Sirtuin 1 interacts with DNA repair molecules during the initiation phase of reprogramming from mouse fibroblasts to mouse induced pluripotent stem cells

Abstract

The reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) involves chromatin remodeling. We have previously shown that a NAD+ dependent histone deacetylase, sirtuin 1 (SIRT1) positively regulated mouse iPSCs formation, partly through deacetylating p53 leading to altered Nanog and p21 expression (1). SIRT1 also deacetylates other proteins like PGC1α [2] and FOXO3 [3]. The present study aimed at identifying the SIRT1 interacting protein during the reprogramming process. Pure population of secondary mouse embryonic fibroblast (MEF) containing doxycycline (DOX) inducible OSKM factors were obtained from chimeric MEF by Fluorescence Activated Cell Sorting. The cells were cultured in the presence of DOX for the first 5 days (initiation phase of reprogramming). The interacting proteins of SIRT1 during reprogramming were isolated by co-immunoprecipitation. The immunoprecipitated proteins were visualized by silver staining, and identified by mass spectrometry. According to the pathway clustering analysis from the Kyoto Encyclopedia of Genes and Genomes (KEGG), a number of them were involved in repair of double strand break (DSB). Homologous recombination DNA repair genes are important for successful reprogramming [4]. We speculated that SIRT1 might promote DNA repair through binding to the complex of DSB repair proteins. Indeed, the levels of the SIRT1 interacting DSB repair proteins (SIRT1-DSB) were up-regulated in the MEF upon DOX induction. To this end, we tried to knock down two of these SIRT1-DSBs at the onset of reprogramming. The result showed that the down-regulation of SIRT1-DSBs suppressed the iPSC colony formation as demonstrated by lower colony number and reduced Nanog expressions in the colonies. These data supported that SIRT1 activated the SIRT1-DSBs in the initial phase of reprogramming process leading to increase in colony formation. Acknowledgement: This work is supported by General Research Fund (HKU775711M) and Seed Funding Scheme to Support Research Projects on Human Embryonic Stem Cells (ESC) and Induced Pluripotent Stem Cells (iPSC), Stem Cell and Regenerative Medicine Consortium (SCRMC), Li Ka Shing Faculty of Medicine, The University of Hong Kong. We thank Professor Andras Nagy (The University of Toronto) for his generous gift of primary iPSCs.