Single cell RNA sequencing provides powerful insight into the factors that determine each cell's unique identity. Previous studies led to the surprising observation that alternative splicing among single cells is highly variable and follows a bimodal pattern: a given cell consistently produces either one or the other isoform for a particular splicing choice, with few cells producing both isoforms. Here we show that this pattern arises almost entirely from technical limitations. We analyze alternative splicing in human and mouse single cell RNA-seq datasets, and model them with a probabilistic simulator. Our simulations show that low gene expression and low capture efficiency distort the observed distribution of isoforms. This gives the appearance of binary splicing outcomes, even when the underlying reality is consistent with more than one isoform per cell. We show that accounting for the true amount of information recovered can produce biologically meaningful measurements of splicing in single cells.
All sequencing data reanalyzed in this study were acquired from GEO.
Single-cell analysis of allelic gene expression in pluripotency, differentiation and X-chromosome inactivationNCBI Gene Expression Omnibus, GSE74155.
Defining the early steps of cardiovascularlineage segregation by single cell RNA-seqNCBI Gene Expression Omnibus, GSE100471.
Pseudo-temporal ordering of individual cells reveals regulators of differentiationNCBI Gene Expression Omnibus, GSE52529.
Single-cell alternative splicing analysis with Expedition reveals splicing dynamics during neuron differentiationNCBI Gene Expression Omnibus, GSE85908.
Olfactory stem cell differentiation: horizontal basal cell (HBC) lineageNCBI Gene Expression Omnibus, GSE95601.
- Carlos F Buen Abad Najar
- Nir Yosef
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- L Stirling Churchman, Harvard Medical School, United States
© 2020, Buen Abad Najar et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
CRISPR technology has made generation of gene knock-outs widely achievable in cells. However, once inactivated, their re-activation remains difficult, especially in diploid cells. Here, we present DExCon (Doxycycline-mediated endogenous gene Expression Control), DExogron (DExCon combined with auxin-mediated targeted protein degradation), and LUXon (light responsive DExCon) approaches which combine one-step CRISPR-Cas9-mediated targeted knockin of fluorescent proteins with an advanced Tet-inducible TRE3GS promoter. These approaches combine blockade of active gene expression with the ability to re-activate expression on demand, including activation of silenced genes. Systematic control can be exerted using doxycycline or spatiotemporally by light, and we demonstrate functional knock-out/rescue in the closely related Rab11 family of vesicle trafficking regulators. Fluorescent protein knock-in results in bright signals compatible with low-light live microscopy from monoallelic modification, the potential to simultaneously image different alleles of the same gene, and bypasses the need to work with clones. Protein levels are easily tunable to correspond with endogenous expression through cell sorting (DExCon), timing of light illumination (LUXon), or by exposing cells to different levels of auxin (DExogron). Furthermore, our approach allowed us to quantify previously unforeseen differences in vesicle dynamics, transferrin receptor recycling, expression kinetics, and protein stability among highly similar endogenous Rab11 family members and their colocalization in triple knock-in ovarian cancer cell lines.
In mammalian cells genes that are in close proximity can be transcriptionally coupled: silencing or activating one gene can affect its neighbors. Understanding these dynamics is important for natural processes, such as heterochromatin spreading during development and aging, and when designing synthetic gene regulation circuits. Here, we systematically dissect this process in single cells by recruiting and releasing repressive chromatin regulators at dual-gene synthetic reporters, and measuring how fast gene silencing and reactivation spread as a function of intergenic distance and configuration of insulator elements. We find that silencing by KRAB, associated with histone methylation, spreads between two genes within hours, with a time delay that increases with distance. This fast KRAB-mediated spreading is not blocked by the classical cHS4 insulators. Silencing by histone deacetylase HDAC4 of the upstream gene can also facilitate background silencing of the downstream gene by PRC2, but with a days-long delay that does not change with distance. This slower silencing can sometimes be stopped by insulators. Gene reactivation of neighboring genes is also coupled, with strong promoters and insulators determining the order of reactivation. Our data can be described by a model of multi-gene regulation that builds upon previous knowledge of heterochromatin spreading, where both gene silencing and gene reactivation can act at a distance, allowing for coordinated dynamics via chromatin regulator recruitment.