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Semih Can Akıncılar1,2, Ekta Khattar1, Priscilla Li Shan Boon 3, Bilal Unal1,2, Melissa
Jane Fullwood3, Vinay Tergaonkar1,2,4*
1Division of Cancer Genetics and Therapeutics, Laboratory of NFκB Signaling, Institute of Molecular and Cell Biology (IMCB), A*STAR (Agency for Science, Technology and Research), Singapore 138673. 2Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore (NUS), Singapore 117597, Singapore 3 Cancer Science Institute, National University of Singapore, Singapore 4 Centre for Cancer Biology, University of South Australia and SA Pathology, Adelaide, Australia. Running Title: Telomerase re-activation by long-range interaction Key words: Telomerase re-activation, long-range interaction, Tert promoter mutation *Correspondence
Vinay TERGAONKAR, [email protected], Institute of Molecular and Cell Biology (A*STAR), Proteos, 61, Biopolis Drive, 138673, Singapore. Ph +65-65869836; Fax +65-67791117. Email: [email protected] Disclosure of Potential Conflicts of Interest No potential conflicts of interest to be disclosed.
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 20, 2016; DOI: 10.1158/2159-8290.CD-16-0177
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 20, 2016; DOI: 10.1158/2159-8290.CD-16-0177
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 20, 2016; DOI: 10.1158/2159-8290.CD-16-0177
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 20, 2016; DOI: 10.1158/2159-8290.CD-16-0177
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 20, 2016; DOI: 10.1158/2159-8290.CD-16-0177
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 20, 2016; DOI: 10.1158/2159-8290.CD-16-0177
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 20, 2016; DOI: 10.1158/2159-8290.CD-16-0177
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 20, 2016; DOI: 10.1158/2159-8290.CD-16-0177
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 20, 2016; DOI: 10.1158/2159-8290.CD-16-0177
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 20, 2016; DOI: 10.1158/2159-8290.CD-16-0177
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 20, 2016; DOI: 10.1158/2159-8290.CD-16-0177
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 20, 2016; DOI: 10.1158/2159-8290.CD-16-0177
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 20, 2016; DOI: 10.1158/2159-8290.CD-16-0177
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 20, 2016; DOI: 10.1158/2159-8290.CD-16-0177
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 20, 2016; DOI: 10.1158/2159-8290.CD-16-0177
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 20, 2016; DOI: 10.1158/2159-8290.CD-16-0177
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 20, 2016; DOI: 10.1158/2159-8290.CD-16-0177
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 20, 2016; DOI: 10.1158/2159-8290.CD-16-0177
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 20, 2016; DOI: 10.1158/2159-8290.CD-16-0177
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 20, 2016; DOI: 10.1158/2159-8290.CD-16-0177
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 20, 2016; DOI: 10.1158/2159-8290.CD-16-0177
two days and after 7 days cells were fixed with 75% ethanol for 30 min and stained
with 0.2% crystal violet dye.
Immunofluorescence: ALT-associated PML bodies were visualized via TRF2 and
PML immunofluorescence staining. Cells were grown on chamber slides (Millipore
EZ slides) and fixed with 4% paraformaldehyde for 10 minutes at room temperature.
After washing with PBS, cells were permeabilized with 0.2% Triton X-100 solution
in PBS for 5 minutes at room temperature and were blocked with PBS supplemented
with 0.2% Triton X-100 and 1% BSA (blocking buffer) for 30 minutes at room
temperature. Following incubation, cells were incubated with primary antibodies
TRF2 (Millipore-05-521) and PML (Santa-Cruz-sc-5621), 1/200 and 1/150 dilutions
respectively, in blocking buffer over night at 4°C. Cells were washed 4 times with
blocking buffer for 8 minutes and incubated with secondary antibodies AF488 and
AF555 (1/2000 dilution) (Invitrogen A11001 and A21428) for 1h at room
temperature. After 4 times washing, image acquisition was performed with Zeiss
LSM800, Plan-Apochromat 63x/1.40 aperture of the objective lenses.
Statistical Analysis: Student’s t-test (two-tailed) was performed to determine the
significance of difference for ChIP-qPCR qPCR and RT-TRAP experiments. Results
of each ChIP, gene expression, 4C, 3C assays were obtained from at least two or three
independent experiments as indicated in the figure legends.
Accession Numbers: All data has been uploaded to GEO (GSE77265).
Acknowledgments
S.C.A and B.U. are supported by the SINGA scholarship. We thank the Agency for Science Technology and Research, Singapore (A*Star) for funding and support to the V.T. laboratory. We thank Phua Qian Hua for her help during the genotyping process and Dr. Shang Li (DUKE-NUS Medical School, Singapore) for the southern blot telomere length experiment.
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 20, 2016; DOI: 10.1158/2159-8290.CD-16-0177
Grant Support This work supported by the core budget from Institute of Molecular and Cell Biology (IMCB), Singapore. P.B. and M.J.F are supported by the National Research Foundation (NRF) Singapore through an NRF Fellowship awarded to M.J.F (NRF-NRFF2012-054), and NTU school of biological sciences start-up funds awarded to M.J.F., as well as by funding given to the Cancer Science Institute, NUS, by the NRF and the Ministry of Education, Singapore under the Research Center of Excellence funding, and the RNA Biology Center at the Cancer Science Institute of Singapore, NUS, as part of funding under the Singapore Ministry of Education’s Tier 3 grants. References 1. Moyzis RK, Buckingham JM, Cram LS, Dani M, Deaven LL, Jones MD, et al. A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes. Proceedings of the National Academy of Sciences of the United States of America. 1988;85:6622-6. 2. Blackburn EH. The end of the (DNA) line. Nature structural biology. 2000;7:847-50. 3. O'Sullivan RJ, Karlseder J. Telomeres: protecting chromosomes against genome instability. Nat Rev Mol Cell Biol. 2010;11:171-81. 4. Yi X, Shay JW, Wright WE. Quantitation of telomerase components and hTERT mRNA splicing patterns in immortal human cells. Nucleic Acids Res. 2001;29:4818-25. 5. Shay JW, Wright WE. Senescence and immortalization: role of telomeres and telomerase. Carcinogenesis. 2005;26:867-74. 6. Horn S, Figl A, Rachakonda PS, Fischer C, Sucker A, Gast A, et al. TERT promoter mutations in familial and sporadic melanoma. Science. 2013;339:959-61. 7. Huang FW, Hodis E, Xu MJ, Kryukov GV, Chin L, Garraway LA. Highly recurrent TERT promoter mutations in human melanoma. Science. 2013;339:957-9. 8. Killela PJ, Reitman ZJ, Jiao Y, Bettegowda C, Agrawal N, Diaz LA, Jr., et al. TERT promoter mutations occur frequently in gliomas and a subset of tumors derived from cells with low rates of self-renewal. Proc Natl Acad Sci U S A. 2013;110:6021-6. 9. Heidenreich B, Nagore E, Rachakonda PS, Garcia-Casado Z, Requena C, Traves V, et al. Telomerase reverse transcriptase promoter mutations in primary cutaneous melanoma. Nat Commun. 2014;5:3401. 10. Akincilar SC, Unal B, Tergaonkar V. Reactivation of telomerase in cancer. Cell Mol Life Sci. 2016;73:1659-70. 11. Bell RJ, Rube HT, Kreig A, Mancini A, Fouse SD, Nagarajan RP, et al. Cancer. The transcription factor GABP selectively binds and activates the mutant TERT promoter in cancer. Science. 2015;348:1036-9. 12. Sui X, Kong N, Wang Z, Pan H. Epigenetic regulation of the human telomerase reverse transciptase gene: A potential therapeutic target for the treatment of leukemia (Review). Oncol Lett. 2013;6:317-22.
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28. Bell RJ, Rube HT, Xavier-Magalhaes A, Costa BM, Mancini A, Song JS, et al. Understanding TERT Promoter Mutations: A Common Path to Immortality. Mol Cancer Res. 2016;14:315-23. 29. Palumbo SL, Ebbinghaus SW, Hurley LH. Formation of a unique end-to-end stacked pair of G-quadruplexes in the hTERT core promoter with implications for inhibition of telomerase by G-quadruplex-interactive ligands. J Am Chem Soc. 2009;131:10878-91. 30. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8:2281-308. 31. Koh CM, Khattar E, Leow SC, Liu CY, Muller J, Ang WX, et al. Telomerase regulates MYC-driven oncogenesis independent of its reverse transcriptase activity. J Clin Invest. 2015;125:2109-22. 32. Ghosh A, Saginc G, Leow SC, Khattar E, Shin EM, Yan TD, et al. Telomerase directly regulates NF-kappaB-dependent transcription. Nat Cell Biol. 2012;14:1270-81. 33. Zhao Y, Cheng D, Wang S, Zhu J. Dual roles of c-Myc in the regulation of hTERT gene. Nucleic acids research. 2014;42:10385-98. 34. Splinter E, de Wit E, van de Werken HJ, Klous P, de Laat W. Determining long-range chromatin interactions for selected genomic sites using 4C-seq technology: from fixation to computation. Methods. 2012;58:221-30. 35. Andrews S. FastQC: a quality control tool for high throughput sequence data. 2010; Available from: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ 36. Buffalo V. Scythe. 2012; Available from: https://github.com/vsbuffalo/scythe 37. Lassmann T. TagDust2: a generic method to extract reads from sequencing data. BMC Bioinformatics. 2015;16:24. 38. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9:357-9. 39. Thongjuea S, Stadhouders R, Grosveld FG, Soler E, Lenhard B. r3Cseq: an R/Bioconductor package for the discovery of long-range genomic interactions from chromosome conformation capture and next-generation sequencing data. Nucleic Acids Res. 2013;41:e132. 40. Klein FA, Pakozdi T, Anders S, Ghavi-Helm Y, Furlong EE, Huber W. FourCSeq: analysis of 4C sequencing data. Bioinformatics. 2015;31:3085-91. 41. Hagege H, Klous P, Braem C, Splinter E, Dekker J, Cathala G, et al. Quantitative analysis of chromosome conformation capture assays (3C-qPCR). Nat Protoc. 2007;2:1722-33. 42. Akincilar SC, Low KC, Liu CY, Yan TD, Oji A, Ikawa M, et al. Quantitative assessment of telomerase components in cancer cell lines. FEBS Lett. 2015;589:974-84.
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Figure Legends Figure 1. Mutant Tert promoter displays active histone marks and distinct long-range interactions: (A) Cell lines that were used in the study with their origin and
Tert promoter status. WT refers to wild type Tert promoter; WT-ALT refers to wild
type Tert promoter with alternate lengthening of telomeres. (B) Schematic view of
were indicated as -146C>T and -124C>T. Tert promoter is shown in three parts
including proximal promoter (up to -1kb), distal promoter (-1kb to -5kb) and long
distance elements. (C) ChIP was performed in A375 and BLM melanoma cells
against histone marks (H3K4Me3 and H3K9Ac). (D) ChIP was performed in primary
melanocytes, Fadu and PC3 cell lines against histone marks (H3K4Me3 and
H3K9Ac). Graph shows qPCR analysis with % Input obtained across various regions
of Tert promoter with the distances indicated in boxes below X-axis. Error bars
indicate the mean ± SD of the two independent experiments. (E) 4C sequencing for
long-range interactions of Tert promoter. Plot generated by r3CSeq of chromatin
interactions of the Tert promoter for A375 and BLM cell lines. The top panel shows
the Refseq genes in chromosome 5. The line plots show detected interactions 500 kb
upstream and downstream of the Tert promoter. X axis indicates the distance from
Tert promoter which was shown as ‘0’. Y axis was the read counts for each
interaction. Different shades of red and blue color dots indicate positive interactions
for A375 and BLM cells (average of two replicates) respectively. Darker color
indicates more significant interactions according to q values indicated in the legend. P
values were calculated by two tailed Student’s t test method.
Figure 2. Reversing the Tert promoter mutation to wild-type reverses the active chromatin marks and alters long-range chromatin interactions. (A) CRISPR/Cas9 mediated reversal strategy of mutated -146C>T residue (shown as red
color) to wild type -146C residue (shown as orange color) by repair template
harboring -146C residue. We obtained BLM cells which have undergone CRISPR
process and are mutant for -146C residue and are labeled as BLM6. BLM cells which
have undergone CRISPR process and mutated back to wild-type for -146T residue to -
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 20, 2016; DOI: 10.1158/2159-8290.CD-16-0177
146C residue are labeled as BLM14. (B) DNA chromatograms spanning the Tert
promoter region in BLM6 and BLM14 are shown. ChIP was performed in BLM6 and
BLM14 cells against histone marks (H3K4Me3 and H3K9Ac). Graph shows qPCR
analysis with % Input obtained across various regions of Tert promoter with distances
indicated in boxes below x-axis. Error bars indicate the mean ± SD of the two
independent experiments. P values were calculated by two tailed Student’s t test
method. (C-E) Plot generated by FourCSeq of the differential intra-chromosomal
interactions of the Tert promoter between replicates of BLM6 and BLM14. The green
line shows the distance dependent fit and the blue dashed line indicates the fit of z-
score > 2. Interactions detected by z-score > 2 (p-adjusted value < 0.05) for at least
one replicate are shown as red dots. Fragments not called as interactions and that do
not show significant change between conditions are shown as black dots. (E) The
calculated log2 fold change is shown above the hg 19 Refseq genes in the regions 1
megabase from the Tert promoter. (F) Quantifications of 5 interactions, obtained from
(C-E), were measured by 3C-qPCR in BLM6 cells. (G) Quantification of Tert
promoter interaction with 300kb upstream (chr5:1,556,087-1,558,758) DNA region
(T-INT1) measured by 3C-qPCR in BLM6 and BLM14 cells. Error bars indicate the
mean ± SD of the 3 independent experiments. P values were calculated by two tailed
Student’s t test method.
Figure 3. Silencing GABPA expression dampens the active chromatin marks as well as long-range interactions in mutated Tert promoter. (A-B) ChIP was
performed in BLM6 and BLM14 cells against GABPA and Pol 2 followed by qPCR
with primers specific for Tert promoter region proximal to TSS. Graph shows qPCR
results with fold recruitment over IgG or % input method as indicated in y-axis. (C) GABPA expression analysis in BLM6 cells transfected siControl (siCont) and
siGABPA. (D) ChIP was performed in BLM6 cells transfected with siCont and
siGABPA against IgG, GABPA and Pol 2 followed by qPCR with primers specific
for Tert promoter region proximal to TSS. (E) Tert expression analysis in BLM6 cells
transfected with siCont and siGABPA. (F) ChIP was performed in BLM6 cells
transfected siCont and siGABPA against histone marks (H3K4Me3 and H3K9Ac)
followed by qPCR with primers specific for Tert promoter region indicated in boxes
below x-axis. (G) Quantification of Tert promoter interaction with 300kb upstream
(chr5:1,556,087-1,558,758) DNA region measured by 3C-qPCR in BLM6 cells upon
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 20, 2016; DOI: 10.1158/2159-8290.CD-16-0177
GABPA knockdown. (H) ChIP was performed in BLM6 and BLM14 cells against
BRD4 followed by qPCR with primers specific for Tert promoter region proximal to
TSS. (I) ChIP was performed in BLM6 cells transfected with siControl (siCont) and
siGABPA against BRD4 followed by qPCR with primers specific for Tert promoter
region proximal to TSS. (J) BRD4 expression analysis in BLM6 and BLM14 cells
transfected siCont and siBRD4. (K) GABPA expression analysis in BLM6 and 14
cells transfected with siCont and siBRD4. (L) ChIP was performed against IgG and
BRD4 in BLM6 cells followed by qPCR with primers specific for GABPA promoter.
(M) Tert expression analysis in BLM6 and BLM14 cells transfected with siCont and
siBRD4. (N) ChIP was performed against histone marks (H3K4Me3 and H3K9Ac)
followed by qPCR with primers specific for proximal Tert promoter region in BLM6
cells transfected with siCont and siBRD4. (O) Quantification of Tert promoter
interaction with 300kb upstream T-INT1 region measured by 3C-qPCR in BLM6 and
BLM14 cells with BRD4 knockdown. Error bars indicate the mean ± SD of the two
independent experiments. P values were calculated by two tailed Student’s t test
method.
Figure 4: Reversing the Tert promoter mutation to wild type in A375 melanoma and T98G glioblastoma cell lines reduces the active chromatin marks and affects long-range chromatin interactions. We obtained A375 cells which have undergone
CRISPR process and are mutant for -146C residue and are labeled as A375 -146C>T.
A375 cells which have undergone CRISPR process and mutated back to wild type for
-146T residue to -146C residue are labeled as A375 -146C . (A) Graph shows qPCR
analysis of Tert expression normalized to actin levels. (B) Graph shows telomerase
activity (TRAP) in A375 -146C>T and A375 -146C cells. (C-F) ChIP was performed
in A375 -146C>T and A375 -146C cells against histone marks (H3K4Me3 and
H3K9Ac), Pol2, GABPA and BRD4. Graph shows qPCR analysis with primers
specific to Tert promoter region proximal to TSS. Results were calculated with %
input or fold recruitment over IgG as indicated in y-axis. (G) 3C-qPCR assay was
performed in A375 -146C>T and A375 -146C cells. Quantification of Tert promoter
interaction with T-INT1 region is shown. (H-J) ChIP was performed in T98G -
146C>T and T98G -146C cells against histone marks (H3K4Me3 and H3K9Ac), Pol2
and GABPA. Graph shows qPCR analysis with % input obtained with primers
specific to Tert promoter region proximal to TSS. (K) 3C-qPCR assay was performed
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in T98G -146C>T and T98G -146C cells. Quantification of Tert promoter interaction
with chr5:1,556,087-1,558,758 region (T-INT1) is shown. n=3 for all the
experiments, error bars indicate mean±SD of 3 independent experiments. P values
were calculated by student’s t test method.
Figure 5: Introducing -146C>T Tert promoter mutation in HCT116 cells increases the active chromatin marks and enables long-range chromatin interaction. (A) CRISPR/Cas9 mediated conversion of -146C residue (orange color)
to mutant -146C>T residue (red color) by repair template harboring -146T nucleotide
is shown. We obtained HCT116 -146C cells which have undergone CRISPR process
and are wild type for -146C residue and are labeled as HCT116 -146C. HCT116 cells
which have undergone CRISPR process and are mutated for -146C residue to -146T
residue and are labeled as HCT116 -146C>T. (B) Graph shows qPCR analysis of
(TRAP) in HCT116 -146C and HCT116 -146C>T cells. (D-G) ChIP was performed
in HCT116 -146C and HCT116 -146C>T cells against histone mark H3K4Me3,
H3K9Ac, Pol 2, GABPA and BRD4. Graph shows qPCR analysis with % input
obtained with primers specific to Tert promoter region proximal to TSS. (H) 3C-
qPCR assay was performed in HCT116 -146C and HCT116 -146C>T cells.
Quantification of Tert promoter interaction with T-INT1 region is shown. n=3 for all
the experiments, error bars indicate mean±SD of 3 independent experiments. P values
were calculated by student’s t test method. (I) Figure summarizes the results of
GABPA enrichment, formation of long-range interaction, enrichment of active
histone marks and Pol2, Tert expression and telomerase activity that were obtained
from isogenic cell lines generated by CRISPR/Cas9 editing. “↑” indicates increase
and “↓” indicates decrease.
Figure 6: Removal of T-INT1 region in melanoma and glioblastoma cell lines reverses the active chromatin marks and decreases Tert expression and telomerase activity. (A) Removal of T-INT1 region strategy in -146C>T mutant cell
line is shown by CRISPR/Cas9 editing (left). Removal of T-INT1 region strategy in -
124C>T mutant cell line is shown by CRISPR/Cas9 editing (right). Red dots indicate
putative GABPA motifs in T-INT1 region. We obtained T-INT1 WT cells which have
undergone CRISPR process and are wild type for Chr5:1,556,087-1,558,758 region
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