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Characterization of Allele-Specific Regulation of Telomerase Reverse Transcriptase in Promoter Mutant Thyroid Cancer Cell Lines
- Source :
- Thyroid
- Publication Year :
- 2020
-
Abstract
- Background: Telomerase reverse transcriptase (TERT) promoter mutations play a role in carcinogenesis and are found in both tumors and cancer cell lines. TERT promoter methylation, transcription factor binding, chromatin remodeling, and alternative splicing are also known to play an integral role in TERT regulation. Methods: Using nanopore Cas9 targeted sequencing, we characterized allele-specific methylation in thyroid cancer cell lines heterozygous for the TERT promoter mutation. Furthermore, using chromatin immunoprecipitation followed by Sanger sequencing, we probed allele-specific binding of the transcription factors GABPA (GA binding protein transcription factor subunit alpha) and MYC, as well as the chromatin marks H3K4me3 and H3K27me3. Finally, using coding single nucleotide polymorphisms and the long-read sequencing, we examined complementary DNA for monoallelic expression (MAE). Results: We found the mutant TERT promoter allele to be significantly less methylated than wild type, while more methylated in the gene body in heterozygous TERT mutant cell lines. We demonstrated that the transcriptional activators GABPA and MYC bind only to the mutant TERT allele. In addition, the activating and repressive chromatin marks H3K4me3 and H3K27me3, respectively, bind mutant and wild-type alleles exclusively. Finally, in heterozygous mutant cell lines, TERT exhibits MAE from the mutant allele only. Conclusions: In summary, by employing new long-read sequencing methods, we were able to definitively demonstrate allele-specific DNA methylation, histone modifications, transcription factor binding, and the resulting monoallelic transcription in cell lines with heterozygous TERT mutations.
- Subjects :
- Telomerase
Heterozygote
DNA, Complementary
Endocrinology, Diabetes and Metabolism
Mutant
030209 endocrinology & metabolism
Biology
medicine.disease_cause
Polymorphism, Single Nucleotide
Histones
Proto-Oncogene Proteins c-myc
03 medical and health sciences
0302 clinical medicine
Endocrinology
Transcription (biology)
CRISPR-Associated Protein 9
Cell Line, Tumor
medicine
Transcriptional regulation
Humans
Immunoprecipitation
Telomerase reverse transcriptase
Thyroid Neoplasms
Promoter Regions, Genetic
Alleles
Methylation
Thyroid Cancer and Nodules
DNA Methylation
Molecular biology
GA-Binding Protein Transcription Factor
Chromatin
Gene Expression Regulation, Neoplastic
030220 oncology & carcinogenesis
DNA methylation
Mutation
CpG Islands
Carcinogenesis
Protein Binding
Transcription Factors
Subjects
Details
- ISSN :
- 15579077
- Volume :
- 30
- Issue :
- 10
- Database :
- OpenAIRE
- Journal :
- Thyroid : official journal of the American Thyroid Association
- Accession number :
- edsair.doi.dedup.....555c7b20fb17091ed923f3e904dd9ee8