown is a representative experiment of two independent biological replicates. (c) Percentage of cells displaying normal karyotype consisting of 40 chromosomes. More than 80% of the cells from the clones stably expressing TALE-mClover_telomere displayed normal karyotype, similarly to the parental ES cell line, indicating that chromosome segregation was not affected by expression of the TALE-mClover_telomere. n indicates the number of cells analyzed for each clone. (d and e) Relative telomere length is analyzed by Q-FISH. (d) Representative images of indicated cell-line showing fluorescent signal for telomere Q-FISH (magenta) and DAPI (gray). (e) Relative telomere length is shown with s.d. n indicates number of chromosome spread analyzed. *, P = 0.047, ** P = 0.087 (Student's t-test). (f) Quantitative RT-PCR analysis for TALE-mClover_telomere in indicated cell-lines. PCR was normalized using Gapdh mRNA levels. Shown are the mean ± s.d., n = 3.

Supplementary Figure 4 Detection of SNPs with TALEs.

(a) We analyzed sequencing data from 16 clones of Mus spretus minor satellite using Tandem repeat finder (http://tandem.bu.edu/trf/trf.html)31 and found that the minor satellite sequence has a basic higher order repeat of 120 bp, composed of two 61 bp (top) and 59 bp (bottom) monomers. The sequence conservations between sequence reads of these monomers are shown as the height of a stack of letters at each base position. The sequence data is provided in Supplementary Table 2. (b) Alignment of minor satellite sequence of Mus musculus (GenBank: Z22170.1) and Mus spretus is shown. Asterisks mean the position of sequence differences. (c) Alignment of minor satellite sequence of Mus musculus (top) and Mus spretus (bottom). Asterisks indicate the positions of SNPs between them. Arrows (mi01-mi09) indicate targeting sequence of TALEs. mi01-06 are directed against Mus spretus sequence. mi07 was designed against Mus musculus sequence. mi08 and mi09 are directed against the identical sequences. (d and e) Alignments of minor satellite sequence of Mus musculus and Mus spretus are indicated at the top, together with the targeting sequence used to design the TALE-VP64 (arrows). 293T cells were transfected with the indicated TALE-VP64 plasmids and the firefly reporter plasmid containing the TALE targeting sequence. Relative luciferase activity representing the binding affinity of TALEs is shown at the bottom. The mean ± s.d. of three independent biological replicates is shown. (d) Binding affinity of TALEs targeting MinSat sequence of Mus musculus and Mus spretus harboring two SNPs is shown. TALE specifically binds to corresponding origin of MinSat, indicating these TALEs distinguish two nucleotide differences. (e) Binding affinities of TALEs against mi08 and mi09 harboring one SNP is shown. Both TALEs bound specifically to MinSat of Mus spretus, suggesting that TALEs can recognize a single nucleotide difference. (f) Representative images of the pericentromeric region of mitotic chromosomes derived from Mus musculus (top) and Mus spretus (bottom) in SF1 cells expressing TALE-mClover (green) recognizing the MajSat sequence and TALE-mRuby2 (magenta) recognizing the indicated MinSat sequence. Large fluorescent foci of TALE-mClover were used as reference to distinguish Mus musculus or Mus spretus chromosomes, respectively. Target genome and number of SNPs for indicated TALEs are indicated above and below the images, respectively. (g) Representative images of mitotic chromosome of SF1 cells expressing the indicated TALE-mRuby2 against MinSat sequence (red) together with the TALE-mClover against the MajSat sequence (green). Scale bar, 2 μm.

Supplementary Figure 5 Characterization of SF1 cell lines expressing fluorescent TALEs.

(a) Representative images of SF1 cell-lines (SF13C-03, -04, and -05) stably expressing TALE-mClover (green) against MajSat seqeuence, TALE-mRuby2 (red) against Mus spretus MinSat sequence (mi01 in Supplementary Fig. 4), and H2B-tdiRFP (gray). (b) The images on the left show representative chromosome spreads of SF13C-03, 04, and 05 cells. The table on the right represents the percentage of cells displaying normal karyotype consisting of 40 chromosomes. More than 75% of cells for all the clones displayed normal karyotype, indicating that chromosome segregation does not seem to be affected by expression of these TALEs. n indicates number of cell analyzed. Scale bar, 5 μm.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 3433 kb)

Supplementary Table 1

Supplementary Table 1 (XLSX 47 kb)

Supplementary Table 2

Supplementary Table 2 (XLSX 25 kb)

Supplementary Table 3

Supplementary Table 3 (XLSX 32 kb)

Live imaging of mouse ES cells transiently expressing mClover-fused TALE_MajSat.

Live imaging of mouse ES cells transiently expressing mClover-fused TALE_MajSat (green) and H2B-mRFP (red). Images were acquired every 8 min over 90 frames (12 hrs). (AVI 369 kb)

Time-lapse imaging of ES cell line (E142C-05) stably expressing TALE-mClover against MajSat and H2B-tdiRFP.

Time lapse imaging of ES cell-line (E142C-05) stably expressing TALE-mClover against MajSat (green) and H2B-tdiRFP (red). Images were acquired every 3 min over 200 time frames (10 hrs). (AVI 7241 kb)

Serial section of confocal images of mitotic ES cells expressing TALE-mClover_MajSat and TALE-mRuby2 _MinSat.

Serial section of confocal images of mitotic ES cells expressing TALE-mClover (green) and TALE-mRuby2 (red) against MajSat and MinSat sequences, respectively. DNA was stained with DAPI (gray). (AVI 3287 kb)

Serial section of confocal images of mitotic ES cells expressing TALE-mClover_MajSat and TALE-mRuby2 _telomere.

Serial section of confocal images of mitotic ES cells expressing TALE-mClover (green) and TALE-mRuby2 (red) against MajSat and telomere sequences. DNA was stained with DAPI (gray). (AVI 4788 kb)

Live imaging of SF1 ES cells transiently expressing TALE-mClover_MajSat, TALE-mRuby2_MinSat and H2B-tdiRFP.

Live imaging of SF1 ES cells transiently expressing TALE-mClover_MajSat (green), TALE-mRuby2_MinSat (red, m01 in Supplementary Fig. 4), and H2B-tdiRFP (gray). Images were acquired every 10 min over 72 frames (12 hrs). (AVI 3395 kb)

Live imaging of SF1 ES cells-line (SF13C-05) stably expressing TALE-mClover_MajSat, TALE-mRuby2_MinSat and H2B-tdiRFP.

Live imaging of SF1 ES cells-line (SF13C-05) stably expressing TALE-mClover_MajSat (green), TALE-mRuby2_MinSat (red, m01 in Supplementary Fig. 4), and H2B-tdiRFP (gray). Images were acquired every 4 min over 180 frames (12 hrs). (AVI 6516 kb)

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Miyanari, Y., Ziegler-Birling, C. & Torres-Padilla, ME. Live visualization of chromatin dynamics with fluorescent TALEs. Nat Struct Mol Biol 20, 1321–1324 (2013). https://doi.org/10.1038/nsmb.2680

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