Fine-tuned KDM1A alternative splicing regulates human cardiomyogenesis through an enzymatic-independent mechanism

Veronica Astro, Gustavo Ramirez-Calderon, Roberta Pennucci, Jonatan Caroli, Alfonso Saera-Vila, Kelly Cardona-Londoño, Chiara Forastieri, Elisabetta Fiacco, Fatima Maksoud, Maryam Alowaysi, Elisa Sogne, Andrea Falqui, Federico Gonzàlez, Nuria Montserrat, Elena Battaglioli, Andrea Mattevi, Antonio Adamo*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

The histone demethylase KDM1A is a multi-faceted regulator of vital developmental processes, including mesodermal and cardiac tube formation during gastrulation. However, it is unknown whether the fine-tuning of KDM1A splicing isoforms, already shown to regulate neuronal maturation, is crucial for the specification and maintenance of cell identity during cardiogenesis. Here, we discovered a temporal modulation of ubKDM1A and KDM1A+2a during human and mice fetal cardiac development and evaluated their impact on the regulation of cardiac differentiation. We revealed a severely impaired cardiac differentiation in KDM1A−/− hESCs that can be rescued by re-expressing ubKDM1A or catalytically impaired ubKDM1A-K661A, but not by KDM1A+2a or KDM1A+2a-K661A. Conversely, KDM1A+2a−/− hESCs give rise to functional cardiac cells, displaying increased beating amplitude and frequency and enhanced expression of critical cardiogenic markers. Our findings prove the existence of a divergent scaffolding role of KDM1A splice variants, independent of their enzymatic activity, during hESC differentiation into cardiac cells.

Original languageEnglish (US)
Article number104665
JournaliScience
Volume25
Issue number7
DOIs
StatePublished - Jul 15 2022

Bibliographical note

Funding Information:
We thank Francesco Rusconi for performing the collection of the embryonal heart tissues. We thank Carol Buitrago-López for her support with graphical abstract preparation. We thank the genomic unit of the KAUST BioCoreLab for technical support with RNA-Seq libraries preparation. We thank the KAUST imaging and characterization facility and ARCL animal facility for support with image acquisition and teratoma formation assays. We thank Sudharshan Elangovan for the generation of the pTP6-Flag-ubKDM1A, pTP6-Flag-KDM1A+2a, pTP6-Flag-ubKDM1A-K661A, and pTP6-Flag-KDM1A+2a-K661A constructs. The two KDM1A inhibitors GSK-090 and MC2652 tranylcypromine were a kind gift of Prof. Antonello Mai, University“"La Sapienza, Rome”" This work was funded by baseline funding (BAS 1077-01-01) to A.A. and by Competitive Research Fund (CRG URF/1/4012) to A.A. A.M. and E.B. A.V. generated all cell lines, performed the differentiation experiments, live imaging acquisitions, and time-lapse analysis, carried out the iPs, mass spectrometry, and RNA-seq analysis. G.R.C. performed most of the experiments required by reviewers, including differentiation experiments, western blots, and apoptosis analyses at FACS. R.P. optimized the differentiation protocol into cardiac cells, performed real-time PCR, ChIP experiments, and generated ChIP-seq and RNA-seq libraries. J.C. and A.M. performed the in vitro biochemical analysis and interpreted the results. A.S.-V. analyzed the ChIP-seq data and assisted with generating the RNA-seq analysis. K.C.L. performed the transcriptomic analysis on the human heart fetal RNA-seq. C.F. and E.B. collected heart mice samples and performed real-time PCR on KDM1A isoforms. E.F. carried out the teratoma formation assays and the cell preparation for karyotype analysis. F.M. performed real-time PCR and RNA-seq libraries. M.A. performed real-time PCRs. E.S. and A.F. performed the S.E.M. acquisitions. F.G. and N.M. trained V.A. for the generation of the genome-edited cell lines. A.V. J.C. A.M. E.B. and A.A. interpreted the results and wrote the manuscript. A.A. designed the study, conceived and supervised the project. The authors declare no competing interests.

Funding Information:
We thank Francesco Rusconi for performing the collection of the embryonal heart tissues. We thank Carol Buitrago-López for her support with graphical abstract preparation. We thank the genomic unit of the KAUST BioCoreLab for technical support with RNA-Seq libraries preparation. We thank the KAUST imaging and characterization facility and ARCL animal facility for support with image acquisition and teratoma formation assays. We thank Sudharshan Elangovan for the generation of the pTP6-Flag-ubKDM1A, pTP6-Flag-KDM1A+2a, pTP6-Flag-ubKDM1A-K661A, and pTP6-Flag-KDM1A+2a-K661A constructs. The two KDM1A inhibitors GSK-090 and MC2652 tranylcypromine were a kind gift of Prof. Antonello Mai, University“"La Sapienza, Rome”" This work was funded by baseline funding ( BAS 1077-01-01 ) to A.A. and by Competitive Research Fund ( CRG URF/1/4012 ) to A.A., A.M., and E.B.

Publisher Copyright:
© 2022 The Author(s)

Keywords

  • Cell biology
  • Molecular mechanism of gene regulation
  • Omics
  • Stem cells research

ASJC Scopus subject areas

  • General

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