Human stem cell-specific epigenetic signatures control transgene expression.

in Biochimica et biophysica acta. Gene regulatory mechanisms by Chulhwan S Kwak, Furkan E Oflaz, Jiamin Qiu, Xinnan Wang

TLDR

  • The study investigates how stem cells can be used to study tissue differentiation and disease mechanisms. The study uses biochemical and cell biological methodologies such as omics, autophagy, and organelle dynamics. However, stem cells have a barrier to applying genetically encoded tools due to epigenetic silencing. The study investigates the molecular mechanisms underlying exogenously expressed gene silencing in human induced pluripotent stem cells (iPSCs), glioblastoma cells (GBM), and embryonic kidney cells (HEK). The study discovers that all promoters tested are highly methylated on the CpG island regions with lower protein expression in iPSCs. The study provides useful information for designing anti-silencing strategies to increase transgene expression in iPSCs.

Abstract

Human stem cell-derived models have emerged as an important platform to study tissue differentiation and disease mechanisms. Those models could capitalize on biochemical and cell biological methodologies such as omics, autophagy, and organelle dynamics. However, epigenetic silencing in stem cells creates a barrier to apply genetically encoded tools. Here we investigate the molecular mechanisms underlying exogenously expressed gene silencing by employing multiple commonly used promoters in human induced pluripotent stem cells (iPSCs), glioblastoma cells (GBM), and embryonic kidney cells (HEK). We discover that all promoters tested are highly methylated on the CpG island regions with lower protein expression in iPSCs, as compared to non-iPSCs. Elongation factor 1 alpha short (EF1α short or EFS) promoter, which has fewer CpG island number compared to the other promoters, can drive relatively higher gene expression in iPSCs, despite CpG methylation. Adding a minimal A2 ubiquitous chromatin opening element (minimal A2 UCOE or miniUCOE) upstream of a promoter inhibits CpG methylation and enhances gene expression in iPSCs. Our results demonstrate stem cell type-specific epigenetic modification of transgenic promoter region and provide useful information for designing anti-silencing strategies to increase transgene expression in iPSCs.

Overview

  • The study investigates the molecular mechanisms underlying exogenously expressed gene silencing in human induced pluripotent stem cells (iPSCs), glioblastoma cells (GBM), and embryonic kidney cells (HEK).
  • The study employs multiple commonly used promoters in these cell types to analyze the impact of epigenetic silencing on gene expression. The study tests the EF1α short (EFS) promoter, which has fewer CpG island number compared to the other promoters, and the minimal A2 ubiquitous chromatin opening element (minimal A2 UCEO or miniUCEE) upstream of a promoter inhibits CpG methylation and enhances gene expression in iPSCs. The study aims to provide useful information for designing anti-silencing strategies to increase transgene expression in iPSCs.

Comparative Analysis & Findings

  • The study discovers that all promoters tested are highly methylated on the CpG island regions with lower protein expression in iPSCs, as compared to non-iPSCs. The EF1α short (EFS) promoter, which has fewer CpG island number compared to the other promoters, can drive relatively higher gene expression in iPSCs, despite CpG methylation. Adding a minimal A2 ubiquitous chromatin opening element (minimal A2 UCEO or miniUCEE) upstream of a promoter inhibits CpG methylation and enhances gene expression in iPSCs.

Implications and Future Directions

  • The study's findings suggest that stem cell type-specific epigenetic modification of transgenic promoter regions plays a crucial role in exogenously expressed gene silencing. The study provides useful information for designing anti-silencing strategies to increase transgene expression in iPSCs. Future research could explore the use of these strategies in other stem cell types and investigate the impact of epigenetic modifications on disease mechanisms in stem cells.
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