Transcription factor dynamics in the nucleus

Transcription

1 Transcription factor dynamics in the nucleus March 21, 2018 Luca Giorgetti

2 Transcription factors bind to enhancer and promoter regions Adapted from Streubel & Bracken EMBO J 2015 Bind with high specificity to cognate DNA motifs Recruit co-activator or co-repressor complexes Recruit chromatin remodelers

3 In vitro assays: structural basis and thermodynamics of TF binding X-ray crystallography EMSA, SELEX, Protein Binding Microarrays, Surface Plasmon Resonance etc MyoD Siggers et al Mol Sys Biol 2011 Mammalian transcription factors:! " = 10 &' 10 &) nm

4 What is the kinetics of TF binding in vivo TF 1 TF 2 Pol II TSS TF binding sites core promoter How do TFs and co-factors assemble and disassemble at regulatory regions How do they recruit Pol II How long does it take to find a target site

5 How do TF find their target sites (A) The eukaryotic nucleus is a dense environment: excluded volumes effect are likely to be important TFs can bind to very large numbers of non-specific or lower-affinity binding sites Chromosomes are folded into complex structures Adapted from Mirny et al, J Phys A 2009

6 In vivo target search must be fast to ensure proper transcriptional activation Transcription of NF-kB early immediate target genes is synchronous with nuclear translocation Giorgetti et al Mol Cell 2010

7 In vivo target search is faster than expected by pure diffusion of the TF Transcription of NF-kB early immediate target genes is synchronous with nuclear translocation LacI search for LacO sites in E.coli is much faster than allowed by diffusion only Theory! " #$ = 4'( #$ ) /0 1 /0 Experiment LacI LacO! " 234 = /0 1 /0 = 100! " #$ Giorgetti et al Mol Cell 2010 Riggs et al J Mol Biol 1970

8 Facilitated diffusion Minireview: Facilitated Target Locationin Biological Systems,,-. Schematic view of lac renteracting with a large opntaining DNA molecule in ution. (The DNAmolecules # parated into domains under ## itions.) The (upper)expanded s repressor bound to a segment 1I rator DNA, on which it can Interdomain de or engage in intradomain Association n-association processes in specific (operator) target site. r) expanded view shows a reinterdomain olecule double bound to two Dissociation ments; this corresponds to the te state in the intersegment ocess. 677 c. C -,,I p@ I Repressor \ Intersegment Transfer von Hippel and Berg, J Biol Chem 1989 ix within the domain. This transientdouble bound state of n is disrupted by subsequent diffusion apart from the DNA resulting (50%of the time if both binding interactions are ong) in thedirect transfer of the protein from one nonspeng site to another that is quite remote along the DNA step that is detected by the assay useds6 For such systems studied in vitro any or all of the following observations indicate that facilitated diffusion is involved. (i) The apparent reaction rate is larger than seems reasonable for a simple (three-dimensional) diffusion-controlled macromolecular in-

9 Measuring TF binding in vivo: Chromatin immunoprecipitation Population-averaged Crosslinking-based: no time resolution Semi-quantitative Park, Nat Rev Genetics 2009

10 New paradigm: Live-cell imaging of fluorescently labeled TFs Single-cell resolution Time resolved Quantitative Sox2-HALOtag in mouse ESC Chen et al, Cell 2014 Two enabling technological breakthroughs: 1. Fluorescent labeling 2. Live-cell microscopy

11 Fluorescent Recovery After Photobleaching (FRAP) Fluorescent protein TF fusions TF GFP Confocal microscope immobile fraction jic.ac.uk

12 FRAP measurements of nuclear protein mobility H2B: slow (hours) Kimura & Cook J Cell Biol 2001 HP1: fast (10s of sec) Cheutin et al Cell 2003 NF-kB: faster (few sec) Sung et al PLoS One 2009 FRAP drawbacks: Averaged over large populations of molecules Critically relies on model interpretation to extract binding and diffusion rates

13 What is the kinetics of TF binding in vivo TF 1 TF 2 Pol II TSS TF binding sites core promoter ü How fast do TFs and co-factors assemble and disassemble at regulatory regions Few seconds How fast do they recruit Pol II How long does it take to find a target site

14 Direct observation of single transcription factors Problem #1: high density of nuclear milieu Turn on a few labeled TFs only Photoconvertible protein TF fusions TF pfp

15 Direct observation of single transcription factors Problem #2: Single molecules are faint, and move fast A new generation of high-speed, low-background, live imaging microscopes, such as: HILO microscope Tokunaga et al Nat Meth 2007 Multifocal microscope Abrahamson et al Nat Meth 2013 Bessel beam microscope Gao et al Nat Meth 2014

16 Single-particle imaging of Sox2 in mouse ES cells wt Sox2 Sox2-TAD Specific binding Sox2 HALO-TMR The search for a specific DNA target follows a trial-and- error sampling mechanism: each TF undergoes multiple rounds of short-lived nonspecific chromatin-binding events (tns= s) punctuated by 3D diffusion episodes (t3d = s) before eventually encountering a specific DNA target to which it binds more stably (ts ~10s) Chen et al, Cell 2014

17 Single-particle imaging of Sox2 in mouse ES cells Minireview: Facilitated Target Locationin Biological Systems Single-particle tracking of single Sox2 molecules reveals a mixture of 3D FIG. diffusion and sub-diffusive motion that is DNA compatible with facilitated diffusion,,-. 1. Schematic view of lac repressor interacting with a large operator-containing molecule in dilute solution. (The DNAmolecules # are well separated into domains under ## these conditions.) The (upper)expanded view shows repressor bound to a segment 1I of non-operator DNA, on which it can Interdomain either slide or engage in intradomain Association dissociation-association processes in seeking its specific (operator) target site. The (louer) expanded view shows a reinterdomain pressor molecule double bound to two Dissociation DNA segments; this corresponds to the intermediate state in the intersegment transfer process. Search time ~ 380 seconds, which increases to 700 seconds in the absence of the DNA binding domain ( pure diffusion) 677 c. C -,,I p@ double helix within the domain. This transientdouble bound state of the protein is disrupted by subsequent diffusion apart from the DNA segments, resulting (50%of the time if both binding interactions are equally strong) in thedirect transfer of the protein from one nonspecxcbinding site to another that is quite remote along the DNA I Repressor \ Intersegment Transfer step that is detected by the assay useds6 For such systems studied in vitro any or all of the following observations indicate that facilitated diffusion is involved. (i) The apparent reaction rate is larger than seems reasonable for a simple (three-dimensional) diffusion-controlled macromolecular in- Chen et al, Cell 2014

18 What is the kinetics of TF binding in vivo TF 1 TF 2 Pol II TSS TF binding sites core promoter ü How fast do TFs and co-factors assemble and disassemble at regulatory regions Few seconds How fast do they recruit Pol II ü How long does it take to find a target site Few minutes

19 Super-resolution live-cell imaging in the nucleus Stochastic Optical Reconstruction Microscopy (STORM) Photo-Activation Localization Microscopy (PALM) hmmi.org Yang et al Chem Soc Rev 2016 hmmi.org

20 Live-cell PALM on PolII b-actin RNA PolII PolII Dendra2 PolII clusters have an average lifetime of 8 seconds Cluster lifetime is correlated with the number of RNAs produced Cho et al, elife 2016

21 What is the kinetics of TF binding in vivo TF 1 TF 2 Pol II TSS TF binding sites core promoter ü ü ü How fast do TFs and co-factors assemble and disassemble at regulatory regions How fast do they recruit Pol II How long does it take to find a target site Few seconds Few seconds Few minutes

22 What is the kinetics of TF binding in vivo How does it relate to enhancer-promoter dynamics TF 1 TF 2 Pol II TSS TF binding sites core promoter

23 What is the kinetics of TF binding in vivo How does it relate to enhancer-promoter dynamics Xite-CuO (CymR-dTomato) Chic1-TetO (TetR-EGFP) Chic1 ~50 kb time Xite 1 frame = 10 seconds more on chromosome architecture and dynamics on May 23

24 Next lesson: Felix Naef, EPFL Lausanne March 28 at 13:00 in room 5.30, at the FMI "Transcriptional bursting and promoter cycles in mammalian cells & tissues Followed at 15:00 by a research seminar Systems Chronobiology in the liver: propagation of rhythmic information with gene expression No paper presentation by students next week. Papers will be presented on April 4!