Journal: Journal of Biological Chemistry

Article Title: Growth Hormone Receptor Ubiquitination, Endocytosis, and Degradation Are Independent of Signal Transduction via Janus Kinase 2

doi: 10.1074/jbc.m103583200

Figure Lengend Snippet: FIG. 5. Visualization of Cy3-GH up- take by confocal microscopy. ts20 cells expressing wtGHR (A–C) or GHR4P-A (D–F) were incubated for 1 h at 30 °C (A and D) or 42 °C (C and F) or incubated with 20 M MG132 at 30 °C (B and E). Cy3-GH was then added for 30 min, the cells were washed and fixed, and the flu- orescence was visualized by confocal microscopy.

Article Snippet: Microscopy—Cy3-GH was prepared using a FluoroLink Cy3 label kit according to the supplier’s instructions (Amersham Pharmacia Biotech).

Techniques: Confocal Microscopy, Expressing, Incubation

Journal: Nucleic Acids Research

Article Title: Single-molecule imaging reveals a direct role of CTCF’s zinc fingers in SA interaction and cluster-dependent RNA recruitment

doi: 10.1093/nar/gkae391

Figure Lengend Snippet: CTCF does not block transcription by T7 polymerase. ( A ) T7-Pol bulk transcription assay on a PCR product containing a T7-promoter as well as 4× CBSs with the downstream motif pointing towards the promoter. The length and amount of produced RNA was measured for three independent experiments with and without CTCF and no significant reduction in amount of produced RNA was observed. ( B ) Same as (A) but with upstream motif pointing towards the promoter. ( C ) Illustration of in-vitro transcription assays and representative kymograms for T7-Pol moving down the DNA during transcription with 1 mM nucleotide concentrations (cyan = Cy3- RNA) measured at 100 ms illumination time. ( D ) same as (C) but after enrichment of 10 nM Alexa Fluor 660-CTCF on 4× CBSs. CTCF (green) and T7-Pol (cyan, RNA) are moving mutually. ( E ) same as (C) but after enrichment of SA-CTCF complexes containing 10 nM unlabeled CTCF and 100 nM SA-LD655 (magenta) on 4× CBSs. CTCF-SA complexes are pushed by T7-Pol. ( F ) Mean transcription velocities of T7-Pol alone and T7-Pol pushing different CTCF variants or CTCF-SA complexes. WT CTCF reduced transcription velocity, while no significant difference was found for ΔN, ΔC, SA1-CTCF and SA2-CTCF compared to T7-Pol velocities. ( G ) Representative kymograms of continuous transcription (top), pausing and stopping events (middle), and snapback of polymerases (bottom). ( H ) Fraction of processive transcription in case of single or multiple transcription events on one λ-DNA molecule ( N = 49/118 T7 control, 40/147 WT, 33/134 ΔN and 34/177 ΔC for single T7/multiple T7 transcription events). All CTCF variants impair transcription significantly in both cases of single and multiple T7 polymerases on one DNA. In absence of CTCF, multiple T7-Pols on one DNA impair transcription also significantly.

Article Snippet: For RNA recruitment experiments, 100 bp Cy3-UTP labeled RNA was generated using a PCR-product containing a T7-promoter site and the HiScribe T7 High Yield RNA Synthesis Kit (NEB) as well as Cy3-UTP (Jena Bioscience).

Techniques: Blocking Assay, Transcription Assay, Produced, In Vitro, Control

Journal: Nucleic Acids Research

Article Title: Single-molecule imaging reveals a direct role of CTCF’s zinc fingers in SA interaction and cluster-dependent RNA recruitment

doi: 10.1093/nar/gkae391

Figure Lengend Snippet: RNA recruitment by oligomeric CTCF. ( A ) 100 bp Cy3-UTP labeled RNA-loading (25 ng/μl) on CBS-enriched Alexa-Fluor 660-CTCF. Left: Experimental workflow. Right: TIRF microscopy at 100 ms illumination time of CBS-bound CTCF (green) before RNA load (top) and of Cy3-UTP labeled RNA (cyan) after RNA load (bottom). No RNA capture was observed. ( B ) Representative intensity curve of a two-step CTCF photobleaching event. Bleaching steps are illustrated by a black line. ( C ) Histogram of CTCF bleaching steps on 4× CBSs. Same as , added here for comparison. ( D ) 100 bp Cy3-UTP labeled RNA-loading (25 ng/μl) on clustered Alexa-Fluor 660-CTCF. Left: Experimental workflow. Right: TIRF microscopy at 100 ms illumination time of CTCF clusters (green) before RNA load (top) and of Cy3-UTP labeled RNA (cyan) after RNA load. RNA is recruited to CTCF clusters (white arrows). ( E ) Representative intensity curve of multi-step CTCF bleaching event of a CTCF-RNA cluster. Steps are illustrated by a black line. ( F ) Histogram of CTCF bleaching steps in CTCF-RNA clusters. ( G ) 10 nM ATTO-643-CTCF loading at RNA transcripts. Left: Experimental workflow. RNA transcripts were formed by loading T7-Pol and facilitating transcription as before. Right: TIRF microscopy at 100 ms illumination time of Cy3-UTP labeled RNA in RNA transcripts (cyan) before CTCF load (top) and of CTCF (green) after loading (bottom). CTCF is partially recognizing RNA transcripts (orange arrows), but is mainly coating the DNA (white arrows). ( H ) Enrichment of CTCF on 4× CBSs is not significantly different in presence or absence of RNA transcripts. ( I ) CTCF lifetime on RNA transcripts is similar to lifetimes on λ-DNA and significantly smaller than on 4× CBSs.

Article Snippet: For RNA recruitment experiments, 100 bp Cy3-UTP labeled RNA was generated using a PCR-product containing a T7-promoter site and the HiScribe T7 High Yield RNA Synthesis Kit (NEB) as well as Cy3-UTP (Jena Bioscience).

Techniques: Labeling, Microscopy, Comparison

Journal: Science Advances

Article Title: Condensed but liquid-like domain organization of active chromatin regions in living human cells

doi: 10.1126/sciadv.adf1488

Figure Lengend Snippet: ( A ) Model of Cy3-labeled DNA structure with a side and top view (provided by Y. Joti at Spring-8). ( B ) FACS profiles of HeLa cells synchronized at the G1-S boundary, using a double thymidine block, and released in fresh medium at the indicated time point after release. ( C ) HeLa cell DNA replication foci labeled with a pulse of Cy3-dCTP (second row) 1 hour (first column) and 5 hours (second column) after release from a double thymidine block. The nuclei were stained with DAPI (top row, blue). ( D ) A scheme of Cy3-dCTP–labeled DNA immuno-purification and analysis. Synchronized HeLa cells in early and mid S phase were pulse-labeled with Cy3-dCTP. DNA from each cell fraction was purified, fragmented, and immunoprecipitated with an anti-Cy3 antibody. The enriched DNA fractions are indexed, amplified, and sequenced. ( E ) Visualization of Cy3-dCTP–labeled regions on chromosome 10 using the Integrative Genomics Viewer (IGV) Browser. ( F ) Cy3-labeled genome regions are enriched with active histone marks and excluded from an inactive mark: view from chr6_5512490–8384778 (also see fig. S3, A and B). ( G ) Venn diagrams show overlapped size (in bp) between Cy3-labeled regions and contact domains (left) and Cy3-labeled regions and A-compartment (right).

Article Snippet: For each assay, 200 ng of an enhanced green fluorescent protein (EGFP) gene polymerase chain reaction (PCR) fragment amplified by the Cy3 PCR labeling kit (PP-301S-CY3; Jena Bioscience) and the linearized plasmid (pGEM-T Easy plasmid digested with EcoRI) were resuspended in PBS buffer containing 300 ng of anti-Cy3 antibody (sc-166894; Santa Cruz Biotechnology).

Techniques: Labeling, Blocking Assay, Staining, Purification, Immunoprecipitation, Amplification

Journal: Science Advances

Article Title: Condensed but liquid-like domain organization of active chromatin regions in living human cells

doi: 10.1126/sciadv.adf1488

Figure Lengend Snippet: ( A ) Schematic of replication foci labeling with fluorescent nucleotides at high concentrations (left) and single-nucleosome labeling with a low concentration of fluorescent nucleotides (right). The inset shows that single-nucleosome labeling is based on the incorporation of a single nucleotide conjugated with fluorescent dye. This cartoon is simplified and fluorescent labeling can also be in the linker DNA region. ( B ) Experimental scheme for labeling and imaging. ( C ) Image of single-nucleosome dots labeled with Cy3-dCTP in a live HeLa nucleus. The bar represents 1 μm. See also movies S1 and S2. ( D ) Oblique illumination microscopy system. Only a thin optical layer within the nucleus (red) was illuminated with a low background using a sheet light (green). The cartoon was reproduced and modified with permission from . ( E ) Representative single-step photobleaching of a Cy3-labeled nucleosome dot. The vertical axis represents the fluorescence intensity and the horizontal axis is the tracking time series. A.U., arbitrary units. ( F ) Three representative trajectories of single-nucleosomes. The bar represents 100 nm.

Article Snippet: For each assay, 200 ng of an enhanced green fluorescent protein (EGFP) gene polymerase chain reaction (PCR) fragment amplified by the Cy3 PCR labeling kit (PP-301S-CY3; Jena Bioscience) and the linearized plasmid (pGEM-T Easy plasmid digested with EcoRI) were resuspended in PBS buffer containing 300 ng of anti-Cy3 antibody (sc-166894; Santa Cruz Biotechnology).

Techniques: Labeling, Concentration Assay, Imaging, Microscopy, Modification, Fluorescence

Journal: Science Advances

Article Title: Condensed but liquid-like domain organization of active chromatin regions in living human cells

doi: 10.1126/sciadv.adf1488

Figure Lengend Snippet: ( A ) Displacement (movement) distributions of single-nucleosomes labeled with Cy3-dCTP at active chromatin (euchromatic) regions in live HeLa cells for 50 ms ( n = 27 cells). ( B ) MSD plots (±SD among cells) of Cy3-labeled nucleosomes (red, n = 27 cells) and H2B-Halo–labeled nucleosomes [blue, data were reproduced from ] in living HeLa cells from 0 to 3 s. The Kolmogorov-Smirnov test was used to determine P values. * P < 0.05 for Cy3-dCTP versus H2B-Halo ( P = 0.049). *** P < 0.0001 for Cy3 nucleosomes versus FA-fixed Cy3 nucleosomes ( P = 2.1 × 10 −13 ), H2B-Halo nucleosomes versus FA-fixed H2B-Halo nucleosomes ( P = 5.7 × 10 −6 ). Not significant (N.S.) for FA-Cy3 nucleosomes versus FA-H2B-Halo nucleosomes ( P = 0.37). R c values (estimated radius of constraint of the nucleosome motion) are also shown. R c values of Cy3 and H2B-Halo nucleosomes are also significantly different: P = 0.049 by Kolmogorov-Smirnov test. ( C ) Log-log plots of MSD data shown in (B). The indicated straight lines were fitted using the data from 0.05 to 0.5 s. The plots cannot be fitted linearly beyond this time range, suggesting the motion mode changes over 0.5 s. ( D ) Schematic for nucleosome motion angle distribution analysis . Schematics were reproduced from . ( E ) Angle distributions of Cy3 (left) and H2B-Halo nucleosomes (right). Moving angles of single nucleosomes are biased toward 180° and the AC is negative, indicating that they are often pulled back to their original positions. H2B-Halo data were reproduced from .

Article Snippet: For each assay, 200 ng of an enhanced green fluorescent protein (EGFP) gene polymerase chain reaction (PCR) fragment amplified by the Cy3 PCR labeling kit (PP-301S-CY3; Jena Bioscience) and the linearized plasmid (pGEM-T Easy plasmid digested with EcoRI) were resuspended in PBS buffer containing 300 ng of anti-Cy3 antibody (sc-166894; Santa Cruz Biotechnology).

Techniques: Labeling

Journal: Science Advances

Article Title: Condensed but liquid-like domain organization of active chromatin regions in living human cells

doi: 10.1126/sciadv.adf1488

Figure Lengend Snippet: (A) Diagram of dual-color visualization of single-nucleosome dynamics. (B) Two hypothetical models of higher-order chromatin structure, condensed organization (left), or extended loops (right). In both models, cohesin holds the chromatin fiber to make loop(s). Correlation analysis of the two single-nucleosome movements [G (green) and R (red); their hypothetical movements are shown on the right] can be used to distinguish between these two possible models in living cells. ( C ) Scheme for Alexa488-dUTP (green) and Cy3-dCTP (red) incorporations during early DNA replication for dual-color labeling of nucleosomes. (D) Schematic for dual-color imaging with a beam splitter system (W-VIEW GEMINI, Hamamatsu Photonics). The images of two single-nucleosomes with different colors were acquired with a single sCMOS camera (left half, green color; right half, red color). ( E ) Representative images of single-nucleosomes labeled with Alexa488-dUTP (left) or Cy3-dCTP (right) in a living HeLa nucleus. The bar represents 1 μm. The box regions are enlarged to the right. The bar represents 500 nm.

Article Snippet: For each assay, 200 ng of an enhanced green fluorescent protein (EGFP) gene polymerase chain reaction (PCR) fragment amplified by the Cy3 PCR labeling kit (PP-301S-CY3; Jena Bioscience) and the linearized plasmid (pGEM-T Easy plasmid digested with EcoRI) were resuspended in PBS buffer containing 300 ng of anti-Cy3 antibody (sc-166894; Santa Cruz Biotechnology).

Techniques: Labeling, Imaging

Journal: Science Advances

Article Title: Condensed but liquid-like domain organization of active chromatin regions in living human cells

doi: 10.1126/sciadv.adf1488

Figure Lengend Snippet: (A) Four representative trajectory sets of two nucleosomes labeled with green (Alexa488-dUTP) and red (Cy3-dCTP). While the upper two sets show trajectories of two closely localized nucleosomes, the lower two represent distant nucleosomes. See also movies S5 to S8. (B) Histograms of congruence coefficient r c calculated between the two single-nucleosomes (green and red) whose averaged distances were in the ranges of 0 to 50 nm (median r c = 0.75), 50 to 100 nm (median r c = 0.48), 100 to 150 nm (median r c = 0.29), 150 to 200 nm (median r c = 0.08), and over 200 nm (median r c = 0.05) for 500 ms and the random control (median r c = 0.02). (C) Frequency plots of nucleosome pairs (green and red) with r c > 0.6 versus the pair distances. The dashed line shows the frequency of randomly collected nucleosome pairs with r c > 0.6. (D) Model of nucleosomes formed into a condensed domain with ~150 nm diameter.

Article Snippet: For each assay, 200 ng of an enhanced green fluorescent protein (EGFP) gene polymerase chain reaction (PCR) fragment amplified by the Cy3 PCR labeling kit (PP-301S-CY3; Jena Bioscience) and the linearized plasmid (pGEM-T Easy plasmid digested with EcoRI) were resuspended in PBS buffer containing 300 ng of anti-Cy3 antibody (sc-166894; Santa Cruz Biotechnology).

Techniques: Labeling, Control

Journal: Science Advances

Article Title: Condensed but liquid-like domain organization of active chromatin regions in living human cells

doi: 10.1126/sciadv.adf1488

Figure Lengend Snippet: ( A ) Schematic for two-point MSD. ( B ) Two-point MSD plots (±SD among ≥3 clusters) between Cy3- and Alexa488-nucleosomes whose averaged distances ( d ) were 0 to 50 nm in living siRAD21-treated HeLa cells (red, 54 pairs), untreated cells (black, 84 pairs), and TSA-treated cells (blue, 42 pairs). Ten or more pairs were grouped as a cluster. ( C ) Two-point MSD plots (±SD among ≥3 clusters; d , 50 to 150 nm) in living siRAD21-treated HeLa cells (red, 33 pairs), untreated cells (black, 95 pairs), and TSA-treated cells (blue, 35 pairs). ( D ) Schematic for replication foci labeled with Cy3-dCTP at high concentrations (left) and their representative images in a living HeLa nucleus (right). The square region is magnified at lower right. Note that each dot corresponds to one replication focus, consisting of one or a few contact domains. Also see movie S15. ( E ) Schematic for single-nucleosome imaging and their typical representation. Note that each dot corresponds to a single-nucleosome. ( F ) MSD plots (±SD among cells) of early replication foci (light blue, n = 12 cells) and single-nucleosomes at the early replicated regions (orange, n = 19 cells) in living HeLa cells. ** P < 0.001 by Kolmogorov-Smirnov test for foci versus single nucleosomes ( P = 4.8 × 10 −4 ). ( G ) Schematic for FRAP. ( H ) Representative images of Cy3-labeled early replication foci before, just after and 30 min after photobleaching. Photobleached region is shown by a yellow dotted line. XY -movement of the cell is corrected. ( I ) Mean normalized fluorescence intensity over time (fluorescence recovery curve) for Cy3-labeled early replication foci, n = 10 cells. ( J ) Mean normalized fluorescence intensity of photobleached region before, just after, and 30 min after photobleaching ( n = 11 cells). Also see movie S16.

Article Snippet: For each assay, 200 ng of an enhanced green fluorescent protein (EGFP) gene polymerase chain reaction (PCR) fragment amplified by the Cy3 PCR labeling kit (PP-301S-CY3; Jena Bioscience) and the linearized plasmid (pGEM-T Easy plasmid digested with EcoRI) were resuspended in PBS buffer containing 300 ng of anti-Cy3 antibody (sc-166894; Santa Cruz Biotechnology).

Techniques: Labeling, Imaging, Fluorescence