Germline cis variant determines epigenetic regulation of the anti-cancer drug metabolism gene dihydropyrimidine dehydrogenase (DPYD)
- Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, United States
- Department of Clinical Chemistry, Inselspital, Bern University Hospital, University of Bern, Switzerland
- Graduate School for Cellular and Biomedical Sciences, University of Bern, Switzerland
- Eshelman School of Pharmacy, Division of Pharmacotherapy and Experimental Therapeutics, University of North Carolina at Chapel Hill, United States
- Division of Hematology, Department of Medicine, Mayo Clinic, United States
- Department of Visceral Surgery and Medicine, Inselspital, Bern University Hospital, University of Bern, Switzerland
- Department of Pathology, University of Iowa Carver College of Medicine, University of Iowa, United States
- Holden Comprehensive Cancer Center, University of Iowa Carver College of Medicine, University of Iowa, United States
Figures
Figure 1 with 1 supplement
Identification of a novel dihydropyrimidine dehydrogenase (DPYD) enhancer.
(A) Candidate DPYD enhancer regions were selected for further study using data from GeneHancer and Ensembl Regulatory Build. Coordinates are based on GRCh37/hg19. Regions are termed E9, E16, and E20 …
Figure 1—figure supplement 1
CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) screen to identify dihydropyrimidine dehydrogenase (DPYD) cis-regulatory elements in HCT116 cells.
For CRISPRi, DPYD expression was measured in HCT116 cells expressing dCas9-KRAB following transfection with guide-RNAs specific to the E9 (A), E16 (B), and E20 (C) regions. For CRISPRa, DPYD …
Figure 2 with 1 supplement
Epigenetic changes at the E9 region induced by CRISPR interference (CRISPRi)/CRISPR activation (CRISPRa).
Lentiguide vectors encoding guide-RNAs targeting the E9 region (E9 gRNAs) or empty vector control (Lentiguide) were transduced into HepG2 cells expressing dCas9-KRAB (A) or dcas9-P300 (B) and HCT116 …
Figure 2—figure supplement 1
Genomic context of rs4294451.
Rs4294451 is located within a putative enhancer region showing evidence for regulatory activity in Ensembl Regulatory Build data and within transcription factor binding sites in ENCODE Factorbook …
Figure 3 with 1 supplement
The rs4294451 T allele is associated with elevated dihydropyrimidine dehydrogenase (DPYD) expression.
(A) Luciferase reporter constructs were generated by cloning the E9 region containing the reference rs4294451 T allele or the variant rs4294451 A allele into reporter vectors containing the DPYD …
Figure 3—figure supplement 1
Rs4294451 A allele is associated with epigenetic repression at the E9 region in human liver specimens.
Chromatin enrichment of H3K27ac (A) and H3K9me3 (B) was measured using chromatin immunoprecipitation (ChIP) coupled with quantitative PCR (ChIP-qPCR) of liver specimens obtained from human donors …
Figure 4
The rs4294451 T allele is associated with increased interaction between E9 and the dihydropyrimidine dehydrogenase (DPYD) promoter.
(A) Schematic of HindIII restriction enzyme sites (vertical bars) and primers (arrows) used for chromatin conformation capture (3 C) relative to the DPYD transcription start site (TSS) and E9 …
Figure 5
T allele is associated with higher occupancy of CEBPB at E9 and the dihydropyrimidine dehydrogenase (DPYD) promoter.
(A) Expression plasmids for CEBPB or GFP (control) were co-transfected into HEK293T cells with the luciferase reporter plasmids depicted in Figure 3A. (B) Chromatin immunoprecipitation (ChIP) …
Figure 6 with 2 supplements
CEBPB-mediated upregulation of dihydropyrimidine dehydrogenase (DPYD) is dependent on rs4294451 T allele.
(A) Immunoblot showing knockdown of CEBPB expression in HCT116 cells carrying different rs4294451 genotypes transduced with lentiviral particles encoding two independent shRNAs against CEBPB (sh1 …
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Figure 6—source data 1
Original file for western blot analysis in Figure 6A (anti-CEBPB).
- https://cdn.elifesciences.org/articles/94075/elife-94075-fig6-data1-v1.tif
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Figure 6—source data 2
Original file for western blot analysis in Figure 6A (anti-tubulin).
- https://cdn.elifesciences.org/articles/94075/elife-94075-fig6-data2-v1.tif
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Figure 6—source data 3
PDF containing Figure 6A and annotated western blots used to make figure, including highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/94075/elife-94075-fig6-data3-v1.pdf
Figure 6—figure supplement 1
Disruption of CEBPB reduces enrichment at the E9 region and dihydropyrimidine dehydrogenase (DPYD) promoter in rs4294451 T/T and A/T cells, but not in T/T cells.
CEBPB enrichment at the E9 region (A) and the DPYD promoter (B) was measured by chromatin immunoprecipitation (ChIP) coupled with quantitative PCR (ChIP-qPCR) in CEBPB knockdown and scramble (scr) …
Figure 6—figure supplement 2
Allele frequency for rs4294451-T allele in global populations.
(A) Allele frequencies in various populations for the rs4294451-T allele was retrieved from the gnomAD browser v3.1.2. (B) Local ancestry-informed frequency data was retrieved for the rs4294451-T …
Additional files
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Supplementary file 1
Table of primers used for chromatin immunoprecipitation (ChIP), cloning, quantitative PCR (qPCR), and site-directed mutagenesis.
- https://cdn.elifesciences.org/articles/94075/elife-94075-supp1-v1.docx
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Supplementary file 2
Table of sequences and positions of the primers used for 3 C analysis.
- https://cdn.elifesciences.org/articles/94075/elife-94075-supp2-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/94075/elife-94075-mdarchecklist1-v1.docx
