Journal: Journal of Cell Science

Article Title: OptoLoop – an optogenetic tool to probe the functional role of genome organization

doi: 10.1242/jcs.264574

Figure Lengend Snippet: Optogenetic manipulation of proximity between repetitive genomic loci. (A) Scheme of OptoLoop consisting of a fusion between dCas9 and the optogenetic protein CRY2. OptoLoop is targeted to specific genomic loci by introducing specific sgRNAs. CRY2–CRY2 interactions activated by blue light bridge targeted loci to form a chromatin loop. (B) Left panel, region of chromosome 19 showing sgIDR3 and sgTCF3 target sites, representative Hi-C contact map (data from ) and BACs used in DNA-FISH to label the IDR3 (magenta) and TCF3 loci (green). Right panel, mCherry channel images of U2OS dCas9–3XmCherry–CRY2 cells transfected with sgIDR3 and sgTCF3, kept in dark or illuminated with blue light for 3 h (1 s pulses every 10 s), and fixed. Scale bars: 5 µm. (C) Left panel, representative image of DNA-FISH for IDR3 and TCF3 with specific BAC FISH probes in U2OS cells. Right panel represents a single cell highlighted in left panel (yellow box); the expansion shows a single allele in this cell. Dashed line denotes the distance between the two FISH signals. Scale bars: 20 µm (left panel), 5 µm (right panel), 1 µm (expansion). (D) IDR3–TCF3 distances, calculated for U2OS dCas9–mCherry–CRY2 polyclonal cells transfected with indicated combinations of sgIDR3 and sgTCF3, kept under dark or illuminated for 3 h (1 s pulses every 10 s). Violin plot corresponds to a representative experiment, with black lines representing median distances. Bar plot represents means of two independent experiments. Each dot represents the median of typically 5000–10,000 alleles analyzed per experiment. (E) Fraction of alleles with IDR3-TCF3 distance <0.27 µm measured from DNA-FISH images for U2OS dCas9–mCherry–CRY2 polyclonal cells and three clones of U2OS dCas9–3XmCherry–CRY2 cells, transfected with indicated combinations of sgIDR3 and sgTCF3, and kept in dark or illuminated for 3 h (1 s pulses every 10 s). Each dot represents the fraction of typically 5000–10,000 alleles analyzed per experiment. Bars represent means of two or three independent experiments. (F) Measurement of cell-to-cell heterogeneity in loop formation. Bars with green shades: observed fraction of cells with none, one or both alleles with IDR3–TCF3 distance <0.27 µm obtained from a representative experiment shown in E with 2500–5000 cells analyzed per sample. Bars with magenta shades: expected fraction of cells with none, one or both alleles with IDR3–TCF3 distance <0.27 µm assuming that alleles from a same cell are independent between each other (Eqn 2). * P <0.05; *** P <0.001; ns, not significant [two-way ANOVAs followed by post-hoc Tukey tests (D,E); paired two-tailed t -test (E); chi-squared test (F)].

Article Snippet: NIH3T3 (mouse fibroblasts, ATCC #CRL-1658), U2OS (from human osteosarcoma, ATCC #HTB-96), HeLa (from human cervical adenocarcinoma, ATCC #CRM-CCL-2) and Lenti-X HEK-293T (from human embryonic kidney, cat. #632180 from Takara Bio, Japan) cell lines were cultured in Dulbecco's modified Eagle's medium (Gibco, Waltham, MA, USA) supplemented with 10% (15% for NIH3T3) fetal bovine serum (Gibco, Waltham, MA, USA) plus 100 IU/ml penicillin and 100 μg/ml streptomycin (Gibco, Waltham, MA, USA) at 37°C in a humidified atmosphere with 5% CO 2 .

Techniques: Hi-C, Transfection, Single Cell, Clone Assay, Two Tailed Test

Journal: Journal of Cell Science

Article Title: OptoLoop – an optogenetic tool to probe the functional role of genome organization

doi: 10.1242/jcs.264574

Figure Lengend Snippet: Benchmarking OptoLoop against a previous optogenetic manipulation tool. (A) Scheme of LADL consisting of soluble CRY2wt and a fusion between dCas9 and the CRY2 partner CIBN. dCas9–CIBN tethers specific genomic loci and light-activation induces both CRY2–CRY2 and CRY2–CIBN interactions bridging the targeted loci to form a loop. (B) Images of U2OS cells expressing dCas9–3XGFP–CIBN and mCherry fused to CRY2wt or CRY2olig, transfected with sgIDR3 and sgTCF3, and fixed after being kept under dark or illuminated with blue light pulses for 3 h (1 s pulses every 10 s). White arrows indicate FISH signals corresponding to IDR3–TCF3 loci. Scale bar: 5 µm. (C) Fraction of alleles with IDR3–TCF3 distance <0.27 µm measured from DNA-FISH images of U2OS cell lines stably expressing dCas9-3XGFP-CRY2 and mCherry-CRY2olig (LADL, clone #7) or dCas9-3XmCherry-CRY2 (OptoLoop, clone #3), transfected with sgIDR3 and sgTCF3, and kept under dark or illuminated with blue light for 3 h (1 s pulses every 10 s). Each dot represents the fraction of 5000–7000 alleles analyzed per experiment. Bars represent the means of three independent experiments. * P <0.05; ** P <0.01; *** P <0.001 (two-way ANOVA followed by post-hoc Tukey test).

Article Snippet: NIH3T3 (mouse fibroblasts, ATCC #CRL-1658), U2OS (from human osteosarcoma, ATCC #HTB-96), HeLa (from human cervical adenocarcinoma, ATCC #CRM-CCL-2) and Lenti-X HEK-293T (from human embryonic kidney, cat. #632180 from Takara Bio, Japan) cell lines were cultured in Dulbecco's modified Eagle's medium (Gibco, Waltham, MA, USA) supplemented with 10% (15% for NIH3T3) fetal bovine serum (Gibco, Waltham, MA, USA) plus 100 IU/ml penicillin and 100 μg/ml streptomycin (Gibco, Waltham, MA, USA) at 37°C in a humidified atmosphere with 5% CO 2 .

Techniques: Activation Assay, Expressing, Transfection, Stable Transfection