@article{3cc2aa9aea5943f4a9b3718ac1a33226,
title = "Responsiveness to perturbations is a hallmark of transcription factors that maintain cell identity in vitro",
abstract = "Identifying the particular transcription factors that maintain cell type in vitro is important for manipulating cell type. Identifying such transcription factors by their cell-type-specific expression or their involvement in developmental regulation has had limited success. We hypothesized that because cell type is often resilient to perturbations, the transcriptional response to perturbations would reveal identity-maintaining transcription factors. We developed perturbation panel profiling (P3) as a framework for perturbing cells across many conditions and measuring gene expression responsiveness transcriptome-wide. In human iPSC-derived cardiac myocytes, P3 showed that transcription factors important for cardiac myocyte differentiation and maintenance were among the most frequently upregulated (most responsive). We reasoned that one function of responsive genes may be to maintain cellular identity. We identified responsive transcription factors in fibroblasts using P3 and found that suppressing their expression led to enhanced reprogramming. We propose that responsiveness to perturbations is a property of transcription factors that help maintain cellular identity in vitro. A record of this paper's transparent peer review process is included in the supplemental information.",
keywords = "cardiac myocytes, cell identity, fibroblasts, gene regulation, induced pluripotent stem cells, reprogramming, systems biology, transdifferentiation",
author = "Mellis, {Ian A.} and Edelstein, {Hailey I.} and Rachel Truitt and Yogesh Goyal and Beck, {Lauren E.} and Orsolya Symmons and Dunagin, {Margaret C.} and {Linares Saldana}, {Ricardo A.} and Shah, {Parisha P.} and P{\'e}rez-Bermejo, {Juan A.} and Arun Padmanabhan and Wenli Yang and Rajan Jain and Arjun Raj",
note = "Funding Information: We thank members of the Raj lab, Jain lab, and Penn iPSC Core, particularly Eduardo Torre, Allison C?te, Aman Kaur, Ben Emert, and Kate Alexander, as well as the late John Gearhart for insightful discussions related to this work. We thank Deepak Srivastava and members of the Srivastava lab, including Palmer Yu, for providing 7F vectors, immHCF cells, and advice on cardiac transdifferentiation protocols and iPSC-CM knockdown experiments. We thank Bruce Conklin for advice on iPSC-CM knockdown experiments. We thank Davide Cacchiarelli and Alex Meissner for providing hiF-T cells and for advice on reprogramming protocols. We thank Mitchell Guttman, Sofia Quinodoz, and Mario Blanco for advice on RNAtag-seq. We thank Edward Wallace for making PyRNAtagSeq scripts publicly available. We thank Russ Carstens for Platinum-A cells. We thank Junwei Shi for their assistance with flow cytometry. We thank the Penn iPSC Core for assistance with iPSC-derived cardiac myocytes, the Penn high-throughput screening Core for assistance with small-molecule drugs, shRNAs, and cDNA clones, and the Penn DNA Sequencing Core for assistance with large-scale plasmid preps. The Genotype-Tissue Expression (GTEx) Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS. The data used for the analyses described in this manuscript were obtained from the GTEx Portal on 10/05/17. The authors acknowledge grant support for this project from NIH/NINDS F30NS100595 and NIH/NHGRI T32HG000046 to I.A.M.; HFSP LT000919/2016-L to O.S.; grants from Schmidt Science Fellows (in partnership with the Rhodes Trust), the Jane Coffin Childs Memorial Fund for Medical Research, and Burroughs Wellcome Fund Career Awards at the Scientific Interface to Y.G.; NSF 15-48571 and NIH F31HL147416 to R.A.L.S.; the Penn Institute for Regenerative Medicine to W.Y.; NIH (1K08HL157700-01), Tobacco-Related Disease Research Program (578649), Giannini Foundation (P0527061), Michael Antonov Charitable Foundation, and the Sarnoff Cardiovascular Research Foundation to A.P.; Burroughs Wellcome Fund Career Award for Medical Scientists, American Heart Association, Allen Foundation and NIH Director's Transformative Research Award R01GM137425 to R.J.; and R01 CA238237, NIH Director's Transformative Research Award R01 GM137425, R01 CA232256, NSF CAREER 1350601, P30 CA016520, SPORE P50 CA174523, NIH U01 CA227550, NIH 4DN U01 HL129998, NIH Center for Photogenomics (RM1 HG007743), and the Tara Miller Foundation to A.R. A.R. and I.A.M. conceived of P3, and I.A.M. R.J. and A.R. conceived of the larger project with applications to cardiac transdifferentiation and reprogramming. I.A.M. designed, conducted, and analyzed all experiments with supervision by A.R. and R.J. with the exception of iPSC-CM differentiation performed by H.I.E. R.T. and W.Y. O.S. contributed to the design of some transdifferentiation experiments. W.Y. and R.T. contributed to the analysis of reprogramming experiments. Y.G. contributed to some transdifferentiation experiments, analysis, and figure design. M.C.D. P.P.S. and R.A.L.S. contributed to some transdifferentiation experiments. J.A.P.-B. and A.P. contributed to the design and conducting of iPSC-CM knockdown experiments. I.A.M. designed all analyses and wrote all software, with some assistance from L.E.B. with supervision by A.R. and R.J. I.A.M. R.J. and A.R. wrote the paper. A.R. receives patent royalties from LGC/Biosearch Technologies related to Stellaris RNA FISH probes. Funding Information: We thank members of the Raj lab, Jain lab, and Penn iPSC Core, particularly Eduardo Torre, Allison C{\^o}te, Aman Kaur, Ben Emert, and Kate Alexander, as well as the late John Gearhart for insightful discussions related to this work. We thank Deepak Srivastava and members of the Srivastava lab, including Palmer Yu, for providing 7F vectors, immHCF cells, and advice on cardiac transdifferentiation protocols and iPSC-CM knockdown experiments. We thank Bruce Conklin for advice on iPSC-CM knockdown experiments. We thank Davide Cacchiarelli and Alex Meissner for providing hiF-T cells and for advice on reprogramming protocols. We thank Mitchell Guttman, Sofia Quinodoz, and Mario Blanco for advice on RNAtag-seq. We thank Edward Wallace for making PyRNAtagSeq scripts publicly available. We thank Russ Carstens for Platinum-A cells. We thank Junwei Shi for their assistance with flow cytometry. We thank the Penn iPSC Core for assistance with iPSC-derived cardiac myocytes, the Penn high-throughput screening Core for assistance with small-molecule drugs, shRNAs, and cDNA clones, and the Penn DNA Sequencing Core for assistance with large-scale plasmid preps. The Genotype-Tissue Expression (GTEx) Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health and by NCI , NHGRI , NHLBI , NIDA , NIMH , and NINDS . The data used for the analyses described in this manuscript were obtained from the GTEx Portal on 10/05/17. The authors acknowledge grant support for this project from NIH/ NINDS F30NS100595 and NIH/ NHGRI T32HG000046 to I.A.M.; HFSP LT000919/2016-L to O.S.; grants from Schmidt Science Fellows (in partnership with the Rhodes Trust), the Jane Coffin Childs Memorial Fund for Medical Research, and Burroughs Wellcome Fund Career Awards at the Scientific Interface to Y.G.; NSF 15-48571 and NIH F31HL147416 to R.A.L.S.; the Penn Institute for Regenerative Medicine to W.Y.; NIH ( 1K08HL157700-01 ), Tobacco-Related Disease Research Program ( 578649 ), Giannini Foundation ( P0527061 ), Michael Antonov Charitable Foundation , and the Sarnoff Cardiovascular Research Foundation to A.P.; Burroughs Wellcome Fund Career Award for Medical Scientists, American Heart Association , Allen Foundation and NIH Director{\textquoteright}s Transformative Research Award R01GM137425 to R.J.; and R01 CA238237 , NIH Director{\textquoteright}s Transformative Research Award R01 GM137425 , R01 CA232256 , NSF CAREER 1350601 , P30 CA016520 , SPORE P50 CA174523 , NIH U01 CA227550 , NIH 4DN U01 HL129998 , NIH Center for Photogenomics ( RM1 HG007743 ), and the Tara Miller Foundation to A.R. Publisher Copyright: {\textcopyright} 2021 Elsevier Inc.",
year = "2021",
month = sep,
day = "22",
doi = "10.1016/j.cels.2021.07.003",
language = "English (US)",
volume = "12",
pages = "885--899.e8",
journal = "Cell Systems",
issn = "2405-4712",
publisher = "Cell Press",
number = "9",
}