Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

How telomeres protect the ends of our chromosomes - Jack Griffith

258 views

Published on

Advances in complex systems July 3-7, 2017

Published in: Science
  • Be the first to comment

  • Be the first to like this

How telomeres protect the ends of our chromosomes - Jack Griffith

  1. 1. Oya Bermek Cristina Nadalutti Lindsey Constantini Anirban Kar Smaranda Willcox Tony Cesare Brian Bower Rachel Stancel Ozlem Arat Sarah Compton Nicole Fouche Lubomir Tomaska Comenius University Titia de Lange Rockefeller University Ylli Doksani Katza Krantz HOW TELOMERES PROTECT THE ENDS OF OUR CHROMOSOMES Advances in complex systems Lake Como, Italy, July 2017 Jack Griffith, Lineberger Cancer Center, University of North Carolina at Chapel Hill
  2. 2. There are 6 x 1024 human telomeres on Earth today (as calculated by Lubomir Tomaska, Comenius University) First identified in early studies of McClintock and others as elements that inhibit end to end fusions of chromosomes.
  3. 3. Telomeres • Tandem array of repetitive sequences • Single strand 3’-overhang of the G-rich strand • Replicated by the cellular replication machinery but extended in some cells by telomerase • Bound by general cellular and telomere-specific proteins: shelterins histones DNA repair factors. 5’-TTAGGG-3’ 3’-AATCCC-5’ Centromere Telomere 3’-overhang
  4. 4. A human telomere compacted by histones, shelterin and repair factors is 120 nm – 180 nm in diameter, similar to the diameter of a Herpes Virus capsid. HSV-1 DNA is 150kb in length which is 1/1000th the length of DNA of many human chromosomes.
  5. 5. In search for species with unusual telomeres, Lubomir Tomaska captures an example with a telomere at only one end.
  6. 6. The mammalian repeat TTAGGG is evolutionarily very old, dating at least to the 1950’s Telomeric sequences
  7. 7. Telomeric sequences Humans: (TTAGGG)n 5-10 kb Mice (TTAGGG)n 20-40 kb X laevis (TTAGGG)n 10-50 kb Drosophila: long viral-like retroposons A thalliana (TTTAGGG)n 2-6 kb Peas (TTTAGGG)n 50-120 kb Barley (TTTAGGG)n up to 300 kb Onions arrays of ribosomal repeats T brucei (TTAGGG)n 5-10 kb T thermophila (TTGGGG)n 1-2 kb O nova (TTTTGGGG)n 20 bp/long S pombe (TTACAG) 1-8 200-300 bp S cerevisiae (TG 1-3) ~350 bp Y lipolytica (TTA CTGA GGG)n 300-500 bp Selection through evolution for a motif in which one strand is rich in 3-4 G residues which we know have the ability to Self-compact into G quadruplexes
  8. 8. Telomeres • Tandem array of repetitive sequences • Single strand 3’-overhang of the G-rich strand • Replicated by the cellular replication machinery but extended in some cells by telomerase • Bound by general cellular and telomere-specific proteins: shelterins histones DNA repair factors. 5’-TTAGGG-3’ 3’-AATCCC-5’ Centromere Telomere 3’-overhang
  9. 9. Telomerase, ALT and extending our age/telomeres Telomerase has been seen as an excellent target for cancer therapy as it is over expressed in >80% of common cancers. However– it is not expressed in the ALT phenotype and blocking telomerase could induce an end run by ALT Hence we need to know much more about the ALT pathway Some cancers show are neither activation of telomerase or ALT If you could reactivate your telomerase in your cells would you live longer? On line drugs claim to do this (TA-65 etc) Current studies (Denmark) indicate that longer telomeres may be marginally beneficial for lowering cardiac issues but may predispose for cancer Problem: mouse models are problematic since mice never die of short telomere issues
  10. 10. Telomere-specific proteins TRF1- first telomere DNA binding protein found by de Lange. TRF2- found by homology search to myb domain binds ds telomeric DNA (de Lange) strong preference for replication forks, Holliday junctions and ds/ss junctions via a p53 like basic domain on N terminus (Griffith). Pot1- Baumann and Cech, binds to the ss overhang Rap1- binds TRF2 as dimer TIN2, TPP1 bridge Pot1 and the rest of the complex Full shelterin complexes Isolated from mouse cells by affinity tagging bound to telomeric DNA. Large ball in Top left is 440 kDa for size reference In addition to the full complex there are numerous sub-complexes de Lange and Griffith labs
  11. 11. TRF1 and TRF2 basic acidic homodimerization 30% identity Myb domain (telobox) 58% identity TRF2 TRF1 45 67 245 264 442 378 500 439 mTRF1 41% 83% 38% 84%
  12. 12. Cellular effects due to TRF2 manipulation No Treatment Fibroblasts = senescence in ~ 80 PD after telomere erosion to ~ 7kb Immortalized cells = infinite cell division with no telomere erosion dominant negative TRF2 Fibroblasts = rapid induction of senescence without telomere shortening HeLa cells = rapid p53/ATM dependent apoptosis without telomere shortening wtTRF2 overexpression Fibroblasts = Increased telomere shortening per cell division Conclusion: A change in telomere structure, not length, triggers senescence induction
  13. 13. Based on what we know about recA and rad 51 will TRF2 form a strand Invaded loop? Our proposal in 1998 based on yeast studies and basic homologous recombination reactions driven by recA protein: telomeric DNA should loop back on itself.
  14. 14. Rachel Stancel incubates Her model telomere DNA with TRF2 purified in the de Lange lab and sees looped molecules. No looping with TRF1 or with non-telomeric DNAs
  15. 15. AluI (5’AGCT3’) MboI (5’GATC3’) Nuclear Extract Gel Filtration Chromatography Observe high molecular weight DNA by EM for t-loops Psoralen/UV crosslink Restriction enzyme digestion
  16. 16. 18 kb loop T loop from HeLa cells 18 kb loop
  17. 17. telomeric DNA from garden peas arranged as a t-loop pBlue 80 kb loop pBlue 80 kb loop
  18. 18. Telomere loops and homologous recombination-dependent telomeric circles in a Kluyveromyces lactis telomere mutant strain. Cesare AJ, Groff-Vindman C, Compton SA, McEachern MJ, Griffith JD. t-Loops in yeast mitochondria. Tomaska L, Makhov AM, Griffith JD, Nosek J. Telomeric DNA in ALT cells is characterized by free telomeric circles and heterogeneous t-loops. Cesare AJ, Griffith JD. Taz1 binding to a fission yeast model telomere: formation of telomeric loops and higher order structures. Tomaska L, Willcox S, Slezakova J, Nosek J, Griffith JD. Closed chromatin loops at the ends of chromosomes. Nikitina T, Woodcock CL. t-loops at trypanosome telomeres. Muñoz-Jordán JL, Cross GA, de Lange T, Griffith JD. Telomeres of polytene chromosomes in a ciliated protozoan terminate in duplex DNA loops. Murti KG, Prescott DM. Telomere looping in P. sativum (common garden pea). Cesare AJ1, Quinney N, Willcox S, Subramanian D, Griffith JD. Mammalian telomeres end in a large duplex loop. Griffith JD, Comeau L, Rosenfield S, Stansel RM, Bianchi A, Moss H, de Lange T. C. elegans telomeres contain G-strand and C-strand overhangs that are bound by distinct proteins. Raices M1, Verdun RE, Compton SA, Haggblom CI, Griffith JD, Dillin A, Karlseder J.
  19. 19. Efforts to eliminate TRF2 and determine effect on t-loops Observing t-loops in cells by EM is a great experiment but a miserable assay: time consuming expensive hard to score very large numbers of DNAs. Numerous efforts with the de Lange lab (Eros Denichi, Agata Smogorzewska) knocking out TRF2 in mouse cells only late understood that this must be done in an ATM minus background to avoid destruction of the telomeric DNA Enter Yilli Doksani (de Lange lab) and Xiaowei Zhang (Harvard) with STORM imaging
  20. 20. The TRF2 conundrum Rachel’s in vitro experiments show that TRF2 will form t-loops on a model DNA with a long 3’ ss overhang However– the efficiency is low (~15% of the templates are looped) No looping so far with the full shelterin complex Forming D loops requires helicase action and TRF2 is not a helicase. At this level of efficiency (15%) bubbles or breathing of the DNA could contribute to loop formation. Doksani’s experiments show that TRF2 is required to observe t-loops, meaning that TRF2 could either form the loops or rather stabilize them once formed by a variety of pathways. If so, what mechanisms might be involved in opening the helix to allow looping?
  21. 21. TERRA, R-loops and Homologous Recombination Telomeric R-loops involving TERRA paired to its template strand have been detected in yeast, human cells, human cancer cells and ALT cells (Balk 2014; Pfeiffer 2013; Arora 2014; Yu 2014). In ALT cells a correlation has been observed between: enhanced HR, elevated TERRA levels and repression of RNase H1 (Arora 2014). Overexpression of RNase H1 in ALT cells leads to lowered TERRA levels and loss of ALT via HR (Arora 2014). These R loops are very likely responsible for opening the helix and providing sites for recombination events. Could transcription of TERRA open the helix and stimulate t-loop formation?
  22. 22. BsmBI T7 (TTAGGG)96 (CCCTAA)96 BbsI T3 ScaI (TTAGGG)96 T7 (TTAGGG)9 T7 T7 3 kb pGEM backbone pRST5 TEMPLATES TERRA
  23. 23. Transcription followed by Proteinase K/SDS treatment shifts the DNA to a smear which is resolved into a ladder of multimer bands by RNase A 2 3 4 DNA transcribed no + RNAseA RNaseA
  24. 24. The smear: TRANSCRIPTION OF pRST5 WITH T7 RNA POLYMERASE RESULTS IN 92% OF THE DNA (N=200) SHOWING RNA BUNDLES AT ONE END
  25. 25. RNaseA treatment following transcription and deproteinization reveals an abundance of t-loops at one end of the DNA: an intramolecular HR product
  26. 26. The ladder of bands: Transcription generates an abundance of multimer DNAs joined at their ends: intermolecular HR products
  27. 27. Transcription also generates “DNA bouquets” in which many DNAs have undergone intermolecular recombination at their telomeric ends.
  28. 28. A human telomere compacted by histones, shelterin and repair factors is 120 nm – 180 nm in diameter, similar to the diameter of a Herpes Virus capsid. HSV-1 DNA is 150kb in length which is 1/1000th the length of DNA of many human chromosomes.
  29. 29. CONTROLS: T-loops are NOT formed if: the incubation lacks RNA polymerase includes RNA polymerase but lacks triphosphates the DNA is incubated with Mg++, triphosphates, and purified TERRA but NO RNA polymerase If the repeats are transcribed, but reside in the center of the DNA T-loops are formed if: T3 RNA polymerase transcribes the repeats present at the other end of the DNA
  30. 30. TRANSCRIPTION-MEDIATED LOOPING IS HIGHLY EFFICIENT pRST5 DNA with 54 nt 3’ ss overhang, 30 min at 37 o with T7 RNA polymerase and triphosphates; +RNase A, 1h at 37C, then Proteinase K/SDS followed by agarose bead filtration to purify the DNA for EM. Fields of molecules scored at the EM. In one series of 6 experiments with this template 40% loops at one end 44% 47% 50% 52% 61% background: mock transcription but no RNA polymerase 4% loops (n=338) mock transcription but no ribo G 1% loops (n=201) (790 molecules scored)
  31. 31. T7 T7 Transcription of the mini-chromosome with telomeres at both ends generates mini-chromosomes with 2 t-loops
  32. 32. t-loop formation does NOT require a 3’ single strand overhang T loops formed with a blunt ended telomere pRST5 with a 4 nt 5’ extension 55% loops (n=165) 45% loops (n=150) pRST5 blunt ended 38% loops (n=165) pRST5 with telomeric tract at the opposite end and a 4nt 5’ extension transcribed with T3 RNA polymerase 3 experiments (n=470) 35% loops 46% loops 47% loops Looping does not require a 3’ overhang and occurs equally well with two different RNA polymerases
  33. 33. The observation that t-loops are formed with BLUNT ended DNA argues that BOTH strands must be inserted at the t-loop and the junction is more complex than a simple D-loop This raises two issues: Are these t-loops more stable than ones generated by insertion of just a 3’ ss DNA tail? What is the appearance of the junction at higher EM resolution?
  34. 34. TRANSCRIPTION-MEDIATED T-LOOPS ARE VERY STABLE EXPERIMENT: Transcribe pRST5 containing a 54 nt 3’ overhang, then crosslink with psoralen and UV, process for EM 61% loops (n=109) no crosslinking, treat with RNase A, Pr K/SDS, leave at 4 degrees for 60 hr then crosslink and prepare for EM 48% loops (n=100) Thus the t-loops are very stable without having to be crosslinked. EXPERIMENT: Transcribe pRST5 with a 54 nt overhang, store for 72 hr at 4 degrees, then treat with RNase A, and prepare for EM. 57% loops The loops may be further stabilized by the presence of TERRA.
  35. 35. T-loop with no discontinuity at the junction T-loops with small 11x13 nm beads at the loop junction Transcription-mediated t-loops frequently contain a small nucleic acid bead at the junction The beads remain after extensive Proteinase K/SDS and RNase A treatment but are diminished ~50% in number by RNase H We believe they may contain both TERRA and ss DNA The percentage of junctions with a bead has varied from 25-75% over many experiments
  36. 36. Very long single stranded TTAGGGn DNA appears as a chain of large particles, and single molecule magnetic tweezers pulling reveals discrete steps. Griffith and Fishel labs (unpublished data)
  37. 37. Non è possibile visualizzare l'immagine. TERRA REMAINS AT THE JUNCTIONS DESPITE RNase TREATMENT T7 transcription reactions were carried out with biotin-16 UTP in the reaction followed by purification including Rnase A treatment. Iron nano-particles coated with streptavidin were then added to detect and remaining RNA.
  38. 38. The t-loop junctions frequently show extruded DNA stems typical of chicken foot structures A loop formed by invasion of both strands has features of both replication forks and a Holliday junctions
  39. 39. GEN1 binds preferentially at the t-loop junction GEN1 Courtesy of West lab
  40. 40. In search for species with unusual telomeres, Lubomir Tomaska captures an example with a telomere at only one end.
  41. 41. p53 binds to the t-loop junction and also to sites of residual R loops, here at the telomere-plasmid junction
  42. 42. Synthesis of high molecular weight telomeric ss and ds DNA and TERRA
  43. 43. 41 kb 27 kb 22 kb Double stranded telomeric DNA (TTAGGG)n beginning with a T7 promoter
  44. 44. EXAMPLES OF T-LOOPS GENERATED ON THE LONG TELOMERIC DNA 11.1 kb 8.6 kb 7.4 kb
  45. 45. EXAMPLES OF T-LOOPS GENERATED ON THE LONG TELOMERIC DNA 5.3 kb 3.8 kb4.4 kb 2.6 kb
  46. 46. Template % looped molecules G-rich DNA 5(TTAGGG) 28.5, 27, 28 G-rich DNA (no polymerase) 4, 4.5, 4 G-rich DNA (no triphosphates) 6.5, 5, 4.5 G-rich DNA (+ S1 nuclease) 27, 26, 26 Quadruplex mutant DNA 5(TTAGTG)n3’ 12, 11, 9.5 Sequence mutant DNA 5(TGAGTG)n3 16, 11, 14 CONTROLS WITH THE LONG DNAs
  47. 47. POSSIBLE IMPLICATIONS Transcription of telomere promotes HR, providing another pathway of forming t-loops. These loops are very stable due to a more complex junction than a simple D-loop. Loop formation is DNA-driven --- reflecting the G-rich nature of the telomeric DNA sequence as contrasted to being protein- driven. The complex junction may provide a means of extending the telomere in the absence of telomerase and hence the unique nature of the telomeric repeats may have evolved for this purpose prior to the appearance of telomerase. Ability to form these loops in vitro will facilitate studies of junction cleavage and protection.
  48. 48. The mammalian repeat TTAGGG is evolutionarily very old, dating at least to the 1950’s Telomeric sequences

×