Ataxia telangiectasia is a neurodegenerative, inherited disease that is very rare, affecting 1 out of 40000 children worldwide each year. Ataxia refers to loss of motor control. Telangiectasia refers to the spidery veins that often appear in subjects with the disease due to dilation of blood vessels near the surface of the skin or mucous membrane. Other symptoms of A-T include tumor formation, so many A-T subjects develop cancers.
In particular, A-T impairs the cerebellum by affecting the Purkinje cells, causing deficits in motor coordination. The disease occurs due to the deficiency of ATM protein, which is essential for repair of broken DNA.
Possible questions: -Why does telangiectasia occur in A-T? There aren’t conclusive studies on why A-T causes telangiectasia. We know that acquired telangiectasia is often due to venous reflux. And we know that in A-T, the deficiency in ATM protein means a deficiency in DSB repair if the DNA is ever damaged. Accumulation of these breaks leads to genome instability. Since A-T affect various cell types that rely on ATM and other PIKK kinases for DNA repair, including endothelial cells lining the vein walls, endothelial cells lining the vein walls are likely weakened in A-T subjects and allow for venous reflux, creating the telangiectasia we see. -If A-T affects all cells, why do we mainly see it affecting Purkinje neurons? Neurons are inherently more susceptible to damage because they already experience excitotoxicity.
Fairly large 370 kDa protein Serine/threonine protein kinase that is a member of the phosphatidylinositol 3-kinase related kinase family; proteins in this family are often considered housekeeping and are important for DNA repair regulation ATM regulates the DNA damage response cascade by activating Chk2 Checkpoint Kinase 2: important for activating further proteins for cell cycle arrest at the G1-S phase for DNA repair. Electron microscopy and 3D reconstructions of a human ATM kinase. The images shows that the kinase uses an arm-like domain to clamp around DNA, so the arm-like domain will wrap around the DNA in regions of DSBs to start the DNA damage response and recruit other proteins to the site of damage for repair.
First, there needs to be damage to the DNA for the repair response to start. The damage is usually in the form of DSBs induced by ionizing radiation, such as X-rays. A complex called MRN recruits ATM to the site of damage. The recruited ATM then phosphorylates the histone isoform H2A.X to γH2A.X, and γH2A.X binds to the DSBs. This binding causes ATM to further activate via autophosphorylation at the S1981 site, though there are other sites of autophosphorylation on ATM required for kinase activation. The activated pATM then activates Chk2 at the T68 site. The pChk2 goes on to activate further downstream targets, namely p53 the tumor suppressor, all to eventually arrest the cell cycle at the G1 phase in order for DNA repair to occur before replication.
So knowing the importance of ATM kinase in DNA repair explains why we see a pleiotropic phenotype in subjects with A-T, who have neurological abnormalities, immunodeficiency, and predisposition to cancer.
EXTRA: The onset of DNA damage repair involves a cascade of substrate phosphorylation that begins with the activation of H2AX after radiation damage. The Mre11-Rad50-Nbs1 (MRN) complex recruits ATM to sites of DNA damage, where it phosphorylates as many as 700 substrates involved in DNA repair and cell cycle checkpoint activation ATM phosphorylates the histone H2A isoform H2AX at Serine-139 to gH2AX, a 15 kDa protein that binds to DNA double strand breaks (DSBs), or damage caused by irradiation (not shown in image). The binding of gH2AX to DSBs further activates ATM via autophosphorylation at various sites, though most notably at Serine-1981, Serine-367, and Serine-1893 (Smith et al., 2010). The activated ATM in turn phosphorylates Checkpoint Kinase 2 (Chk2) on Threonine-68 (Ahn et al., 2000). Once phosphorylated, two Chk2 molecules homodimerize, leading to intermolecular autophosphorylation and full activation (Ahn et al., 2002; Cai et al., 2009). This activated Chk2 molecule dissociates from sites of damage, disperses throughout the nucleus to act on multiple substrates involved in cell cycle progression, and arrests the cell cycle at the G1-S phase for DSB repair by activating transcription factor function of p53 tumor suppressor protein and p21CIP1, a cyclin-dependent kinase inhibitor and downstream target of p53 (Lukas et al., 2003; Kulkarni and Kumuda, 2008; Kastan and Bartek, 2004).
G1 Phase: cell grows in size and synthesizes mRNA and proteins in preparation for subsequent steps leading to mitosis. It’s important to note that the ATM cascade stops the cell cycle at the G1 phase because DNA damage should be repaired before replication of DNA, as well as to prevent transcription of mRNA with the wrong sequence, which ultimately leads to dysfunctional protein.
In order to study this disease state in the lab, I culture induced pluripotent stem cells that come from patients with A-T. iPSCs are mature cells that are reprogrammed to become pluripotent by the introduction of transcription factors, including Oct4, Sox2, c-Myc, and Klf4. One benefit to using iPSCs for studies is disease modeling, which is what we currently do at our lab. We can take cells from a subject with the disease, culture them to become pluripotent, and then study the progression of the disease as the cells differentiate. Since iPSCs can be reprogrammed from cells obtained from the patient, it is also a promising outlet for personalized clinical therapy.
EXTRA: Transcription Factors: 1. Oct3/4: A transcription factor that is associated with many target genes implicated in maintenance of pluripotency 2. Sox2: A transcription factor necessary for embryonic development and for preventing ES cell differentiation 3. C-Myc: A transcription factor with many cellular functions; important for proliferation. An issue is that C-Myc is oncogenic, so there may be some risk of cells transformed by C-Myc. 4. Klf4: A transcription factor whose overexpression inhibits differentiation of ES cells
iPSCs were reprogrammed from the lymphocytes of subjects. CAR3 cells, or the carrier, are from a blood sample of a carrier of A-T at Johns Hopkins Hospital. Q3 cells, or the A-T cells, are from a blood sample of the child of the carrier. Q1 cells were obtained from another subject unrelated to Q3 and CAR3 who has A-T.
Oct3/4: A transcription factor that is associated with many target genes implicated in maintenance of pluripotency. Oct-4 antibody in immunofluoresence recognizes a protein that is critically involved in the self-renewal of undifferentiated ES cells. It is a nuclear stain, as seen in the image on the slide.
Tra-1-60: The TRA-1-60 antibody recognizes a protein expressed on undifferentiated human embryonic stem cells and germ cells. The epitope that the antibody recognizes is lost upon cell differentiation.
Now that we have confirmed pluripotency in our cell cultures, we want to make sure the cells also have the mutations that we see in the subjects from which they came from.
The CAR3 cells, which are from the carrier of A-T, have a frameshift mutation on one allele, as seen in the top row of the gene mutation map in the top right. The purple regions indicate the wild type sequence in ATM+/+ control cells called SC1, which are from a random sample of healthy subjects. While my experiments focus on the A-T cells, you will see later on that some of my results include these ATM+/+ cells as an additional positive control. The yellow line on this one allele indicates the position of the deletion of two bases. The other ATM allele has no mutations, so functional ATM kinase can still be produced in the CAR3 cells.
Our second cell line Q3 are cells obtained from a subject with A-T, namely the child of the CAR3 subject. Here, you see the same frameshift mutation caused by a deletion of two bases that we see in the CAR3 subject. Additionally, on the other allele there is a point mutation from cytosine to thymine, shown here by the yellow-shaded region.
A protein domain is a conserved part of a given protein sequence and structure that can evolve, function, and exist independently of the rest of the protein chain. The PIK domain sequence is found in all proteins of the PIKK family. Consequently, the coding of the PIK domain, shown in the image in red, is necessary for the translation of functional ATM.
Q1SA: One allele has a frameshift that results in a nonsense mutation. The PIK domain sequence is not coded for, resulting in the formation of a dysfunctional, truncated protein. One allele contains a substitution mutation that results in a missense mutation. The sequence in this area is normally responsible for the S2394 phosphorylation site on ATM. So while the PIK domain is coded for and a full protein can be made in Q1SA, it is dysfunctional due to the mutation at the S2394 site.
Q3: -One allele has a frameshift that results in a nonsense mutation. Again, the PIK domain sequence is not coded for, resulting in a truncated protein. -The other allele has a substitution that leads to a pre-mature stop codon before the PIK domain sequence, resulting in the formation of a truncated protein.
Since we have confirmed the allele mutations in our A-T cell lines, we would expect that functional ATM kinase would not be present in these cells. Consequently, we should not see any downstream targets of ATM in these cells when we induce DNA damage via irradiation.
EXTRA: PIKKs proteins contain the following four domains: 1. N-terminus FRAP-ATM- TRRAP (FAT) domain 2. C-terminus FAT-C-terminal (FATC) -FAT is named after the main groups of protein that share the domain (FRAP, ATM, and TRRA) -FATC is named for the same reason as FAT (it’s a domain shared by FRAP, ATM, and TRRA). The C indicates that the domain is located on the C-terminus. 3. These domains have been shown to participate in the activation of ATM
Pfam is a database that is used to view multiple domains in relation to one another
PIK domain wedged between FAT and FATC
However, when we probe for pChk2, a downstream target of ATM as seen in the previous diagram, through Western blotting, we see an unexpected band in Q3SC cells that have been irradiated here on the left blot. So our question was, why do we see no ATM activity in one cell line here (Q3SA), but we do see ATM activity in a separate cell line (Q3SC) derived from the same individual?
Noting the presence of ATM activity in the Q3SC cell line, we then decided to add an ATM inhibitor called KU-55933 to the different cell lines. The inhibitor acts by reversibly binding to the ATP binding site on ATM. While we do see a fainter band in the ATM+/+ control (SC1), there is still ATM activity, as seen by the presence of pChk2, in the Q3SC cell lines after irradiation. This indicates that we need to do a proper dose response curve to find a more effective concentration of KU to bring about inhibition of ATM activity, and I will touch upon this more when I discuss future directions.
So what could this mean? Well, the mutated ATM gene in the Q3SC cell line has reverted. How? Well, the mutation mapping shown in one of the previous slides was done around the fourth passage of the Q3 iPSCs. Shortly after, we believe spontaneous mutagenesis occurred in the Q3SC cell line, reverting the previously mutated ATM gene so that functional ATM kinase can be produced in these cells. We say spontaneous because at that point, we had not done any experiments to the Q3SC cells that would induce gene rearrangement.
Possible question: -Second band could be explained by some degradation of the protein in the cells during protein prep for the Western blot.
To confirm this idea, we ran a second activity assay by staining for γH2A.X, a more direct target of the DNA Damage Response, via immunofluoresence. As expected, we see γH2A.X in the CAR3 cells (since that one normal allele is able to produce functional ATM), while little to no γH2A.X in the Q1SA and Q3SA cells. Similar to what we saw in the Western blots, there is a high number of γH2A.X in the Q3SC cells. This confirms the robust ATM activity seen in the Q3SC cells in contrast to the little activity seen in the other A-T cell lines.
Possible Question: -Why do we see a small dot of γH2A.X in the image? It is possible that a few cells have indeed undergone spontaneous reversion of the ATM gene at that point. It is clear, however, that we see more signs of γH2A.X in the double-irradiated Q3SA cells from the mutagenesis experiment, supporting the hypothesis that spontaneous mutagenesis is exacerbated by the absence of DNA repair after irradiation.
One of the worries we had after seeing these data is that we may have accidentally cross-contaminated cell lines over time. So we turned to sequencing for SNPs present in each A-T cell line. A Single Nucleotide Polymorphism is a DNA sequence variation occurring commonly within a population, in which a single nucleotide in a particular sequence differs between members of the same species, in this case humans. So we would expect a larger fraction of SNPs that are identical between family members than between unrelated subjects. We looked at 700,000 different nucleotides known for their high variability in the human genome in our cell lines. The heat map generated above by my lab peer Gary Hoffman shows the fraction of shared SNPs between the various cell lines. The orange region we see between CAR3 and Q3 cells indicates that the two lines share a fraction of identical SNPs, while the red region we see between Q1 and Q3 cells indicates an even smaller fraction of shared SNPs. This makes sense: while CAR3 is a different subject from Q3, the two are related as mother-child, so they would share a larger fraction of SNPs. Q1 is a subject from a different family altogether, so Q1 would share a smaller fraction of SNPs from Q3.
The off-white region we see among the Q3SA iPSCs, Q3SC iPSCs, and original Q3 T cells from which they derived from indicate they share 100% of their SNPs. This shows that despite having different ATM activity responses, Q3SA and Q3SC cells perfectly match in their SNPs to the original Q3 lymphocytes from which they derived, confirming that the cultures have not been mixed up through various passages.
1. Plate Q1SA, Q3SA, Q3SC iPSCs on two 96-well plates (20,000 cells/well) 2. Wait 2-3 days for cells to re-form into iPSC colonies 3. Introduce DNA damage to the cells through X-Irradiation to investigate the effects on DNA damage repair capability by irradiating plate at 1 Gy (100 V for 1 min.) – cheat: for this presentation, we assume no control and only one plate of cells 4. Incubate both plates after irradiation and continue to feed cells for one week to allow time for reversion to potentially occur in the A-T cell line genomes 5. One week after the first irradiation, plate was irradiated again at 1 Gy to elicit pATM formation 6. Incubate plates for 30 minutes after second irradiation before fixing cells with 4% PFA 7. Continue with ICC to stain for pATM
After inducing the ATM -/- cell lines with X-irradiation twice, we do see a small fraction of pATM in the Q3SA cells, and a slightly larger fraction in the Q1SA cells. Contrast this to what we saw in the western blot and immunofluoresence assays, in which there was little to no pChk2 or γH2A.X, respectively, after irradiation in either the Q1SA or Q3SA cells.
As expected for Q3SC, which we believe to have reversion as seen by the presence of pChk2 in the western blot and γH2A.X in the immunofluoresence, there is an even larger fraction of nuclei with pATM after irradiation.
The results we see from the mutagenesis experiment further support that reversion of the ATM gene has occurred in the Q3SC cells. The presence of pATM in the Q3SA cells, although small, also points to the possibility of reversion having occurred.
We can say the same for Q1SA as well. The small presence of pATM seen after irradiation is potential sign of reversion. Alternatively, however, the formation of pATM in specifically the Q1SA cells can also be explained by leaky autophosphorylation of the ATM kinase. Remember from the gene mutation map shown on a previous slide, reproduced on this slide, that a point mutation on one allele leads to a dysfunctional S2394 autophosphorylation site in Q1 cells. The pATM antibody we used in the experiment recognizes pATM that has been phosphorylated at S1981. So it is possible that gene reversion did not occur and the ATM kinase is not actually fully active because of the dysfunctional S2394 site caused by the gene mutation specifically in the Q1SA cells, and that the pATM we do see in the previous stain is just due to leaky autophosphorylation at the S1981 site detected by the specific pATM antibody that was used.
In order to confirm whether or not reversion occurred in the Q1SA and Q3SA cell lines, we will repeat the mutagenesis experiment, this time also staining for γH2A.X, which is a more direct target of active ATM in the DNA repair response. The presence of γH2A.X in these ATM -/- cells would indicate irradiation-exacerbated reversion.
We also want to establish the half maximal inhibitory concentration of the ATM inhibitor KU-55933 by doing a dose response experiment. An effective concentration will allow us to use the genomically reverted Q3SC cell line as either a positive control or, with effective ATM inhibition, negative control depending on our needs for future experiments. We would use the IN CELL Analyzer microscope again to image cells treated with varying concentrations of KU inhibitor and stain for downstream targets of ATM as a readout for ATM activity.
EXTRA: -IC50: indicates how much of a particular drug is needed to inhibit a particular biological process by half ***Q3SC is isogenic control of Q3SA for A-T
SURF 2015 Mutagenesis in Ataxia Telangiectasia Induced Pluripotent Stem Cells
Mutagenesis in Ataxia Telangiectasia
Induced Pluripotent Stem Cells
Dr. Ronald P. Hart
Department of Cell Biology and Neuroscience
Rutgers, The State University of New Jersey
Summer Undergraduate Research Fellowship 2015
• Ataxia = loss of motor control; Telangiectasia = spider veins
• Neurodegenerative, inherited disease caused by mutation in ATM
– 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
Llorca et al., 2003
DNA Damage Repair Cascade
Matsuoka et al., 2007
Induced Pluripotent Stem Cells
• Cells that can be reprogrammed from adult
somatic cells through transcription factors to
Bellin, M., Marchetto, M. C., Gage, F. H., and Mummery, C. L., 2013
infection with 4
Sox2, cMyc, Klf4
• CAR3 Cells (carrier): Deletion causing
frameshift on one allele and one normal
• 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
Age Sex Diagnosis Mutations iPSC Lines
JHU_Q1 23 F A-T c.[1564delGA];
JHU_Q3 8 M A-T c.[7792C>T];
JHU_CAR3 42 F Carrier Not tested CAR3-SB
Q1 and Q3 Allele Mutations
Hart, R. P., unpublished
Age Sex Diagnosis Mutations iPSC Lines
JHU_Q1 23 F A-T c.[1564delGA];
JHU_Q3 8 M A-T c.[7792C>T];
High levels of γH2A.X seen in Q3-SC cells
ATM +/- ATM -/- ATM -/- ATM -/-
Q3-SA and Q3-SC Cells are from the
Hoffman, G., unpublished
• 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
–All ATM -/- cells are more sensitive to
reversion than other ATM genotypes
• Introduce DNA damage to the cells
through X-Irradiation to induce
mutagenesis and perhaps
• 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
pATM formation after irradiation seen in
all ATM -/- cell lines
Images and Data Collected by Gary Hoffman, 2015
• Further support of reversion in Q3-SC cells
• Possibility of reversion in Q3-SA cells
• Reversion in Q1-SA cells?
Hart, R. P., unpublished
• Repeat mutagenesis experiment with the
addition of staining for γH2A.X along with
• Establish IC50 of ATM inhibitor for control cells
and genomically reverted A-T cell line Q3-SC
• Ronald P. Hart, PhD
• Alana Toro-Ramos
• Gary Hoffman
• Michael Lazaropoulos
• Eileen Oni
• Mavis Swerdel
• Jennifer Moore, PhD
• Angela Tiethof
• Kunal Garg
• Sri Puli
• Lourdes Serrano, PhD
• Zhiping Pang, PhD
• Lauren Aleksunes,
• Debra L. Laskin, PhD
• National Institutes of Health
• A-T Children’s Project of the
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