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Mutation Processes in 293-Based Clones Overexpressing the DNA Cytosine Deaminase APOBEC3B.

Akre MK, Starrett GJ, Quist JS, Temiz NA, Carpenter MA, Tutt AN, Grigoriadis A, Harris RS - PLoS ONE (2016)

Bottom Line: Targeted sequencing of portions of TP53 and MYC demonstrated greater mutation accumulation in the A3B-eGFP exposed pools.Clones were generated and microarray analyses were used to identify those with the greatest number of SNP alterations for whole genome sequencing.A3B-eGFP exposed clones showed global increases in C-to-T transition mutations, enrichments for cytosine mutations within A3B-preferred trinucleotide motifs, and more copy number aberrations.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, Institute for Molecular Virology, Masonic Cancer Center, University of Minnesota, Minneapolis, MN, United States of America.

ABSTRACT
Molecular, cellular, and clinical studies have combined to demonstrate a contribution from the DNA cytosine deaminase APOBEC3B (A3B) to the overall mutation load in breast, head/neck, lung, bladder, cervical, ovarian, and other cancer types. However, the complete landscape of mutations attributable to this enzyme has yet to be determined in a controlled human cell system. We report a conditional and isogenic system for A3B induction, genomic DNA deamination, and mutagenesis. Human 293-derived cells were engineered to express doxycycline-inducible A3B-eGFP or eGFP constructs. Cells were subjected to 10 rounds of A3B-eGFP exposure that each caused 80-90% cell death. Control pools were subjected to parallel rounds of non-toxic eGFP exposure, and dilutions were done each round to mimic A3B-eGFP induced population fluctuations. Targeted sequencing of portions of TP53 and MYC demonstrated greater mutation accumulation in the A3B-eGFP exposed pools. Clones were generated and microarray analyses were used to identify those with the greatest number of SNP alterations for whole genome sequencing. A3B-eGFP exposed clones showed global increases in C-to-T transition mutations, enrichments for cytosine mutations within A3B-preferred trinucleotide motifs, and more copy number aberrations. Surprisingly, both control and A3B-eGFP clones also elicited strong mutator phenotypes characteristic of defective mismatch repair. Despite this additional mutational process, the 293-based system characterized here still yielded a genome-wide view of A3B-catalyzed mutagenesis in human cells and a system for additional studies on the compounded effects of simultaneous mutation mechanisms in cancer cells.

No MeSH data available.


Related in: MedlinePlus

Summary of somatic mutations detected by WGS.(A) Stacked bar graphs representing total number of C/G and T/A context somatic mutations in the indicated granddaughter subclones (black and white bars, respectively). Sequences from granddaughter clone AG3 were used as a baseline to call mutations in AA3 (i.e., mutations for AG3 are not shown in bar format because WGS data from another control granddaughter clone were not available for comparison). (B) Pie charts representing the proportion of each type of cytosine mutation across the genome in the indicated granddaughter clones. Red, blue, and black wedges represent C-to-T, C-to-A, and C-to-G mutations, respectively. (C) Stacked bar graphs representing the observed percentage of C-context somatic trinucleotide mutations detected in each granddaughter clone from the B panel. (D) Stacked bar graphs representing the extracted mutation signatures from WGS data. (E) The relative proportion that each extracted mutation signature contributes to the overall base substitution spectrum in the indicated granddaughter clones.
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pone.0155391.g004: Summary of somatic mutations detected by WGS.(A) Stacked bar graphs representing total number of C/G and T/A context somatic mutations in the indicated granddaughter subclones (black and white bars, respectively). Sequences from granddaughter clone AG3 were used as a baseline to call mutations in AA3 (i.e., mutations for AG3 are not shown in bar format because WGS data from another control granddaughter clone were not available for comparison). (B) Pie charts representing the proportion of each type of cytosine mutation across the genome in the indicated granddaughter clones. Red, blue, and black wedges represent C-to-T, C-to-A, and C-to-G mutations, respectively. (C) Stacked bar graphs representing the observed percentage of C-context somatic trinucleotide mutations detected in each granddaughter clone from the B panel. (D) Stacked bar graphs representing the extracted mutation signatures from WGS data. (E) The relative proportion that each extracted mutation signature contributes to the overall base substitution spectrum in the indicated granddaughter clones.

Mentions: Using this conservative approach for mutation identification, a total of 6741, 3496, and 3530 somatic mutations occurred at cytosines in granddaughter clones that had been subjected to 10 rounds of A3B-eGFP pulses in comparison to only 910 and 1531 cytosine mutations in the eGFP controls, consistent with the results of the SNP analyses described above (p = 0.018, Student’s t-test; Fig 4A; S1 Table). In particular, the A3B-eGFP pulsed granddaughter clones had higher proportions of C-to-T mutations than the eGFP controls, 59%, 54%, and 52% versus 36% and 47%, respectively (red slices in pie graphs in Fig 4B). The A3B-eGFP pulsed granddaughter clones also had higher proportions of mutations at A/T base pairs suggesting that genomic uracil lesions introduced by A3B may be processed by downstream error-prone repair processes analogous to those involved in AID-dependent somatic hypermutation of immunoglobulin genes [49] (Fig 4A).


Mutation Processes in 293-Based Clones Overexpressing the DNA Cytosine Deaminase APOBEC3B.

Akre MK, Starrett GJ, Quist JS, Temiz NA, Carpenter MA, Tutt AN, Grigoriadis A, Harris RS - PLoS ONE (2016)

Summary of somatic mutations detected by WGS.(A) Stacked bar graphs representing total number of C/G and T/A context somatic mutations in the indicated granddaughter subclones (black and white bars, respectively). Sequences from granddaughter clone AG3 were used as a baseline to call mutations in AA3 (i.e., mutations for AG3 are not shown in bar format because WGS data from another control granddaughter clone were not available for comparison). (B) Pie charts representing the proportion of each type of cytosine mutation across the genome in the indicated granddaughter clones. Red, blue, and black wedges represent C-to-T, C-to-A, and C-to-G mutations, respectively. (C) Stacked bar graphs representing the observed percentage of C-context somatic trinucleotide mutations detected in each granddaughter clone from the B panel. (D) Stacked bar graphs representing the extracted mutation signatures from WGS data. (E) The relative proportion that each extracted mutation signature contributes to the overall base substitution spectrum in the indicated granddaughter clones.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4862684&req=5

pone.0155391.g004: Summary of somatic mutations detected by WGS.(A) Stacked bar graphs representing total number of C/G and T/A context somatic mutations in the indicated granddaughter subclones (black and white bars, respectively). Sequences from granddaughter clone AG3 were used as a baseline to call mutations in AA3 (i.e., mutations for AG3 are not shown in bar format because WGS data from another control granddaughter clone were not available for comparison). (B) Pie charts representing the proportion of each type of cytosine mutation across the genome in the indicated granddaughter clones. Red, blue, and black wedges represent C-to-T, C-to-A, and C-to-G mutations, respectively. (C) Stacked bar graphs representing the observed percentage of C-context somatic trinucleotide mutations detected in each granddaughter clone from the B panel. (D) Stacked bar graphs representing the extracted mutation signatures from WGS data. (E) The relative proportion that each extracted mutation signature contributes to the overall base substitution spectrum in the indicated granddaughter clones.
Mentions: Using this conservative approach for mutation identification, a total of 6741, 3496, and 3530 somatic mutations occurred at cytosines in granddaughter clones that had been subjected to 10 rounds of A3B-eGFP pulses in comparison to only 910 and 1531 cytosine mutations in the eGFP controls, consistent with the results of the SNP analyses described above (p = 0.018, Student’s t-test; Fig 4A; S1 Table). In particular, the A3B-eGFP pulsed granddaughter clones had higher proportions of C-to-T mutations than the eGFP controls, 59%, 54%, and 52% versus 36% and 47%, respectively (red slices in pie graphs in Fig 4B). The A3B-eGFP pulsed granddaughter clones also had higher proportions of mutations at A/T base pairs suggesting that genomic uracil lesions introduced by A3B may be processed by downstream error-prone repair processes analogous to those involved in AID-dependent somatic hypermutation of immunoglobulin genes [49] (Fig 4A).

Bottom Line: Targeted sequencing of portions of TP53 and MYC demonstrated greater mutation accumulation in the A3B-eGFP exposed pools.Clones were generated and microarray analyses were used to identify those with the greatest number of SNP alterations for whole genome sequencing.A3B-eGFP exposed clones showed global increases in C-to-T transition mutations, enrichments for cytosine mutations within A3B-preferred trinucleotide motifs, and more copy number aberrations.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, Institute for Molecular Virology, Masonic Cancer Center, University of Minnesota, Minneapolis, MN, United States of America.

ABSTRACT
Molecular, cellular, and clinical studies have combined to demonstrate a contribution from the DNA cytosine deaminase APOBEC3B (A3B) to the overall mutation load in breast, head/neck, lung, bladder, cervical, ovarian, and other cancer types. However, the complete landscape of mutations attributable to this enzyme has yet to be determined in a controlled human cell system. We report a conditional and isogenic system for A3B induction, genomic DNA deamination, and mutagenesis. Human 293-derived cells were engineered to express doxycycline-inducible A3B-eGFP or eGFP constructs. Cells were subjected to 10 rounds of A3B-eGFP exposure that each caused 80-90% cell death. Control pools were subjected to parallel rounds of non-toxic eGFP exposure, and dilutions were done each round to mimic A3B-eGFP induced population fluctuations. Targeted sequencing of portions of TP53 and MYC demonstrated greater mutation accumulation in the A3B-eGFP exposed pools. Clones were generated and microarray analyses were used to identify those with the greatest number of SNP alterations for whole genome sequencing. A3B-eGFP exposed clones showed global increases in C-to-T transition mutations, enrichments for cytosine mutations within A3B-preferred trinucleotide motifs, and more copy number aberrations. Surprisingly, both control and A3B-eGFP clones also elicited strong mutator phenotypes characteristic of defective mismatch repair. Despite this additional mutational process, the 293-based system characterized here still yielded a genome-wide view of A3B-catalyzed mutagenesis in human cells and a system for additional studies on the compounded effects of simultaneous mutation mechanisms in cancer cells.

No MeSH data available.


Related in: MedlinePlus