Advances in complex systems July 3-7, 2017

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. 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. 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

5. 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.

6. In search for species with unusual telomeres, Lubomir Tomaska
captures an example with a telomere at only one end.

7. The mammalian repeat TTAGGG is evolutionarily very old, dating
at least to the 1950’s
Telomeric sequences

8. 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

9. 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

10. 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

11. 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

12. 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%

13. 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

14. 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.

16. 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

19. 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

20. 18 kb loop
T loop
from
HeLa
cells
18 kb
loop

23. telomeric
DNA from
garden peas
arranged as
a t-loop
pBlue
80 kb loop
pBlue
80 kb loop

24. 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.

25. 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

26. 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?

28. 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?

29. BsmBI
T7
(TTAGGG)96
(CCCTAA)96
BbsI
T3
ScaI
(TTAGGG)96
T7
(TTAGGG)9
T7
T7
3 kb pGEM backbone
pRST5 TEMPLATES
TERRA

30. 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

31. The smear: TRANSCRIPTION OF pRST5 WITH T7 RNA POLYMERASE RESULTS IN 92%
OF THE DNA (N=200) SHOWING RNA BUNDLES AT ONE END

32. RNaseA treatment following transcription and deproteinization reveals an
abundance of t-loops at one end of the DNA: an intramolecular HR product

33. The ladder of bands: Transcription generates an abundance of multimer
DNAs joined at their ends: intermolecular HR products

34. Transcription also generates “DNA bouquets” in which many DNAs
have undergone intermolecular recombination at their telomeric ends.

35. 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.

36. 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

37. 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)

38. T7
T7
Transcription of the mini-chromosome with telomeres at both
ends generates mini-chromosomes with 2 t-loops

39. 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

40. 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?

41. 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.

42. 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

43. 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)

44. 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.

45. 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

46. GEN1 binds preferentially at the t-loop junction
GEN1
Courtesy of
West lab

47. In search for species with unusual telomeres, Lubomir Tomaska
captures an example with a telomere at only one end.

48. p53 binds to the t-loop junction and also to sites of residual
R loops, here at the telomere-plasmid junction

49. Synthesis of high molecular weight telomeric ss and ds DNA and TERRA

50. 41 kb 27 kb 22 kb
Double stranded telomeric DNA (TTAGGG)n beginning with a T7
promoter

51. EXAMPLES OF T-LOOPS GENERATED ON THE LONG TELOMERIC DNA
11.1 kb 8.6 kb 7.4 kb

52. EXAMPLES OF T-LOOPS GENERATED ON THE LONG TELOMERIC DNA
5.3 kb
3.8 kb4.4 kb
2.6 kb

53. 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

54. 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.

55. The mammalian repeat TTAGGG is evolutionarily very old, dating
at least to the 1950’s
Telomeric sequences