Centromere protein A dynamics in human pluripotent stem cell self-renewal, differentiation and DNA damage

Abstract

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.

PRMT5 is Required for Human Embryonic Stem Cell Proliferation But Not Pluripotency

“…Lentiviral vectors were packaged, concentrated by ultracentrifugation at 50 000 g for 90 min, resuspended in hESC media and stored at −80° C as previously described [12]. Tittering was performed on Human Embryonic Kidney 293 FT cells.…”

“…Tittering was performed on Human Embryonic Kidney 293 FT cells. Transduction in hESCs line was performed at MOI 1 as previously described [12]. …”

“…H1 hESCs were transduced with either control or PRMT5 KD virus as previously described [12]. Cells were allowed to recover for 24 hrs after lentiviral transduction before selection in with hygromycin for 3 days.…”

Flexibility of centromere and kinetochore structures

“…Interestingly, decreased levels of kinetochore proteins cause different responses in different cell types. For example, depletion of CENP-A protein in fibroblasts eliminates kinetochore function, while the same knockdown permits cell cycle progression in human pluripotent stem cells, presumably because they have stored CENP-A mRNA, further protecting them from CENP-A depletion [81]. …”

The Emerging Role of Centromere/Kinetochore Proteins in Cellular Senescence

“…The senescence that arises from CENP-A depletion may be a self-defense mechanism to suppress the otherwise catastrophic impact upon genome integrity that would arise from kinetochore dysfunction following certain types of stress. It should be noted that reduction of CENP-A does not result in irreversible growth arrest in human pluripotent stem cells (Ambartsumyan et al, 2010). Ambartsumyan et al demonstrated that CENP-A-depleted undifferentiated human pluripotent stem cells were capable of maintaining a functional centromere marks and showed no changes in morphology or proliferation rate relative to control cells, whereas CENP-A-depleted BJ fibroblasts showed arrest in G2/M and underwent apoptosis.…”