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Single-cell perturbation dissects transcription factor control of progression speed and trajectory choice in early T-cell development

Zhou, Wen and Gao, Fan and Romero-Wolf, Maile and Jo, Suin and Rothenberg, Ellen V. (2021) Single-cell perturbation dissects transcription factor control of progression speed and trajectory choice in early T-cell development. . (Unpublished)

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In early T-cell development, single cells dynamically shift expression of multiple transcription factors (TFs) during transition from multipotentiality to T-lineage commitment, but the functional roles of many TFs have been obscure. Here, synchronized in vitro differentiation systems, scRNA-seq with batch indexing, and controlled gene-disruption strategies have unraveled single-cell impacts of perturbing individual TFs at two stages in early T-cell development. Single-cell CRISPR perturbation revealed that early-acting TFs Bcl11a, Erg, Spi1 (PU.1), Gata3, and Tcf7 (TCF1) each play individualized roles promoting or retarding T-lineage progression and suppressing alternative trajectories, collectively determining population dynamics and path topologies. Later, during T-lineage commitment, cells prevented from expressing TF Bcl11b ‘realized’ this abnormality not with a developmental block, but by shifting into a divergent path via bZIP and Sox TF activation as well as E protein antagonism, finally exiting the T-lineage trajectory. These TFs thus exert a network of impacts to control progression kinetics, trajectories, and differentiation outcomes of early pro-T cells.

Item Type:Report or Paper (Discussion Paper)
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URLURL TypeDescription Paper ItemData ItemData
Zhou, Wen0000-0003-0357-2744
Gao, Fan0000-0001-6832-3402
Romero-Wolf, Maile0000-0002-8024-7198
Rothenberg, Ellen V.0000-0002-3901-347X
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license. This version posted September 6, 2021. We thank Jeff Park and Sisi Chen from the Caltech Single Cell Profiling and Engineering Center for providing support for processing 10X Chromium samples, Rochelle Diamond and members of the Caltech Flow Cytometry and Cell Sorting facility for sorting, Ingrid Soto for mouse care, Maria Quiloan for mouse genotyping and supervision, and Igor Antoshechkin and Vijaya Kumar of the Caltech Jacobs Genomics Facility for bulk RNA sequencing. We also thank Gay Crooks and Amélie Montel-Hagen (UCLA) for sharing the mATO system with us. Support for this project came from USPHS grants (R01AI135200, R01HL119102, and R01HD100039) to E.V.R., The Beckman Institute at Caltech for support of all the Caltech facilities, the Biology and Biological Engineering Division Bowes Leadership Chair Fund, the Louis A. Garfinkle Memorial Laboratory Fund, and the Al Sherman Foundation. E.V.R. gratefully acknowledges past support from the Albert Billings Ruddock Professorship. Author Contributions: Conceptualization: WZ, EVR; Methodology: WZ, FG; Investigation: WZ, FG, MRW, SJ; Funding acquisition: EVR; Supervision: EVR; Writing – original draft: WZ, EVR; Writing – review & editing: WZ, FG, EVR; WZ designed the project, carried out the experiments, analyzed the data, and wrote the paper. FG wrote the in-house bioinformatic pipeline for perturb-seq and hashtag alignment and assignment, helped with analysis and edited the manuscript. MRW and SJ performed preliminary experiments. EVR supervised research, guided the design of the project, analyzed the data and wrote the paper. Competing Interests: WZ is now employed by 10X Genomics. EVR is a member of the Scientific Advisory Board for Century Therapeutics and has advised Kite Pharma and A2 Biotherapeutics. Data and Materials Availability: All data, code, and materials used in the analysis will be made available to researchers for reproducing or extending the analysis, upon request to the Corresponding Author, without requiring more than a Simple Letter Agreement or the Uniform Biological Materials Transfer Agreement of the NIH Office of Technology Transfer. All genomic sequencing data have been deposited in Gene Expression Omnibus under accession numbers GSE165835 and GSE183026.
Funding AgencyGrant Number
Caltech Beckman InstituteUNSPECIFIED
Caltech Division of Biology and Biological EngineeringUNSPECIFIED
Louis A. Garfinkle Memorial Laboratory FundUNSPECIFIED
Al Sherman FoundationUNSPECIFIED
Albert Billings Ruddock ProfessorshipUNSPECIFIED
Record Number:CaltechAUTHORS:20210909-213904290
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Official Citation:Single-cell perturbation dissects transcription factor control of progression speed and trajectory choice in early T-cell development. Wen Zhou, Fan Gao, Maile Romero-Wolf, Suin Jo, Ellen V. Rothenberg. bioRxiv 2021.09.03.458944; doi:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:110796
Deposited By: Tony Diaz
Deposited On:09 Sep 2021 22:14
Last Modified:16 Nov 2021 19:42

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