Centromere protein A (CENP-A) dynamics in human pluripotent stem cell self renewal, differentiation and dna repair

Abstract

OBJECTIVE: Accurate chromosome segregation is dependent on the expression of histone H3 variant CENP-A. Reduced expression of CENP-A in somatic cells results in chromosome misalignment and apoptosis. Compared to somatic cells, CENP-A is highly expressed in the human oocyte and in human embryonic stem cells (hESCs). Our objective is to study the significance of high CENP-A mRNA levels in hESCs and in human induced pluripotent stem cells (hIPSCs) to elucidate its role in early human development. DESIGN: All experiments were compared to somatic cells and performed with and without differentiation of stem cells. CENP-A levels were reduced using shRNA lentiviral technology and assays were compared to lentiviral controls. MATERIALS AND METHODS: Two independent HESC and hIPSC lines were used. BJ fibroblasts were used as control. PCR and Western blot were done for CENP-A expression. Propidium Iodide for Cell cycle profile and Annexin assay for apoptosis were used. Immunohistochemistry was used to localize CENP-A. Two-sample student t-test was used for statistics. RESULTS: Reduction of CENP-A in hESCs and hIPSCs is not lethal and does not result in aneuploidy suggesting lower CENP-A need to maintain a functional centromere. CENP-A depleted cells undergo apoptosis upon differentiation suggesting higher CENP-A need to maintain a functional centromere with differentiation. Upon DNA damage, CENP-A moves from centromeres into multiple small nuclear foci, and hESCs with depleted CENP-A do not to mount an effective response to DNA damage. CONCLUSION: Our results indicate that human pluripotent cells may have evolved a novel mechanism for maintaining genomic integrity by lowering amount of CENP-A required for a functional centromere. We also show that CENP-A mRNA reserve is required to maintain functional centromere with differentiation and to repair DNA damage. These results highlights the unique biology of the early human embryo which may have evolved to support rapid cell divisions while maintaining genomic integrity.