Journal
ONCOTARGET
Volume 7, Issue 29, Pages 46545-46556Publisher
IMPACT JOURNALS LLC
DOI: 10.18632/oncotarget.10234
Keywords
CRISPR-dCas9; TET1; BRCA1; DNA demethylation; gene activation
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Funding
- W.M. Keck Foundation grant
- National Science Foundation [1249315]
- Indiana Clinical and Translational Sciences Institute from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award [UL1TR001108]
- Women's Global Health Institute (WGHIMEER)
- Purdue University Centre for Cancer Research Undergraduate Research Program
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [1249315] Funding Source: National Science Foundation
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DNA hypermethylation at the promoter of tumour-suppressor genes is tightly correlated with their transcriptional repression and recognized as the hallmark of majority of cancers. Epigenetic silencing of tumour suppressor genes impairs their cellular functions and activates a cascade of events driving cell transformation and cancer progression. Here, we examine site-specific and spatiotemporal alteration in DNA methylation at a target region in BRCA1 gene promoter, a model tumour suppressor gene. We have developed a programmable CRISPR-Cas9 based demethylase tool containing the deactivated Cas9 (dCas9) fused to the catalytic domain (CD) of Ten-Eleven Translocation (TET) dioxygenase1 (TET1CD). The fusion protein selectively demethylates targeted regions within BRCA1 promoter as directed by the designed single-guide RNAs (sgRNA), leading to the transcriptional up-regulation of the gene. We also noticed the increment in 5-hydroxymethylation content (5-hmC) at the target DNA site undergoing the most profound demethylation. It confirms the catalytic activity of TET1 in TET1-dCas9 fusion proteins-mediated demethylation at these target sequences. The modular design of the fusion constructs presented here allows for the selective substitution of other chromatin or DNA modifying enzymes and for loci-specific targeting to uncover epigenetic regulatory pathways at gene promoters and other selected genomic regions.
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