Centromere protein A dynamics in human pluripotent stem cell self-renewal, differentiation and DNA damage.
Human pluripotent stem cells (hPSCs) hold significant promise for use in regenerative medicine, or as a model to understand human embryo development. However, the basic mechanisms required for proliferation and self-renewal of hPSCs have not been fully uncovered. Proliferation in all eukaryotes is dependent upon highly regulated expression of the histone H3 variant Centromere protein A (CENP-A). In the current study, we demonstrate that hPSCs have a unique messenger ribonucleic acid (mRNA) reserve of CENP-A not found in somatic fibroblasts. Using short hairpin RNA technology to reduce but not ablate CENP-A, we show that CENP-A-depleted hPSCs are still capable of maintaining a functional centromeric mark, whereas fibroblasts are not. However, upon induction of differentiation or DNA damage, hPSCs with depleted CENP-A arrest in G2/M and undergo apoptosis. Analysis of CENP-A dynamics following DNA damage in hPSCs reveals that 60 min after irradiation, CENP-A is found in multiple small nuclear foci that are mutually exclusive to γH2AX as well as CENP-C. Furthermore, following irradiation, hPSCs with depleted CENP-A mount a normal apoptotic response at 6 h; however at 24 h, apoptosis is significantly increased in CENP-A-depleted hPSCs relative to control. Taken together, our results indicate that hPSCs exhibit a unique mechanism for maintaining genomic integrity by possessing the flexibility to reduce the amount of CENP-A required to maintain a functional centromere under self-renewing conditions, and maintaining a reserve of CENP-A mRNA to rebuild the centromere following differentiation or DNA damage.
Ambartsumyan
Gayane
G
Department of Molecular Cell and Developmental Biology, University of California Los Angeles, Los Angeles, CA 90095, USA.
Gill
Rajbir K
RK
Perez
Silvia Diaz
SD
Conway
Deirdre
D
Vincent
John
J
Dalal
Yamini
Y
Clark
Amander T
AT
D017209:Apoptosis; D001324:Autoantigens / Q000235:genetics* / Q000378:metabolism*; D015153:Blotting, Western; D002503:Centromere / Q000502:physiology*; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D016195:G2 Phase / Q000235:genetics; D005786:Gene Expression Regulation; D006657:Histones / Q000378:metabolism; D006801:Humans; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics
D001324:Autoantigens; C000619700:CENPA protein, human; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D006657:Histones; D012333:RNA, Messenger; D034741:RNA, Small Interfering