wilcoxon rank sum test  (MathWorks Inc)

Journal: bioRxiv

Article Title: Mechanisms of transcription control by distal enhancers from high-resolution single-gene imaging

doi: 10.1101/2023.03.19.533190

Figure Lengend Snippet: ( A ) Schematic of the Klf4 locus integrated with 24×MS2 (green rectangle) and representative image of a nucleus with an actively transcribed locus. Black rectangles are exons and gray rectangles are 5′ and 3′ UTR. The stem loops formed by MS2 RNA can be recognized by MCP-mNeonGreen. The yellow arrow points to Klf4 nascent transcription site. ( B ) Rad21-Halo staining showing the time-course of treatment with dTAG-13 (400nM). Control cells are treated with DMSO for 6 hrs. Scale bar = 3μm. The left panel shows quantification of relative Rad21 nuclear levels, normalized to DMSO. ( C ) Distribution of Klf4 bursts in DMSO- ( n =119 traces from 303 cells) and dTAG-treated ( n =110 traces from 780 cells) Rad21-FKBP F36V -halo KMG cells. Each data point represents the probability that a certain number of bursts, n bursts , was observed in 1 hr. Solid lines indicate fits to a Poisson distribution for the data with n bursts ≥1. The resulting two parameters (A, r ) are: DMSO (0.6, 2.29), dTAG (0.28, 1.59). ( D ) Box plots of burst amplitude and duration. Each gray dot represents one burst. Data points are from 3 independent experiments with total n = 160 and 117 bursts for DMSO and dTAG, respectively. p-values are calculated based on a Wilcoxon rank-sum test. ( E ) Two intensity traces of transcription sites from DMSO- and dTAG-treated Rad21-FKBP F36V -Halo KMG cells. ( F ) Profiles of H3K27ac, Rad21 and CTCF ChIPseq signals at the Klf4 Locus (published datasets GSM1526287, GSM2418859 and GSM2418860, respectively.). The red and yellow rectangles indicate the positions of DNA FISH probes (tagging promoter and −55kb enhancer regions, respectively). Each set of probes spans around 3kb. ( G ) 3D promoter-enhancer distances based on OligoPAINT FISH, for DMSO vs. dTAG treated KMG cells (mean ± SD: WT 240±132, dTAG 305 ± 140). n = 280 and 257, respectively. ( H ) Left: Cumulative distribution of enhancer–promoter 3D distances in Rad21-FKBP F36V -halo KMG cells at the Klf4 loci. Right: relative contact frequency distribution estimated from the DMSO/dTAG ratio of cumulative distance distributions. Grey shaded area indicates binomial 95% confidence intervals. ( I ) Schematic of 42 kb truncation between Klf4 promoter and +55 kb downstream enhancer (42kDel). (J) Left: 3D enhancer-promoter distances (mean ± SD: WT 237±111, 42kDel 205 ± 121). Right: relative contact frequency, estimated by the ratio 42kDel/WT of cumulative distance distributions. Grey shaded area indicates binomial 95% confidence intervals. ( K ) (Top): Two intensity traces of transcription sites from WT and 42kDel KMG cells. (Bottom): Probability of observing certain number of bursts in an 1 hr observation time, in WT ( n =93 traces from 85 cells) and 42kDel ( n =110 traces from 104 cells) KMG cells. Solid lines: fits to Poisson distribution, for the data with n bursts ≥1.

Article Snippet: For significance testing, Wilcoxon rank sum statistical tests were performed in MATLAB.

Techniques: Staining, Control

Journal: bioRxiv

Article Title: Mechanisms of transcription control by distal enhancers from high-resolution single-gene imaging

doi: 10.1101/2023.03.19.533190

Figure Lengend Snippet: ( A ) Two intensity traces of transcription sites from DMSO- and dTAG-treated Tbp-Trf2-FKBP F36V KMG cells. ( B ) Probability of observing certain number of bursts in an 1 hr observation time, in DMSO- ( n =114 traces from 161 cells) and dTAG-treated ( n =72 traces from 738 cells) Tbp-Trf2-FKBP F36V KMG cells. Solid lines: fits to Poisson distribution, for the data with n bursts ≥1. The resulting two parameters (A, r ) are: DMSO (0.44 and 1.91), dTAG (0.12 and 0.49). ( C ) Box plots of burst amplitude and duration. Each gray dot represents one burst. Data points are from two independent experiments with total n = 175 and 73 bursts for DMSO and dTAG, respectively. p-values are calculated based on a Wilcoxon rank-sum test. ( D ) Heat-map of fold-change of bursting parameters (non-bursting population, frequency, amplitude and duration) from different perturbations in KMG cells, including dTAG induced degradation (Rad21, Sox2, Tbp-Trf2, Taf1, Med19, Gtf2f1 and CDK7), Halo-PROTAC-E induced partial degradation (40nM) of Rpb1, small amount of inhibitors [100nM THZ1 (CDK7i) and triptolide (XPBi), 5nM NVP-2(CDK9i)], and JQ1. Data are normalized by Log2 (control/treatment). The data include 2–3 independent experiments. ( E ) Multi-step burst initiation cascade schematic, including the early phases of the Pol II transcription cycle.

Article Snippet: For significance testing, Wilcoxon rank sum statistical tests were performed in MATLAB.

Techniques: Control

Journal: bioRxiv

Article Title: Mechanisms of transcription control by distal enhancers from high-resolution single-gene imaging

doi: 10.1101/2023.03.19.533190

Figure Lengend Snippet: ( A ) Two intensity traces of transcription sites from DMSO- and dTAG-treated Rad21-Taf1-FKBP F36V KMG cells. ( B ) Probability of observing certain number of bursts in an 1 hr observation time, in DMSO- ( n =135 traces from 152 cells) and dTAG-treated ( n =51 traces from 935 cells) Rad21/Taf1-FKBP F36V KMG cells. Solid lines: fits to Poisson distribution, for the data with n bursts ≥1. The resulting two parameters (A, r ) are: DMSO (0.56 and 1.69), dTAG (0.086 and 0.38). ( C ) Box plots of burst amplitude and duration. Each gray dot represents one burst. Data points are from two independent experiments with total n= 189 and 42 bursts for DMSO and dTAG, respectively. p-values are calculated based on a Wilcoxon rank-sum test. The data include two independent experiments. ( D ) Heat-map of fold-change of bursting parameters (non-bursting population, frequency, amplitude and duration) with double perturbations in KMG cells, including double dTAG degrons (Rad21 and Taf1, Med19, or Gtf2f1), dTAG combined with inhibitors (Rad21 and XPBi, or CDK7i). Data are normalized by Log2 (control/treatment). ( E ) Fold-change of control/treatment in single and double perturbations. Black and red boxes are the predicted additive and multiplicative fold-change, respectively. The white boxes show the experimental data, which are larger than both predicted additive and multiplicative effects. The data are from 2–3 independent experiments.

Article Snippet: For significance testing, Wilcoxon rank sum statistical tests were performed in MATLAB.

Techniques: Control