Glenn, R. | bioRxiv Science

bioRxiv2024

Genetic variation modulates susceptibility to aberrant DNA hypomethylation and imprint deregulation in naive pluripotent stem cells

Naive pluripotent stem cells (nPSC) frequently undergo pathological and not readily reversible loss of DNA methylation marks at imprinted gene loci. This abnormality poses a hurdle for using pluripotent cell lines in biomedical applications and underscores the need to identify the causes of imprint instability in these cells. We show that nPSCs from inbred mouse strains exhibit pronounced strain-specific susceptibility to locus-specific deregulation of imprinting marks during reprogramming to pluripotency and upon culture with MAP kinase inhibitors, a common approach to maintain naive pluripotency. Analysis of genetically highly diverse nPSCs from the Diversity Outbred (DO) stock confirms that genetic variation is a major determinant of epigenome stability in pluripotent cells. We leverage the variable DNA hypomethylation in DO lines to identify several trans-acting quantitative trait loci (QTLs) that determine epigenome stability at either specific target loci or genome-wide. Candidate factors encoded by two multi-target QTLs on chromosomes 4 and 17 suggest specific transcriptional regulators that contribute to DNA methylation maintenance in nPSCs. We propose that genetic variants represent candidate biomarkers to identify pluripotent cell lines with desirable properties and might serve as entry points for the targeted engineering of nPSCs with stable epigenomes. HighlightsO_LINaive pluripotent stem cells from distinct inbred mouse strains exhibit variable DNA methylation levels at imprinted gene loci. C_LIO_LIThe vulnerability of pluripotent stem cells to loss of genomic imprinting caused by MAP kinase inhibition strongly differs between inbred mouse strains. C_LIO_LIGenetically diverse pluripotent stem cell lines from Diversity Outbred mouse stock allow the identification of quantitative trait loci controlling DNA methylation stability. C_LIO_LIGenetic variants may serve as biomarkers to identify naive pluripotent stem cell lines that are epigenetically stable in specific culture conditions. C_LI

developmental biology↗

bioRxiv2023

Dynamic network-guided CRISPRi screen reveals CTCF loop-constrained nonlinear enhancer-gene regulatory activity in cell state transitions

Comprehensive enhancer discovery is challenging because most enhancers, especially those affected in complex diseases, have weak effects on gene expression. Our network modeling revealed that nonlinear enhancer-gene regulation during cell state transitions can be leveraged to improve the sensitivity of enhancer discovery. Utilizing hESC definitive endoderm differentiation as a dynamic transition system, we conducted a mid-transition CRISPRi-based enhancer screen. The screen discovered a comprehensive set of enhancers (4 to 9 per locus) for each of the core endoderm lineage-specifying transcription factors, and many enhancers had strong effects mid-transition but weak effects post-transition. Through integrating enhancer activity measurements and three-dimensional enhancer-promoter interaction information, we were able to develop a CTCF loop-constrained Interaction Activity (CIA) model that can better predict functional enhancers compared to models that rely on Hi-C-based enhancer-promoter contact frequency. Our study provides generalizable strategies for sensitive and more comprehensive enhancer discovery in both normal and pathological cell state transitions.

genetics↗