Study / S4KLMTB4W2022-11-30
Efficient Selection of Enhancers and Promoters from MIA PaCa-2 Pancreatic Cancer Cells by ChIP-lentiMPRA
Kirill Nikitich Kashkin, Elena Sergeevna Kotova, Irina Vasilievna Alekseenko, Svetlana Sergeevna Bulanenkova, Sergey Borisovich Akopov et al.
About this study
A library of active genome regulatory elements (putative promoters and enhancers) from MIA PaCa-2 pancreatic adenocarcinoma cells was constructed using a specially designed lentiviral vector and a massive parallel reporter assay (ChIP-lentiMPRA). Chromatin immunoprecipitation of the cell genomic DNA by H3K27ac antibodies was used for primary enrichment of the library for regulatory elements. Totally, 11,264 unique genome regions, many of which are capable of enhancing the expression of the CopGFP reporter gene from the minimal CMV promoter, were identified. The regions tend to be located near promoters. Based on the proximity assay, we found an enrichment of highly expressed genes among those associated with three or more mapped distal regions (2 kb distant from the 5′-ends of genes). It was shown significant enrichment of genes related to carcinogenesis or Mia PaCa-2 cell identity genes in this group. In contrast, genes associated with 1–2 distal regions or only with proximal regions (within 2 kbp of the 5′-ends of genes) are more often related to housekeeping functions. Thus, ChIP-lentiMPRA is a useful strategy for creating libraries of regulatory elements for the study of tumor-specific gene transcription.
Full author list & citation
Kirill Nikitich Kashkin, Elena Sergeevna Kotova, Irina Vasilievna Alekseenko, Svetlana Sergeevna Bulanenkova, Sergey Borisovich Akopov, Eugene Pavlovich Kopantzev, Lev Grigorievich Nikolaev, Igor Pavlovich Chernov, Dmitry Alexandrovich Didych. Efficient Selection of Enhancers and Promoters from MIA PaCa-2 Pancreatic Cancer Cells by ChIP-lentiMPRA. 2022-11-30. https://doi.org/10.3390/ijms232315011
Experiments 1
E5ICN1XCE
H3K27ac-enriched chromatin fragments from MIA PaCa-2 cells were cloned next to a minimal CMV promoter in the pLVPGm.1-mP lentiviral CopGFP reporter, integrated into MIA PaCa-2 cells, and selected by puromycin followed by FACS for high GFP fluorescence. The two independently prepared output libraries (L1 and L2) were sequenced and merged by the authors into 11,264 unique mapped hg38 regions; the table provides those region-level MPRA selection outputs and genomic annotations.