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Characterization of Allele-Specific Regulation of Telomerase Reverse Transcriptase in Promoter Mutant Thyroid Cancer Cell Lines.

Authors
  • McKelvey, Brittany A1, 2
  • Gilpatrick, Timothy3
  • Wang, Yongchun1
  • Timp, Winston2, 3
  • Umbricht, Christopher B1, 4, 5
  • Zeiger, Martha A6
  • 1 Department of Surgery, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
  • 2 Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
  • 3 Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
  • 4 Department of Oncology, and The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
  • 5 Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
  • 6 Surgical Oncology Program, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.
Type
Published Article
Journal
Thyroid
Publisher
Mary Ann Liebert
Publication Date
Oct 01, 2020
Volume
30
Issue
10
Pages
1470–1481
Identifiers
DOI: 10.1089/thy.2020.0055
PMID: 32228178
Source
Medline
Keywords
Language
English
License
Unknown

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.

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