DNA damage repair (DDR) is a safeguard for genome integrity maintenance. Increasing DDR efficiency could increase the yield of induced pluripotent stem cells (iPSC) upon reprogramming from somatic cells. The epigenetic mechanisms governing DDR during iPSC reprogramming are not completely understood. Our goal was to evaluate the splicing isoforms of histone variant macroH2A1, macroH2A1.1, and macroH2A1.2, as potential regulators of DDR during iPSC reprogramming. GFP-Trap one-step isolation of mtagGFP-macroH2A1.1 or mtagGFP-macroH2A1.2 fusion proteins from overexpressing human cell lines, followed by liquid chromatography-tandem mass spectrometry analysis, uncovered macroH2A1.1 exclusive interaction with Poly-ADP Ribose Polymerase 1 (PARP1) and X-ray cross-complementing protein 1 (XRCC1). MacroH2A1.1 overexpression in U2OS-GFP reporter cells enhanced specifically nonhomologous end joining (NHEJ) repair pathway, while macroH2A1.1 knock-out (KO) mice showed an impaired DDR capacity. The exclusive interaction of macroH2A1.1, but not macroH2A1.2, with PARP1/XRCC1, was confirmed in human umbilical vein endothelial cells (HUVEC) undergoing reprogramming into iPSC through episomal vectors. In HUVEC, macroH2A1.1 overexpression activated transcriptional programs that enhanced DDR and reprogramming. Consistently, macroH2A1.1 but not macroH2A1.2 overexpression improved iPSC reprogramming. We propose the macroH2A1 splicing isoform macroH2A1.1 as a promising epigenetic target to improve iPSC genome stability and therapeutic potential.

Central European Institute of Technology Masaryk University Brno Czech Republic

Department of Biology Faculty of Medicine Masaryk University Brno Czech Republic

Department of Chemistry and Biotechnology Tallinn University of Technology Tallinn Estonia

Department of Histology and Embryology Faculty of Medicine Masaryk University Brno Czech Republic

Department of Medical Genetics Medical University of Varna Varna Bulgaria

Department of Translational Stem Cell Biology Research Institute of the Medical University of Varna Varna Bulgaria

Integrative Genomics of Ageing Group Institute of Ageing and Chronic Disease University of Liverpool Liverpool UK

International Clinical Research Center St Anne's University Hospital Brno Czech Republic

IVEX Lab Akadeemia 15 Tallinn Estonia

Laboratory of Bioinformatics Fondazione IRCCS Casa Sollievo della Sofferenza San Giovanni Rotondo Italy

Laboratory of Genome Integrity Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacky University Olomouc Czech Republic

National Centre for Biomolecular Research Masaryk University Brno Czech Republic

Program for Hematology Immunology BMT and Cell therapy St Marina University Hospital Varna Bulgaria

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