1. Volume 162, Issue 4, Pages 911-923 (August 2015)
Identification of Gene Positioning Factors Using High-Throughput Imaging Mapping 
Sigal Shachar, Ty C. Voss, Gianluca Pegoraro, Nicholas Sciascia, Tom Misteli 
Cell 
Volume 162, Issue 4, Pages (August 2015)
DOI: /j.cell
Copyright © 2015 Elsevier Inc. Terms and Conditions

2. Cell  , DOI: ( /j.cell )
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 1
A High-Throughput Imaging-Based Method to Map Gene Positioning
(A) HIPMap outline. Cells are cultured in 384-well imaging plates, and FISH is carried out in a fully automated fashion using directly labeled BAC probes, followed by automated image acquisition using high-throughput microscopy. Image analysis by Acapella segments the nucleus border and detects FISH signals. Normalized radial distances from the nuclear border are measured. Distance measurements distributions are plotted as histograms and/or density curves.
(B) Representative maximal projections of images acquired in three channels. FISH signals are automatically detected inside the nucleus ROI (see Experimental Procedures for details). Scale bar, 10 μm.
(C) Density curves for normalized radial distance distributions for the indicated loci. For each locus, the distance from the nucleus periphery of at least 600 FISH spots was determined.
(D) Detection of radial position shifts by siRNA silencing of lamins. Histograms of the normalized radial distance distributions of control non-targeting siRNA (green) compared to LMNA/C/B1 knockdown (purple) for the indicated locus. Distributions were generated from at least 600 FISH spots per sample. p < 1e−16 for all 3 loci using the two-sample KS test. Lines represent estimated density. See additional information in Experimental Procedures and Figure S1.
Cell  , DOI: ( /j.cell )
Copyright © 2015 Elsevier Inc. Terms and Conditions

4. Figure 2
Identification of Genome Positioning Determinants by RNAi Screening
(A) HIPMap-based siRNA screen outline. Reverse transfection of siRNA was conducted in 384-well plates and locus position was measured by HIPMap. p values for each well were generated by pairwise comparison of the distribution of each sample to a negative control distribution generated by pooling of six wells containing non-targeting siRNA using the KS test. Hits were defined as siRNAs with a p value < 2e−3.
(B) Histograms of representative distributions of hits and non-hits. Distributions represent data from >600 FISH spots. Values represent one of two experimental replicates. Lines indicate the estimated density.
(C) List of 50 high-confidence hits identified following primary and validation screen. Hits were defined as p values < 2e−3 in both screens. See additional information in Experimental Procedures, Figure S2, and Tables S1 and S2.
Cell  , DOI: ( /j.cell )
Copyright © 2015 Elsevier Inc. Terms and Conditions

5. Figure 3 Functional Classification of Genome Positioning Determinants
(A) Venn diagram representing the number of validated repositioning factors for each tested locus.
(B) Classification of hits into functional groups. Hits were grouped according to their assigned function and plotted according to the number of affected loci.
(C) Bar graphs representing the relative contribution of each nuclear pathway to the re-positioning of the indicated locus.
(D) Box plots of normalized radial distances from the nuclear border of the indicated locus following siRNA treatment. Dashed line indicates the median of the control distribution. Boxes show the 25th, 50th (median), and 75th percentile of the distributions and whiskers extend up to 1.5 inter-quantile range, outliers are shown as dots. Asterisks indicate hits identified in the screen using KS test with a p < 2e−3. The same data are presented in Figure S3A as distribution curves.
(E) mRNA expression, as measured by qRT-PCR, of COL1A1 and COX2 following siRNA transfection of the indicated gene or combination of genes for 72 hr. Expression is normalized to hTBP. All expression ratios are relative to non-targeting siRNA control (siNT). Values represent averages from two experiments ± SD.
See additional information in Figure S3.
Cell  , DOI: ( /j.cell )
Copyright © 2015 Elsevier Inc. Terms and Conditions

6. Figure 4 DNA Replication Is a Determinant of Genome Positioning
(A) Histograms of representative distributions of loci following knockdown of the indicated gene. Distributions represent data from >600 FISH spots. Values represent one of three experimental replicates. Lines indicate estimated density.
(B) Statistical testing of distributions using the KS test. p values are calculated by comparing the pooled negative controls distribution to the distribution in cells knocked down for the indicated gene or combination of genes. Values represent one of three independent experimental replicates.
(C) Representative distributions of three loci comparing control (green) and siRNA transfected cells (purple) in cycling cells or cells treated with 2 mM thymidine and the indicated siRNA for 72 hr. Values represent one of three experimental replicates.
(D and E) p value heat maps comparing cycling cells to G1/S arrested cells for three different indicated loci. siRNA silencing of replication factors (D) or chromatin and structural proteins (E). Values represent one of three independent experimental replicates.
See additional information in Figure S3 and Table S3.
Cell  , DOI: ( /j.cell )
Copyright © 2015 Elsevier Inc. Terms and Conditions

7. Figure 5
Normal Progression through Replication Is Required for Accurate Positioning
(A) Distributions of untreated (green) or hydroxyurea (HU) treated cells (200 μM, 24 hr, purple) for the indicated locus. p values were calculated using KS test. Values represent one of three experimental replicates.
(B) p value heat map comparing untreated cells to cells treated with the indicated DNA damaging agent for up to 4 hr for 3 different loci. Values represent one of two experimental replicates.
(C) mRNA expression of COL1A1 and COX2 following knockdown of the indicated gene or set of genes for 72 hr in thymidine arrested cells. Values are normalized to hTBP. All expression ratios are relative to non-targeting siRNA control (siNT). Values represent averages from two experiments ± SD. See additional information in Experimental Procedures and Figure S5.
Cell  , DOI: ( /j.cell )
Copyright © 2015 Elsevier Inc. Terms and Conditions

8. Figure 6 Mitosis Is Not Required to Establish Gene Positioning
(A) Radial distance distributions of control (green) and siRNA transfected cells (purple) for the LADF locus. For each treatment, non-targeting siRNA control (siNT) distributions are plotted together with knockdown of lamin distribution.
(B) p value heat map comparing each treatment to the indicated knockdown and treatment. Data represent one of four experiments.
See additional information in Figure S6.
Cell  , DOI: ( /j.cell )
Copyright © 2015 Elsevier Inc. Terms and Conditions

9. Figure S1 Automated Analysis of HIPMap Assay, Related to Figure 1
(A) Outline of the automated Acapella analysis pipeline. Representative images acquired by the Perkin Elmer Opera microscope. Six z stacks in four channels were acquired and maximum projections were generated (for details see Experimental Procedures).
(B) FISH spots were identified either manually or using the Acapella pipeline in 15 fields from different wells (> 300 cells) and accuracy of automatic detection was determined by cross-comparison of identified spots.
(C) The number of detected FISH spots per cell was calculated for 300 wells treated with different siRNAs. N > 300 cells per well, SD are shown.
(D) Determination of the minimal imaged cell number to achieve statistical significance. Nine thousand FISH spots from nine siControl treated wells were combined into a pool from which two groups with an equal number of spots were randomly extracted and compared using the two sample KS test. The sub-sampling simulation was repeated 10,000 times for each indicated spot number and the fraction of trials generating a p value smaller than was calculated. Based on this analysis, to assure robustness in our analysis at least 600 FISH spots were counted per sample. Dashed red line indicates 0.05.
(E) CDFs of cells treated with non-targeting siRNA (red) compared to CDFs of cells knocked down for LMNA/C/B1 (green) of the indicated locus. Different lines in the same color represent replicate wells (p > 0.05, KS test). Each CDF represents at least 600 FISH spots.
(F) CDFs of the indicated genes in various cell lines treated with a non-targeting siRNA. Distributions represent 6 pooled wells, each replicate containing > 300 cells.
(G) Determination of knock-down efficiency. Expression of the indicated gene after siRNA transfection of the indicated gene was measured using qRT-PCR and normalized to hTBP. All measurements are relative to the non-targeting (siNT) control siRNA. Bars indicate SD.
(H) Knockdown of lamins (green) affects LADF positioning in the indicated cell lines compared to control siRNA treatment (siNT, red). Different distributions in the same color represent replicate wells (p > 0.05). Each CDF represents at least 600 FISH spots.
Cell  , DOI: ( /j.cell )
Copyright © 2015 Elsevier Inc. Terms and Conditions

10. Figure S2 General Features of Selected Hits, Related to Figure 2
(A–C) The indicated nuclear characteristics were determined after siRNA transfection of the indicated genes using the DAPI channel. At least 2000 cells were imaged per well. Values represent averages from three experiments ± SD.
(D) Percentage of γH2AX positive cells was determined by quantitative immunofluorescence. At least 2000 cells per well were imaged in four replicates per condition. Values represent averages from three experiments ± SD.
(E) Cell cycle profiles based on DAPI integrated intensity of CRL-1474 cells after siRNA transfection of the indicated gene for 72 hr. Cell cycle profile was determined as described in (Roukos et al., 2015) (for details see Experimental Procedures). At least 5000 cells were analyzed per condition.
(F) Biological replicates of hits from siRNA screen are similar. CDFs of cells treated with non-targeting siRNA (red) compared to CDFs of cells knocked down for a representative hit (green). Each CDF represents at least 600 FISH spots. p value was calculated using the two sample KS test.
(G) Correlation between cell number (x axis) and p values obtained for 135 hits identified in primary screen (y axis) for each indicated locus. R is Spearman’s rank correlation coefficient.
(H) Gene positioning factors do not affect cellular ROS levels. Cellular ROS was determined using H2DCFDA following siRNA transfection of the indicated gene. H2O2 at 0.003% (1 mM) was used as positive control. Values represent averages from three experiments ± SD.
(I) Measurement of transcription activity upon knockdown of gene positioning factors. Active transcription in cells transfected with the indicated siRNA was determined by measuring the levels of polymerase II CTD phospho-Ser2 using quantitative IF. DRB was used as positive control. Values represent averages from three experiments ± SD.
(J) Measurement of RNA polymerase II levels upon knockdown of gene positioning factors. Expression levels of polymerase II are not correlated with hits. Polymerase II levels were quantified using quantitative IF. Values represent averages from three experiments ± SD.
Cell  , DOI: ( /j.cell )
Copyright © 2015 Elsevier Inc. Terms and Conditions

11. Figure S3
Different Hits Affect Positioning of Loci, Related to Figure 3
(A) Radial distance distributions following siRNA transfection for the indicated gene. Green: control siRNA, purple: siRNA of hits. Bars indicate fraction of spots and lines indicate estimated density. p values for each pairwise comparison are generated using the two sample KS test.
(B) Co-localization between the LADF locus and the nuclear lamina decreases following knockdown of indicated hits. LADF and Lamin A/C are detected simultaneously (FISH followed by IF). The percentage of cells containing 0, 1, or 2 co-localization events of LADF with LaminA/C is determined using Acapella Image analysis. Values represent averages from three experiments ± SD.
Cell  , DOI: ( /j.cell )
Copyright © 2015 Elsevier Inc. Terms and Conditions

12. Figure S4
Cell-Cycle Synchronization and Induction of G1/S Arrest, Related to Figure 4
(A) Outline of the assay to assess the effect of replication on gene positioning. Quiescence was induced by growing cells in high density for 72 hr. Cells were split into normal media or media containing 2 mM thymidine and were concomitantly transfected with siRNA. Following additional 72 hr cell cycle and gene position were determined. Cell cycle profiles were generated using FACS, cells were stained for Propidium iodide.
(B and C) Percentage of Cyclin A (B) or Ki67 (C) positive cells in normally cycling and quiescent cells determined by quantitative immuno-fluorescence. The indicated number of cells was plated per well and 72 hr later IF was performed. At least 2000 cells were imaged in four replicates per condition. Values represent averages ± SD.
(D) Gene knock-down efficiency is similar between cycling and thymidine-arrested cells. mRNA levels were determined by qRT-PCR of the indicated gene in knock-down versus control cells or by quantitative immuno-fluorescence for GAPDH. At least 2000 cells per well were imaged in four replicates per condition. Values represent averages ± SD.
(E) Quiescence does not lead to re-positioning. Radial distributions of LADF, COX2 and COL1A1 were determined by HIPMap after 72 hr in high density (purple) or in normally cycling cells (green).
Cell  , DOI: ( /j.cell )
Copyright © 2015 Elsevier Inc. Terms and Conditions

13. Figure S5
Perturbation of Normal Replication Affects Positioning, Related to Figure 5
(A) Cell cycle profile of cells treated with 200μM of Hydroxyurea (HU) for 24 hr. Cell cycle profiles were determined using the integrated DAPI intensity of the cells in 384-well plates as described in (Roukos et al., 2015) (see Experimental Procedures). At least 5000 cells were analyzed per condition.
(B) DNA damage induction following a four hour treatment with the indicated damaging agents. Representative images of cells treated with different DNA damaging agents (ETPSD= Etoposide) and stained for DAPI and γH2AX.
(C) Percentage of γH2AX positive cells in DNA damaging agents treated cells was determined by quantitative IF. At least 2000 cells per well were imaged in four replicates per condition. Values represent averages ± SD.
(D) Cell cycle profile of cells treated with the indicated damaging agent for up to four hours. Cell cycle profiles were determined as described in A.
(E) The radial distance distribution of each locus is not changed during the cell cycle. Normal cells were synchronized using 2 mM thymidine and released into normal media. CDFs were generated at various time points after release when the population of cells was enriched for the indicated cell cycle phase. Each CDF represents at least 600 FISH spots.
(F) mRNA expression of GAPDH (normalized to hTBP) and hTBP (normalized to GAPDH) following siRNA transfection for 72 hr of the indicated gene or set of genes in normally cycling or thymidine-arrested cells. All expression ratios are relative to non-targeting siRNA control (siNT). Values represent averages from two experiments ± SD.
Cell  , DOI: ( /j.cell )
Copyright © 2015 Elsevier Inc. Terms and Conditions

14. Figure S6
Cells Released from Thymidine Block Continue Cycling, Related to Figure 6
(A and B) Percentage of pH3S10 positive cells (A) or cell number (B) in cells released from thymidine block was determined by quantitative IF. At least 2000 cells were imaged in four replicates per condition. Values represent averages ± SD.
(C and D) Cell cycle profiles of cells released from thymidine block for the indicated number of hours. Cell cycle profiles were determined using the integrated DAPI intensity of the cells in 384-well plates as described in (Roukos et al., 2015) (for details see Experimental Procedures). At least 5000 cells were analyzed per condition.
Cell  , DOI: ( /j.cell )
Copyright © 2015 Elsevier Inc. Terms and Conditions