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A single-cell massively parallel reporter assay detects cell-type-specific gene regulation

Nature Genetics volume 55, pages 346–354 (2023)Cite this article

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Abstract

Massively parallel reporter gene assays are key tools in regulatory genomics but cannot be used to identify cell-type-specific regulatory elements without performing assays serially across different cell types. To address this problem, we developed a single-cell massively parallel reporter assay (scMPRA) to measure the activity of libraries of cis-regulatory sequences (CRSs) across multiple cell types simultaneously. We assayed a library of core promoters in a mixture of HEK293 and K562 cells and showed that scMPRA is a reproducible, highly parallel, single-cell reporter gene assay that detects cell-type-specific cis-regulatory activity. We then measured a library of promoter variants across multiple cell types in live mouse retinas and showed that subtle genetic variants can produce cell-type-specific effects on cis-regulatory activity. We anticipate that scMPRA will be widely applicable for studying the role of CRSs across diverse cell types.

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Fig. 1: scMPRA measures CRS activity at single-cell resolution.
Fig. 2: scMPRA detects cell-type-specific CRS activity.
Fig. 3: scMPRA detects substate-specific CRS activity.
Fig. 4: scMPRA design and workflow in mouse retina.
Fig. 5: scMPRA recapitulates Gnb3 expression patterns.
Fig. 6: Mutations in the Gnb3 promoter display cell-type-specific effects.

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Data availability

Next-generation sequencing data that support the findings of the study are available in the Gene Expression Omnibus using accession code GSE188639.

Code availability

The code that supports the findings of this study is available in Zenodo52.

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Acknowledgements

We thank the members of the Cohen laboratory for their critical feedback on the manuscript. We thank J. Hoisington-Lopez and M. Crosby for assistance with high-throughput sequencing. This work is supported by grants to B.A.C. from the National Institutes of Health (R01 GM140711 and R01 GM092910) and to J.C.C. from the National Institutes of Health (R01 EY030075). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Author notes

  1. Siqi Zhao

    Present address: Ginkgo Bioworks, Boston, MA, USA

Authors and Affiliations

  1. Edison Family Center for Systems Biology and Genome Sciences, Washington University School of Medicine, St. Louis, MO, USA

    Siqi Zhao, Clarice K. Y. Hong, David M. Granas, Michael A. White & Barak A. Cohen

  2. Department of Genetics, Washington University School of Medicine, St. Louis, MO, USA

    Siqi Zhao, Clarice K. Y. Hong, David M. Granas, Michael A. White & Barak A. Cohen

  3. Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA

    Connie A. Myers & Joseph C. Corbo

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Contributions

S.Z. and B.A.C. conceived and designed the project. All experiments and analyses were performed by S.Z. with technical contributions from C.K.Y. H. and D.M.G., except for the electroporation and culturing of mouse retinas, which was performed by C.A.M. J.C.C and M.A.W provided critical input into the design of the Gnb3 promoter variant library. S.Z., C.K.Y.H. and B.A.C. wrote the manuscript with input and feedback from all authors.

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Correspondence to Barak A. Cohen.

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Competing interests

S.Z. and B.A.C. are inventors on a pending patent filed by Washington University in St. Louis which may encompass the methods, reagents and data disclosed in this manuscript. B.A.C. is on the scientific advisory board of Patch Biosciences. The remaining authors declare no competing interests.

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Nature Genetics thanks Bas van Steensel, Rickard Sandberg and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 scMPRA measures cell-type specific CRS activity.

(a) UMAP of the single-cell transcriptome from the mixed-cell experiment. 105 out of 3417 cells (3%) are labeled by both K562 and HEK293 cell genes. (b) UMAP of the mixed-cell experiment with cells marked by other representative markers for K562 and HEK293 cell expression. (c, d) Histogram of the number of plasmids (unique cBC-rBC pairs) transfected into K562 cells and HEK293 cells. (e, f) Histogram of the mean number of rBC per cBC (CRS) per cell for K562 cells and HEK293 cells. (g, h) Correlation of bulk MPRA versus scMPRA where only the scMPRA data has been UMI normalized (i, j) Scatterplot of scMPRA reproducibility for housekeeping and developmental promoters in K562 cells and HEK293 cells.

Extended Data Fig. 2 scMPRA measures CRS activity in K562 cell substates.

(a) Reproducibility for mean expression of core promoters in K562 cells. (b) Correlation of bulk and scMPRA (non-UMI corrected) in K562 cells (c) Different dynamics of expression. For UBA52, the promoter is most highly expressed in S phase, whereas for CSF1, the promoter is most highly expressed in G1 phase. For CXCL10, the promoter is expressed evenly through cell cycle (Stars indicate significance from two-sided Wilcoxon rank sum test, *: p < 0.05) (d) Cells no longer cluster together based on cell cycle genes after the effects of the cell cycle are removed.

Extended Data Fig. 3 Robust measurements of Gnb3 promoter library in ex vivo retina.

(a) Expression of marker genes by scRNA-seq used to identify cell types in the retina. (b) Percentage of the total cells recovered represented by each retinal cell type. (c) Plot showing the relationship between the mean activity of a Gnb3 promoter variant in a given cell type (x-axis) and the proportion of cells in which that promoter variant is silent (y-axis). Individual cells in which a given Gnb3 variant is silent are identified as cells with U6-expressed cBC, but no Gnb3-expressed cBC. (d) The correlation between biological replicates (n = 2) is plotted as a function of the number of cells used in the analysis. The bounds of the box represent the upper and lower quartiles respectively, and the center line represents the median. The whiskers extend to the maxima/minima except for points determined to be outliers using a method that is a function of the interquartile range.

Supplementary information

Supplementary Information

Supplementary Fig. 1.

Reporting Summary

Supplementary Tables

Supplementary Table 1—Mixed-cell experiment expression. Supplementary Table 2—Differential expression of the core promoter library between K562 and HEK293 cells. Supplementary Table 3—K562 cell cycle expression. Supplementary Table 4—K562 cell substate expression. Supplementary Table 5—Gnb3 promoter variant library. Supplementary Table 6—Gnb3 library expression in retina. Supplementary Table 7—Oligos used in this study.

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Zhao, S., Hong, C.K.Y., Myers, C.A. et al. A single-cell massively parallel reporter assay detects cell-type-specific gene regulation. Nat Genet 55, 346–354 (2023). https://doi.org/10.1038/s41588-022-01278-7

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