Supplementary Information
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Supplementary Information to:
MOZ (MYST3, KAT6A) inhibits senescence via the INK4AARF pathway
Bilal N. Sheikh1,3, Belinda Phipson2,4, Farrah El-Saafin1,3, Hannah K. Vanyai1,3, Natalie
L. Downer1, Matthew J. Bird4,5, Andrew J Kueh, Rose E. May1, Gordon K. Smyth2,6,
Anne K. Voss1,3,7, and Tim Thomas1,3,7
1
Division of Development and Cancer, The Walter and Eliza Hall Institute of Medical Research,
Melbourne 3052, Victoria, Australia
2
Division of Bioinformatics, The Walter and Eliza Hall Institute of Medical Research, Melbourne 3052,
Victoria, Australia
3
Department of Medical Biology, University of Melbourne, Melbourne 3010, Victoria, Australia
4
Murdoch Children’s Research Institute, Flemington Rd., Melbourne 3052, Victoria, Australia
5
Department of Paediatrics, University of Melbourne, Melbourne 3010, Victoria, Australia
6
Department of Mathematics and Statistics, University of Melbourne, Melbourne 3010, Victoria,
Australia
7
These authors co-supervised this project and contributed equally
Running title: Moz suppresses senescence via the Ink4a-Arf pathway
Corresponding authors:
Tim Thomas, 1G Royal Parade, Melbourne 3052, Victoria, Australia, e-mail:
tthomas@wehi.edu.au Ph: +61 3 93452477 Fax: +61 3 9347 0852
Anne K. Voss, 1G Royal Parade, Melbourne 3052, Victoria, Australia, e-mail:
avoss@wehi.edu.au Ph: +61 3 93452642 Fax: +61 3 9347 0852
Supplementary Information
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Supplementary Figure 1 – Flow cytometry profiles indicating the amount of 5dodecanoylaminofluorescein di--D-galactopyranoside (C12FDG) hydrolysed by galactosidase. C12FDG only becomes fluorescent once hydrolysed by -galactosidase,
allowing quantification of -galactosidase activity. Consistent with an expected increase
in the number of senescent cells with passage number, a marked increase in C12FDGFITC signal was observed with increased passages. Importantly, the C12FDG-FITC
signal was higher in Moz-/- cultures as compared to control cultures at all time points
analysed.
Supplementary Information
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Supplementary Figure 2 – Moz mRNA levels in primary MEFs.
No differences in Moz mRNA levels relative to passage number were observed in wild
type MEFs. This suggests the MOZ-HAT complex levels are relatively stable and MOZ
is specifically recruited to chromatin as required. n = 4 independent cultures per time
point. Moz expression levels were standardised to housekeeping genes Gapdh and
Hsp90ab1.
Supplementary Information
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Supplementary Figure 3 – Cell cycle analysis of MEFs cultured in 20% O2.
(a) Representative FACS plot showing separation into different phases of the cell cycle:
G0, G1, S, G2 and M, based on Ki67 and DAPI staining. (b-d) Quantification of cells in
the different phases of the cell cycle at (b) passage two, (c) passage four and (d) passage
six. n = 4 wild type, 3 Moz+/- and 5 Moz-/-. * marks a significant difference between the
respective genotypes at p < 0.05.
Supplementary Information
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Supplementary Figure 4 – Flow cytometry plots of H2A.X analysis in MEFs cultured
in atmospheric oxygen (20% O2).
Supplementary Information
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(a) Primary MEFs were separated based on DNA content via DAPI. (b) Positive control
– MEFs were treated with 80 M H2O2 for 30 minutes to induce double stranded DNA
breaks, before being stained with an antibody against H2A.X. As expected H2O2
treatment led to increased H2A.X levels. (c) Primary MEFs were collected at passage
one, three and five, fixed, stained for H2A.X as an indicator of double stranded DNA
breaks, and analysed by flow cytometry. Consistent with the quantification provided in
Figure 2b, H2A.X profiles do not show differences between the wild type, Moz+/- and
Moz-/- MEFs. n = 5 wild type, 4 Moz+/-, and 3 Moz-/- cultures.
Supplementary Figure 5 – Cell cycle analysis of MEFs cultured at physiological levels
of oxygen (3% O2).
Cell cycle analyses were performed by flow cytometry using DAPI and Ki67 at
passages two, five and eight. The cell cycle profile was normal at passage two, while
there was a decrease in the number of Moz-/- MEFs in the G2/M phase at passages five
and eight, and concomitant increase in G0 and/or G1. n = 5 wild type, 3 Moz+/- and 4
Moz-/- cultures. Asterisks indicate a significant difference between marked genotypes at
*p < 0.05 and **p < 0.01.
Supplementary Information
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Supplementary Table 1 – List of the top 500 differently expressed genes between wild
type and Moz-/- cultures. Table is provided in an attached Microsoft Excel file.
Supplementary Table 2 – List of the top 500 differently expressed genes between wild
type and Moz+/- cultures. Table is provided in an attached Microsoft Excel file.
Supplementary Table 3 – List of the top 500 differently expressed genes in a dose
dependent manner – i.e. genes that are different between wild type and Moz-/- cultures
and show intermediate expression in Moz+/- cultures. Table is provided in an attached
Microsoft Excel file.
Supplementary Table 4 – List of Broad Institute datasets that significantly correlate
with the Moz dataset. The list of genes significantly changed in a ‘dose dependent’
manner (Supplementary Table 3) was compared to differentially expressed genes in the
datasets provided by the Broad Institute. Table is provided in an attached Microsoft
Excel file.
Supplementary Information
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Supplementary Figure 6 – Western blot analysis of passage 3 wild type and Moz-/MEFs grown at 20% O2.
Protein levels of senescence marker and mediator p16INK4A were increased as early as
passage 3 in Moz-/- MEFs compared to wild type. In contrast, p15INK4B, p21 and p53
levels were unchanged. Treatment with doxorubicin, which induces DNA damage and
cell death, is provided as a positive control for p53. These data are consistent with
mRNA levels of Ink4a, Arf and p21 in passage 3 MEFs (Figure 4), and Ink4a and Arf
levels in passage 5 MEFs (Figure 1e). n = 4 independent cultures of each genotype.
Actin is provided as a loading control.
Supplementary Information
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Supplementary Figure 7 – Expression analysis of wild type and Moz-/- MEFs.
(a) Moz mRNA was not detected in Moz-/- MEFs (b) Gene expression levels of major
regulators of senescence in wild type and Moz-/- MEFs grown at physiological 3%
oxygen. mRNA levels of genes encoding major inhibitors of senescence that were
decreased in Moz-/- MEFs at 20% O2 (Fig. 5) were also decreased at 3% O2 (here). This
suggests that the reduction in mRNA levels of Cdc6, Skp2, Ezh2, Melk, E2f2 and Nsd2
was independent of DNA damage, which is prevalent in MEFs cultured in 20% O2. (c)
Ink4b mRNA levels in Ink4a-Arf-/-;Moz-/- cells were not different to Ink4a-Arf-/- controls
(d) Ratio of housekeeping genes Hsp90ab1 mRNA to Gapdh mRNA were not different
between wild type and Moz-/- cells at 20% O2. n = 4 independent cultures of each
genotype. Gene expression levels were standardised to housekeeping genes Hsp90ab1
and Gapdh. Asterisks mark statistically significant differences between wild type and
Moz-/- cultures at *p < 0.05, **p < 0.01 and ***p < 0.001.
Supplementary Information
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Supplementary Figure 8 – CDC6 overexpression affords a modest growth advantage
to Moz-/- MEFs. MEFs were virally infected with a Cdc6-GFP vector and control
negative Empty-GFP vector. Infection with a Cdc6-GFP vector lead to a small but
statistically significant increase in Moz-/- cells compared to controls. n = 3 independent
cultures of each genotype. Asterisks mark statistically significant differences between
wild type and Moz-/- cultures at *p < 0.05.
Supplementary Table 5 – qRT-PCR primer sequences
Gene
Arf
Primer Sequence
F 5’ GCCGCACCGGAATCCT 3’
R 5’ TTGAGCAGAAGAGCTGCTACGT 3’
Bmi1
F 5’ GAGCAGATTGGATCGGAAAG 3’
R 5’ GCATCACAGTCATTGCTGCT 3’
Cbx7
F 5’ ATGGAGCTGTCAGCCATAGG 3’
R 5’ TGGCTCCCAGGTGCTATACT 3’
Reference
1
Supplementary Information
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Cbx8
F 5’ TGGTCGCAGAAGTACAGCAC 3’
R 5’ GGTTTTAGGCTTGGGTCCTC 3’
Cdca2
F 5’ ATCGAAGAAGCCACTCCTGA 3’
R 5’ TGTGTGGGAATTTCAAACGA 3’
Cdca8
F 5’ TCAAAAGATGCCTTCCATCC 3’
R 5’ CTCATAGCTGGCGTCACAAA 3’
Cdc6
F 5’ AGGAGCCAGACAGTCCTCAA 3’
R 5’ GGGTCAAAAGCAGCAAAGAG 3’
Csf1
F 5’ ATGGACACCTGAAGGTCCTG 3’
R 5’ GCTGGAGAGGAGTCTCATGG 3’
E2f1
F 5’ GAGGCTGGATCTGGAGACTG 3’
R 5’ GAAGCGTTTGGTGGTCAGAT 3’
E2f2
F 5’ GATGGAGTCCTGGACCTGAA 3’
R 5’ CTGCCTACCCACTGGATGTT 3’
E2f3
F 5’ GCTGTACCCTGGACCTCAAA 3’
R 5’ GGGTCTGTGTGTTTCCGTCT 3’
Eed
F 5’ CAACTGTGGGAAGCAACAGA 3’
R 5’ ATAGAGGGTGGCTGGTGTTG 3’
Ercc3
F 5’ TACTTCGCAGGGGGTAGATG 3’
R 5’ GTGGGCTTCAGGTCAATGTT 3’
Ezh2
F 5’ ATCTGAGAAGGGACCGGTTT 3’
R 5’ TCAGGGTCTTTAACGGGATG 3’
Gapdh
F 5’ TTCACCACCATGGAGAAGGC 3’
R 5’ CCCTTTTGGCTCCACCCT 3’
Golga7
F 5’ CAGAGGTGTGAGCAGTGGAA 3’
R 5’ TGGCAGCAGAGACAAGAGAA 3’
Hsp90ab1
F 5’ ACCTGGGAACCATTGCTAAG 3’
R 5’ AGAATCCGACACCAAACTGC 3’
Igf2
F 5’ ACCTTCCAGCCTTTTCCTGT 3’
R 5’ AAGCACCAACATCGACTTCC 3’
Igfbp5
F 5’ GGCGAGCAAACCAAGATAGA 3’
R 5’ TTTCTGCGGTCCTTCTTCAC 3’
Ink4a
F 5’ CGTACCCCGATTCAGGTGAT 3’
R 5’ TTGAGCAGAAGAGCTGCTACGT 3’
1
Ink4b
F 5’ AGATCCCAACGCCCTGAAC 3’
R 5’ CCCATCATCATGACCTGGATT 3’
1
Ink4c
F 5’ ACGTCAACGCTCAAAATGGA 3’
R 5’ TAGCACCTCTGAGGAGAAGCCT 3’
Ink4d
F 5’ TGAACCGCTTTGGCAAGAC 3’
R 5’ ACTAGTACCGGAGGCATCTTGG 3’
Kdm5a
F 5’ GAAGATCCCTGTGGTGGAGA 3’
R 5’ CGGCTACCCACTTTAGACCA 3’
1
2
Supplementary Information
Page 12
Mel-18
F 5’ TGTGACGTCCAGGTCCATAA 3’
R 5’ CGCCGTTTCATTTCATCTTT 3’
Melk
F 5’ TGGCTCTCTCCCAGTAGCAT 3’
R 5’ GAGTCTTGCTTTGCCACTCC 3’
Moz
F 5’ CTTACACGGATGCCAAAAGG 3’
R 5’ GTTTTATCTGTGCCGCCTTC 3’
c-Myc
F 5’ AGAGCTCCTCGAGCTGTTTG 3’
R 5’ TTCTCTTCCTCGTCGCAGAT 3’
Nek3
F 5’ GTAGCTTGGCTCCACTGGTC 3’
R 5’ CTGGGGTTGTACCAGTGCTT 3’
Nsd2
F 5’ TTTCTGCTGACCCACTCCTT 3’
R 5’ GGGCATCACCAAAGAACTGT 3’
p21
F 5’ GTGGGTCTGACTCCAGCCC 3’
R 5’ CCTTCTCGTGAGACGCTTAC 3’
1
p27
F 5’ AGTGTCCAGGGATGAGGAAGCGAC 3’
R 5’ TTCTTGGGCGTCTGCTCCACAGTG 3’
3
p57
F 5’ GCGCAAACGTCTGAGATGAGT 3’
R 5’ AGAGTTCTTCCATCGTCCGCT 3’
3
Pcgf2
F 5’ TGTGACGTCCAGGTCCATAA 3’
R 5’ CGCCGTTTCATTTCATCTTT 3’
Pcgf3
F 5’ CAGAACCATGCAGGACATTG 3’
R 5’ TCCATGCCCAGTTTGTGATA 3’
Peg3
F 5’ TCGACCATCTCATGCTTTTG 3’
R 5’ GTCTCGAGGCTCCACATCTC 3’
Ring1a
F 5’ CAGCGAAAAGCAGTACACCA 3’
R 5’ ATCATTTTGGGTCCTTCGTG 3’
Ring1b
F 5’ TTGCGCGGATTGTATTATCA 3’
R 5’ GCGCTTCATACTCATCACGA 3’
Scmh1
F 5’ CTCTGAACCTCCCAGCAGTC 3’
R 5’ AGGTTTTGGGATGTGCTGAC 3’
Skp2
F 5’ GCGCTAAAACAGGAGTCTGG 3’
R 5’ CCTGAAGGTGCTTCCTATGC 3’
Stat3
F 5’ TCACTTGGGTGGAAAAGGAC 3’
R 5’ TGGTCGCATCCATGATCTTA 3’
Suz12
F 5’ CTGACCACGAGCTTTTCCTC 3’
R 5’ TGGCAAACTTTCACAAGCAG 3’
Supplementary Information
Page 13
Supplementary Table 6 – ChIP primer sequences. The _5’ primers are between 500 and
750 bp 5’ of the TSS.
Gene
Primer Sequence
Reference
Albumin_TSS
F 5’ GGGGTAGGAACCAATGAAATG 3’
R 5’ ATTTTGCCAGAGGCTAGTGG 3’
4
B2m_TSS
F 5’ TGAACGACCAGATACACCAAAC 3’
R 5’ AAAGGGACTTTCCCATTTTCAG 3’
4
Cdc6_TSS
F 5’ TGTGGCGGGAGAGTTTTTAC 3’
R 5’ GGAGCTTTGCACTCTTCAGG 3’
E2f2_TSS
F 5’ ACGGGAACTAGAGGGGTGAA 3’
R 5’ GGACACTCGTGTGCTCTGAC 3’
Ezh2_TSS
F 5’ AGAGGCGCTTGATAGTGCTG 3’
R 5’ GACTCCACTGCCTTCGATGT 3’
Hgb-_TSS
F 5’ GTAAGGGCCAATCTGCTCAC 3’
R 5’ TGTCTGTTTCTGGGGTTGTG 3’
4
Hsp90ab1_TSS
F 5’ AATTGACATCATCCCCAACC 3’
R 5’ TCGTGCCAGACTTAGCAATG 3’
4
Melk_TSS
F 5’ GCTGCTGGAACTTGAATCCT 3’
R 5’ AGTCTAGCAAAGCCGGAACA 3’
Nsd2_TSS
F 5’ AGGCTGGATGGAATTTAGCA 3’
R 5’ CTGCCAAGGATTTCTGGTGT 3’
Skp2_TSS
F
3’
R 5’
5’ GGGAGTTGTGGGTATCTGGA
CTGCCAAGGATTTCTGGTGT 3’
R 5’ CCTGGGTTCTTCCACTCTG 3’
Cdc6_5’
F 5’ GTTATCAGCTCCTCCCCACA 3’
R 5’ TCCTGTATGGCATGAAGCAA 3’
E2f2_5’
F 5’ GAACAGGTTCCTGGGTTTCA 3’
R 5’ TGACCCTCAAGTGCATTTCC 3’
Ezh2_5’
F 5’ AGAGGCGCTTGATAGTGCTG 3’
R 5’ GACTCCACTGCCTTCGATGT 3’
Melk_5’
F 5’ CCCTATGAGATATGCACACTGC 3’
R 5’ ACCACCCAAAACCCTCTCTC 3’
Nsd2_5’
F 5’ GTGCCTAGTGTCTGTGGAGG 3’
R 5’ TCCCTGCAACTAAACGGTGG 3’
Skp2_5’
F
R 5’
5’ GCACGCTGATTTGATCTTCA
TCCCTGCAACTAAACGGTGG3’3’
R 5’ GCGATCCTTTCCTCACTTGT 3’
Supplementary Information
Page 14
Supplementary References
1.
2.
3.
4.
Li, H. et al. The Ink4/Arf locus is a barrier for iPS cell reprogramming. Nature
460, 1136-1139 (2009).
Sheikh, B.N., Dixon, M.P., Thomas, T. & Voss, A.K. Querkopf is a key marker
of self-renewal and multipotency of adult neural stem cells. Journal of cell
science 125, 295-309 (2012).
Matsumoto, A. et al. p57 is required for quiescence and maintenance of adult
hematopoietic stem cells. Cell Stem Cell 9, 262-271 (2011).
Voss, A.K., Collin, C., Dixon, M.P. & Thomas, T. Moz and retinoic acid
coordinately regulate H3K9 acetylation, Hox gene expression, and segment
identity. Developmental cell 17, 674-686 (2009).