Inactivation of Tumor Suppressor CYLD Inhibits Fibroblast Reprogramming to Pluripotency

Simple Summary Many aspects of the regulatory mechanisms of somatic cell reprogramming-the conversion of any cell type into pluripotent stem cells-still remain elusive. The tumor suppressor CYLD regulates several signaling pathways involved in this process. However, its potential role in reprogramming has not been investigated. In this work, we present evidence that CYLD exerts important regulatory control at the early stages of reprogramming. Loss of CYLD catalytic activity leads to the reduced reprogramming efficiency of mouse embryonic fibroblasts. Whole proteome analysis during early reprogramming stages revealed that CYLD DUB deficiency impedes a vital early reprogramming step known as the mesenchymal-to-epithelial transition (MET). Our findings expand our knowledge of early reprogramming mechanics and reveal a novel role for CYLD as an extracellular matrix regulator.Abstract CYLD is a tumor suppressor gene coding for a deubiquitinating enzyme that has a critical regulatory function in a variety of signaling pathways and biological processes involved in cancer development and progression, many of which are also key modulators of somatic cell reprogramming. Nevertheless, the potential role of CYLD in this process has not been studied. With the dual aim of investigating the involvement of CYLD in reprogramming and developing a better understanding of the intricate regulatory system governing this process, we reprogrammed control (CYLDWT/WT) and CYLD DUB-deficient (CYLD Delta 9/Delta 9) mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells (iPSCs) through ectopic overexpression of the Yamanaka factors (Oct3/4, Sox2, Klf4, c-myc). CYLD DUB deficiency led to significantly reduced reprogramming efficiency and slower early reprogramming kinetics. The introduction of WT CYLD to CYLD Delta 9/Delta 9 MEFs rescued the phenotype. Nevertheless, CYLD DUB-deficient cells were capable of establishing induced pluripotent colonies with full spontaneous differentiation potential of the three germ layers. Whole proteome analysis (Data are available via ProteomeXchange with identifier PXD044220) revealed that the mesenchymal-to-epithelial transition (MET) during the early reprogramming stages was disrupted in CYLD Delta 9/Delta 9 MEFs. Interestingly, differentially enriched pathways revealed that the primary processes affected by CYLD DUB deficiency were associated with the organization of the extracellular matrix and several metabolic pathways. Our findings not only establish for the first time CYLD's significance as a regulatory component of early reprogramming but also highlight its role as an extracellular matrix regulator, which has profound implications in cancer research.