Mutagenesis in Ataxia Telangiectasia Induced Pluripotent Stem Cells
Lucy LinDr. Ronald P. Hart
Department of Cell Biology and NeuroscienceRutgers, The State University of New Jersey
Summer Undergraduate Research Fellowship 2015
Ataxia Telangiectasia• Ataxia = loss of motor control; Telangiectasia = spider veins• Neurodegenerative, inherited disease caused by mutation in ATM
gene– Causes tumor formation– Impairs cerebellum– Prevents repair of broken DNA
• Some symptoms– Nystagmus (rapid involuntary eye movement)– Ocular telangiectasia
ATM Kinase Protein• 370 kilodalton• Serine/threonine protein • Member of the phosphatidylinositol 3-kinase related kinase (PIKK)
family important for DNA repair regulation• Regulates DNA damage response cascade to arrest cell cycle for
DNA repair
Llorca et al., 2003
Induced Pluripotent Stem Cells• Cells that can be reprogrammed from adult
somatic cells through transcription factors to become pluripotent
Bellin, M., Marchetto, M. C., Gage, F. H., and Mummery, C. L., 2013
Retrovial infection with 4 transcription factors: Oct3/4, Sox2, cMyc, Klf4
A-T Cell Lines are PluripotentOct-4 Tra-1-60
Q1-SA
Q3-SA
Q3-SC
CAR3-SB
ATM -/-
ATM -/-
ATM -/-
ATM +/-
• CAR3 Cells (carrier): Deletion causing frameshift on one allele and one normal allele
• Q1 and Q3 Cells (A-T): Compound heterozygote - deletion causing frameshift on one allele and point mutation on other
Hart, R. P., unpublished
A-T Cell Line ATM Allele Mutations
Lazaropoulos, M., unpublished
Subject Code
Age Sex Diagnosis Mutations iPSC Lines
JHU_Q1 23 F A-T c.[1564delGA];[7181C>T]
Q1-SA
JHU_Q3 8 M A-T c.[7792C>T];[217_218delGA]
Q3-SAQ3-SC
JHU_CAR3 42 F Carrier Not tested CAR3-SB
Q1 and Q3 Allele Mutations
Hart, R. P., unpublished
Subject Code
Age Sex Diagnosis Mutations iPSC Lines
JHU_Q1 23 F A-T c.[1564delGA];[7181C>T]
Q1-SA
JHU_Q3 8 M A-T c.[7792C>T];[217_218delGA]
Q3-SAQ3-SC
Hypothesis
• Spontaneous mutagenesis is exacerbated by the absence of ATM-guided DNA repair, which can lead to reversion of the ATM gene to produce functional ATM kinase–All ATM -/- cells are more sensitive to
reversion than other ATM genotypes
Methods• Introduce DNA damage to the cells
through X-Irradiation to induce mutagenesis and perhaps reversion
• Incubate cells for another week to allow time for reversion
• One week after the first irradiation, irradiate cells again to elicit pATM formation
• After fixing and staining cells, images were taken using IN CELL Analyzer 6000, an automated confocal microscope
X-Irradiation
Double Strand Breaks
pATM formation after irradiation seen in all ATM -/- cell lines
Images and Data Collected by Gary Hoffman, 2015
Conclusions• Further support of reversion in Q3-SC cells• Possibility of reversion in Q3-SA cells • Reversion in Q1-SA cells?
Hart, R. P., unpublished
S1981
Future Directions
• Repeat mutagenesis experiment with the addition of staining for γH2A.X along with pATM
• Establish IC50 of ATM inhibitor for control cells and genomically reverted A-T cell line Q3-SC
AcknowledgementsHart Lab• Ronald P. Hart, PhD• Alana Toro-Ramos• Gary Hoffman• Michael Lazaropoulos• Eileen Oni• Mavis Swerdel• Jennifer Moore, PhD• Angela Tiethof• Kunal Garg• Sri Puli
Lab Collaboration• Lourdes Serrano, PhD• Zhiping Pang, PhD
SURF Directors• Lauren Aleksunes,
PharmD, PhD• Debra L. Laskin, PhD
Funding• National Institutes of Health
R25ES020721 Grant• A-T Children’s Project of the
ATCP Foundation
References1. Adams, B.R., Golding, S.E., Rao, R.R., Valerie, K. (2010). Dynamic dependence on ATR and ATM for double-strand break repair
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10. Sedgwick, R.P., and Boder, E. (1960). Progressive ataxia in childhood with particular reference to ataxia-telangiectasia. Neurology 10, 705-715.
11. Serrano, L., Liang, L., Chang, Y., Deng, L., Maulion, C., Nguyen, S., and Tischfield, J.A. (2011). Homologous recombination conserves DNA sequence integrity throughout the cell cycle in embryonic stem cells. Stem Cells Dev. 20, 363-374.
12. Shiloh, Y., and Ziv, Y. (2013). The ATM protein kinase: regulating the cellular response to genotoxic stress, and more. Nat Rev Mol Cell Biol 14, 197-210.
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