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Seminar 20150920.2
- 1. High-throughput single-molecule screen for small-molecule perturbation of splicing and transcription kinetics Chris Day Larson lab - NCI, LRBGE October 2015
- 2. A brief introduction to transcription • Transcription a highly regulated process and can be divided into multiple steps. • A pre-initiation complex (PIC) factors is assembled on DNA. • Polymerase (PolII) exits the PIC and elongates along the gene. • PolII is retained at the 3’ end and nascent transcripts are processed. • Transcript are spliced, co-transcriptionally or post release.
- 3. Transcription initiation Kwak & Lis, Annu Rev Genet, 2013.
- 4. Elongation Miller spread showing DNA and RNA in salamander eggs. Miller & Beatty, Science, 1969.
- 5. 3’ processing Transcripts are cleaved and polyadenylated before release into the nuclease. Brown, Garland Science, 2002.
- 6. Splicing Spliceosome assembly is required for intron removal however, splicing can occur per- or post- trancriptionally. Wahl et al., Cell, 2009
- 7. Methods to study transcription Population • Factors associated with chromatin • Chromatin Immunoprecipitation (ChIP) • Kinetics • Global Run on and Sequencing (GRO-seq) • Real-time quantitative RT-PCR Single Cell • Factors associated with chromatin • Fluorescence recovery after photobleaching (FRAP) • Kinetics • Single-molecule imaging and fluctuation analysis • FRAP Johnson et al., Science, 2007. Danko et al., Mol Cell, 2013. Singh & Padgett, Nature Struct. Mol. Biol, 2009. Stenoien et al., Nature Cell Biol, 2001. Coulon et al., eLife, 2014. Drazacq et al., 2007
- 8. GRO-seq • In inducible genes you can track the ‘wave’ of PolII after induction. • By tracking the wave after specific time points elongation rates can be inferred. Danko et al., Mol Cell, 2013.
- 9. RT-PCR Singh & Padgett, Nature Struct. Mol. Biol, 2009. • By tracking expression of each primer pair over time elongation rates can be inferred.
- 10. Single-molecule imaging and fluctuation analysis • Fluorescent fluctuations in fluorescently tagged mRNA carry a significant amount of information about transcription kinetics. Coulon et al., eLife, 2014.
- 11. How is transcription visualized in mammalian cells? • PP7 and MS2 technologies are used to visualize single RNA’s. Larson et al., Science, 2011.
- 12. PP7 stem loops MS2 stem loops b-globin reporter gene PP7 and MS2 bacteriophage coat proteins are constitutively expressed Fluorescent RNA Using PP7 and MS2 to visualize transcription and splicing in living cells Splicing Bertrand et al, Mol. Cell, 1998. Chao et al. , NSMB, 2008. Larson et al., Science, 2011. PP7 coat protein mCherry MS2 coat protein GFP Reporter mRNA
- 13. Project Aims 1. Develop a high-throughput assay to identify factors that alter transcription kinetics. 2. Follow up hits with live-cell single-molecule measurements.
- 14. Red-MS2 Blue-DAPI Development of the cell line: Integration of the β-goblin reporter • Reporter was introduced by transient transfection. • Cell selection with puromycin. • FISH confirmed reporter gene was transcribed. • Coat protein was introduced with lentivirus.
- 15. Selected line has many insertion sites Coulon et al., eLife, 2014. • 3 possible insertion sites with 4-7 copies distributed among them were mapped using paired end sequencing.
- 16. intron exon merge PP7 and MS2 stem loops integrated into living cells PP7 stem loopsTet-on PP7 stem loops MS2 stem loopsCFP-SKL
- 17. Aim 1 • Develop and execute a high-throughput imaging assay to detect small-molecules that effect transcription kinetics. • The screen was conducted using U2-OS cells with a integrated reporter gene. Cells were imaged with the PerkinElmer Opera system.
- 18. Optimizing original PM51 line for a high-throughput assay • A clonal population was generated by single cell cloning.
- 19. What do we screen with this assay? • All FDA approved compounds. • siRNA library. • Compounds of interest, HAT’s, KAT’s, HMT, HDAC, O-GlcNAc transferase inhibitor or O- GlcNAcase inhibitors. • Small library of compounds targeting chromatin remodelers (epigenetic library).
- 20. Selection of small-molecule perturbation • Small-molecules are fast acting. • siRNA knockdown takes 2 days. • Small-molecules can be highly specific. • PFI-1 targets BRD2/4 interaction with acytlehistome tails. • JQ1 targets BRD4’s bromodomain’s.
- 21. Experimental time line cell cycle 0h 24h 48h 52h Plate 10,000 cell/well Dox induction Small-molecule treatment Fix 4% PFA
- 22. • Fixed cells on a 96 well PE cell carrier plate. • Separate 488 and 596 exposures, 11 1μM Z stack. 18 TS in this field ~2,000 achieved in 100 fields 100 f * 2 c * 11 z = 2,200 images per well! 2,200 * 60 = 132k Achieving statistical significance through high- throughput imaging
- 23. Reducing imaging time by removing z planes • 3 z planes does not result in a significant decrease in spot count. • 5 z steps cuts imaging time in half. # of z planes 7z 3z 1z Spotcount 1000 1200 1400 1600 1800 2000 2200 2400 DMSO SGC-CBP30
- 24. Automated image analysis (Acapella) • Nuclei are segmented by MS2 signal. • Contrast, size and roundness filters applied. • Transcription sites are segmented by PP7 signal. • Size and contrast filers are applied. • Background is subtracted and a 9-pixel kernel extracts fluorescence.
- 25. How do we extracting kinetic information from transcription site fluorescence • Ratiometric? (early positive control data) • Loss of information (termination, splicing), inconsistent. • Works for few TS. • Scatter plot? • How can kinetics be extracted?
- 26. Figure 1
- 27. Monte Carlo simulations reveal elongation rates can be extracted from scatter plots Blue-1.3kb/min Pink-3.9kb/min
- 28. Can transcript elongation be explained mathematically? Intensity of mCherry c(ι1/v+T) Intensity of GFP c(ι2/v+T) Rearranged IR=IG+cιΔ/v Where: m = number of nascent transcripts c = initiation rate v = velocity of Pol II ι = # of bases T = termination time PP7 MS2 ι2ι1 y=mx+b
- 29. Do mathematical results support in silico results? Black- Monte carlo Red- Equation 3
- 30. Monte Carlo simulations reveal splicing kinetics can be extracted from scatter plots Blue- 8 min Pink- 4 min
- 31. Termination times can be extracted from scatter plots • Science Blue- Fast Pink- Slow
- 33. Positive controls • DRB- inhibits 5’ pause site release. • Initiation control • CPT- Topoisomerase inhibitor • Elongation control • Herboxidiene- U2snRNP inhibitor • Splicing control
- 34. Vehicle vs positive hit
- 35. y-intercept results show 4 compounds that perturb elongation • SGC-CBP30 • PFI-1 • Tenovin-1 • Tenovin-6
- 36. Cell counts reveal cytotoxic compounds • Tenovin-6 is highly cytotoxic. • Low # of TS make CPT an unreliable control. Percent of cells transcribing Meancellcount 0 10 20 30 40 50 60 70 0 500 1000 1500 2000 2500 3000 3500 CPT DRB Tenovin-6 Red- DMSO
- 37. Center of mass results show 3 compounds that perturb release • PFI-1 • Tenovin-1 • Tenovin-6 GFP fluorescence intensity (a.u.) mCherryfluorescence intensity(a.u.) 100 200 300 400 500 600 700 50 100 150 200 250 300 350 400 CPT DRB Tenovin-1 PFI-1 Tenovin-6 Piceatannol Red- DMSO
- 38. Slope results • Tenovin-1 Suggests quicker splicing. DMSO Garcinol C646 Delphinidinchloride SGC-CBP30 (+)-JQ1 (-)-JQ1 PFI-1 TrichostatinA SAHA SB204990 CAY10669 buytrolactone3 trans-Resveratrol CAY10591 Splitomicin EX-527 SIRT1/2InhibitorIV Salermide AGK2 Sirtinol Tenovin-1 Tenovin-6 Nicotinamide Piceatannol Herboxidiene CPT DRB Slope -0.1 0.0 0.1 0.2 0.3 0.4 0.5 ** ** * * * * **
- 40. Positive controls success or failure? • DRB success! • Lower rates of transcription observed. • CPT failure. • Too few spots. • Herboxidiene failure. • Slopes results were not robust. Changes in slope were reliably observed between 0.2 and 0.27.
- 41. Aim 2 • Validation of hits by live-cell single-molecule imaging and fluctuation analysis. • Raw images were acquired with a custom built laser-illuminated microscope based on the Zeiss AxioObserver.
- 42. Imaging conditions • 37°C, humidity controlled chamber. • 512 frames, 10 sec intervals, 9 x 0.5μm Z stack, simultaneous 488nmand 594nm exposure. Fluorescentintensity Time (in sec)
- 43. Image processing pipeline (live-cell data) • Import images into ImageJ for hyperstacking. • Call Localize (IDL) to compute the fluorescent intensity of the trace in both channels. • Compute an auto- and cross-correlation, and average correlation curves for multiple transcription sites. • Fit the averaged curves to a model to extract elongation rate, splicing time, termination time, and the fraction of transcripts spliced co-transcriptionaly. See: Coulon et al. eLife (2014)
- 44. Correlation functions Dissecting the transcription cycle with fluctuation analysisIntegratedintensity 0 1000 2000 3000 4000 0 10000 20000 30000 40000 50000 60000 70000 time (s)
- 45. DMSO: Kinetics from the vehicle control Delay (s) rg gr -100 -50 0 50 100 150 200 0.8 0.9 1.0 1.1 G(t)/Grg(0) Fraction of pre-release splicing 100% 0% Elongation rate Termination time • Vehicle control • n=20 traces • Elongation rate 2.64±0.20 kb/min • Pause at 3’ end 149.49±9.04 sec • Co-transcriptional splicing 12.97±4.02 % • Splicing time 358.75±23.75 sec
- 46. SGC-CBP30: multiple targets • Elongation rate 1.85±0.08 kb/min • Pause at 3’ end 190.87±8.20 sec • Co-transcriptional splicing 19.62±2.94 % • Splicing time 387.08±11.85 sec • CBP bromodomain • n= 23 traces G(t)/Grg(0) Delay (s) 0.8 0.9 1.0 1.1 -100 -50 0 50 100 150 200 Control SGC-CBP30 **
- 47. PFI-1: multiple targets • Elongation rate 1.42±0.09 kb/min • Pause at 3’ end 118.78±7.01 sec • Co-transcriptional splicing 31.27±5.06 % • Splicing time 237.36±4.93 sec • BRD2/4 Interaction with histone tails • n=26 traces ** **
- 48. • Elongation rate 1.74±0.14 kb/min • Pause at 3’ end 147.72±11.36 sec • Co-transcriptional splicing 5.41±4.38 % • Splicing time 511.94±79.70 sec • SIRT • n= 14 traces Tenovin: perturbs elongation possibly co-transcriptional splicing G(t)/Grg(0) Delay (s) 0.8 0.9 1.0 1.1 -100 -50 0 50 100 150 200 Control Tenovin-1
- 49. (+)JQ1: slows elongation • Elongation rate 1.74±0.09 kb/min • Pause at 3’ end 160.05±10.19 sec • Co-transcriptional splicing 17.02±3.91 % • Splicing time 364.53±14.46 sec • BRD4 bromodomain • n= 27 traces Delay (s) G(t)/Grg(0) -100 -50 0 50 100 150 200 0.8 0.9 1.0 1.1 Control +JQ1
- 50. (-)JQ1: termination time Delay (s) G(t)/Grg(0) -100 -50 0 50 100 150 200 0.8 0.9 1.0 1.1 Control -JQ1 • Elongation rate 2.14±0.23 kb/min • Pause at 3’ end 108.46±7.20 sec • Co-transcriptional splicing 14.86±4.58 % • Splicing time 272.82±12.70 sec • JQ1 inactive isomer • n= 20 traces ** **
- 51. Kinetics extracted from the correlation curves
- 52. Conclusions from live-cell validation • All hits for elongation (SGC-CBP30, PFI-1, Tenovin-1) were validated. • Hits for termination time (PFI-1, Tenovin-1) were not validated. • SGC-CBP30 did perturb release but was not significant in primary screen. • Correlation analysis suggests Tenovin-1 and PFI-1 perturb splicing rates and times. • Version 2.0 of the screen will need to address this.
- 53. Inconsistent measurements • Opera data suggests quicker splicing in Tenovin-1 treated cells. • Correlation analysis suggests longer splicing. DMSO Garcinol C646 Delphinidinchloride SGC-CBP30 (+)-JQ1 (-)-JQ1 PFI-1 TrichostatinA SAHA SB204990 CAY10669 buytrolactone3 trans-Resveratrol CAY10591 Splitomicin EX-527 SIRT1/2InhibitorIV Salermide AGK2 Sirtinol Tenovin-1 Tenovin-6 Nicotinamide Piceatannol Herboxidiene CPT DRB Slope -0.1 0.0 0.1 0.2 0.3 0.4 0.5 ** ** * * * * **
- 54. Numbers of images consistent between live and fixed cells • For live cell ~20 traces of 512 images are averaged. • This is ~10,000 images of transcription sites (TS). • For fixed cell 6 replicates each consisting of ~1,800 cells are averaged. • This is ~11,000 images of cells. • Depending on the percent of cells transcribing this can be up to 5,500 TS per condition.
- 55. Conclusions from imaging assays • We are able to detect two-fold changes in elongation rates. • Danko et al. only show two fold changes in elongation rates. Could this be the maximum variability seen in gene transcription?
- 56. Acknowledgments The Larson Lab Daniel Larson Murali Palangat Antoine Coulon Huimin Chen Joe Rodriguez Tineke Lenstra Heta Patel Simona Patange Chemical Biology Laboratory, NCI Jordan Meier HiTIF Core Gianluca Pegoraro Laurent Ozbun
Editor's Notes
- Lab of receptor biology and gene expression. I’m going to talk about transcription. So I have a short introduction.
- RNA world DNA to RNA to protein The PIC consists of both sequence-specific and general transcription factors.
- a) Histones d) 5’ pause
- May not relate to this project, but Poly A may be linked to insulin RNA depredation in glucose stress.
- Dnase hypersensitivity Goal of my project was to make kinetic measurements of transcription with single-cell resolution.
- Brings me to my project
- This is how you ‘tag’ RNA
- Intron-mCherry exon-GFP will be used interchangeably
- First step was to develop the cell line
- PCR amplification of genomic DNA to isolate human and rat insulin and amylin promoter sequences. Single colonies were isolated after puryo then infection occurred.
- Tet on!!!!!! Don’t forget
- ~18 Spots Mention Nuclioli make sure TS sites are shown as different Singe Cell Clone
- With 5z it still takes 18hr I should have tested this more carefully – actually looked at scatter plotes
- Ratio- slower elongation more red signal quicker splicing less red signal
- Next couple slides will be scatter plot analysis of data
- The read out we get is PP7 intensity and MS2 intensity. Since we are looking at 1,000s of transcripts the population ratio will change
- 4 min may not be physiologically relevant A slope of 1 would mean 1 intron to 1 exon or NO splicing ∞ splicing time!
- 28 compounds
- CPT failed
- Tenovin-1 Low cell PFI-1 high transcribing
- 1 alone this tells us nothing. 2 points data, line model, we are able to extract kinetic information from this. 3 when fit I extract these kinetics
- Intron retained longer. Retained at 3’ end longer
- We have a nice assay. Inducible genes were used. 2 fold changes detected. Perhaps this is the maximum we can knock down by inhibiting transcription factors.