Abstract
Embryonic stem cells can self-renew and differentiate, holding great promise for regenerative medicine. They also employ multiple mechanisms to preserve the integrity of their genomes. Nucleotide excision repair, a versatile repair mechanism, removes bulky DNA adducts from the genome. However, the dynamics of the capacity of nucleotide excision repair during stem cell differentiation remain unclear. Here, using immunoslot blot assay, we measured repair rates of UV-induced DNA damage during differentiation of human embryonic carcinoma (NTERA-2) cells into neurons and muscle cells. Our results revealed that the capacity of nucleotide excision repair increases as cell differentiation progresses. We also found that inhibition of the apoptotic signaling pathway has no effect on nucleotide excision repair capacity. Furthermore, RNA-Seq-based transcriptomic analysis indicated that expression levels of four core repair factors, xeroderma pigmentosum (XP) complementation group A (XPA), XPC, XPG, and XPF-ERCC1, are progressively up-regulated during differentiation, but not those of replication protein A (RPA) and transcription factor IIH (TFIIH). Together, our findings reveal that increase of nucleotide excision repair capacity accompanies cell differentiation, supported by the up-regulated transcription of genes encoding DNA repair enzymes during differentiation of two distinct cell lineages.
Highlights
Embryonic stem cells can self-renew and differentiate, holding great promise for regenerative medicine
The advantages in utilizing the well-characterized NT2 cells are that nucleotide excision repair in cells at various stages of differentiation can be examined in an identical genetic background, and NT2 cells can differentiate into multiple different types of cells upon treatment with different differentiation agents
We monitored the dynamic changes of nucleotide excision repair capacity at various stages of differentiation of human embryonic carcinoma cells (NT2) into neurons and muscle cells
Summary
To investigate the effects of distinct differentiation stages and lineages on nucleotide excision repair, we first tested the induction of neurons and muscle cells from NT2 cells. For CPD repair, undifferentiated NT2 cells have lower repair capacity, and the difference between undifferentiated and differentiated NT2 cells after 21 days’ RA treatment reaches statistical significance following UV irradiation at 5 and 10 J/m2 doses (Fig. 3, G–L). Low repair activity, we used Z-VAD-FMK [42], an irreversible pan-caspase inhibitor, to validate the inhibition of apoptosis following UV treatment in undifferentiated and differentiated NT2 cells at day 21 of RA treatment, and we measured repair rates for (6 – 4)PPs and CPDs, caused by 10 J/m2 of UV irradiation, within 24 h in undifferentiated and differentiated NT2 cells with or without inhibition of apoptosis. Our results suggest that the up-regulation of XPA, XPC, XPG, and XPF-ERCC1, together with some other nucleotide excision repair-related genes such as CSB and DDB2, may contribute to the gradual increase of nucleotide excision repair capacity during multi-lineage differentiation of NT2 cells. We observed that genes coding for the 10 subunits of TFIIH complex including XPB and XPD were
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