Analyzing a calcium-dependent UV response that results in a global chromatin compaction in mammalian cells


May 2023

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Genomic integrity is challenged every day by both endogenous and exogenous stresses. Once DNA gets damaged, a series of events follow, starting with recognizing the damage by accessing it, then processing and repairing the damaged DNA, and eventually restoring the lesion area to its pre-damage state. However, the structural change in the global chromatin in response to UV radiation and the etiology of these changes are not well known. To visualize a global chromatin structural changes upon UVB radiation, we used a fluorometric detection approach. We observed large-scale chromatin compaction immediately after UVB radiation in mouse embryonic fibroblasts, human cervical cancer cells (HeLa), and primary human epidermal melanocytes (HEMs). Using immunofluorescence we also demonstrated that UVB-induced chromatin compaction protects DNA from further UV damage. Using confocal microscopy, we demonstrated cellular calcium influx after UV radiation without external retinal addition. Our RNA-seq data identified two G-protein coupled receptors (GPCRs), SSTR4 and P2RY6, associated with calcium signaling, whose expression was upregulated after UVB radiation in HEMs. Chemical inhibition of P2RY6 and IP3 receptors resulted in a significant reduction in calcium influx and chromatin compaction after UV irradiation of cells. Inhibition on other candidate calcium channel, TRPA1, which mediates UVR-induced calcium influx and early melanin synthesis, did not lead to significant inhibition of chromatin compaction in HEMs after UVB radiation. Based on these findings, we propose that a non-retinal UVB phototransduction pathway is involved in an increase in intracellular calcium triggered by the opening of IP3 receptors which leads to activation of the P2RY6 receptor and then further Ca2+ influx. This increase in cellular Ca2+ concentration leads to an increase in nuclear chromatin compaction, which has a DNA protective role. We suggest that SSTR4 maintains the steady state of calcium by reducing the calcium level after UVB-induced calcium surge in primary human epidermal melanocytes. RNA-seq data revealed that the highest number of genes that upregulated was at 4 hours, and the lowest number of genes upregulated was at 20 minutes after UVB radiation. We suggest that UV-induced chromatin compaction reduces gene expression, and as time progresses, local unfolding of chromatin happens, which allows more gene to be expressed. However, some genes seem to be overexpressed despite the immediate global chromatin compaction and the general gene repression that follows UV radiation. This finding suggests that there are some mechanisms to keep the activation of essential genes after UV irradiation, despite the general gene repression. A detailed understanding of the molecular mechanisms that allow human skin cells to detect UV radiation and respond by compacting the chromatin and protecting the DNA will aid in the search for novel therapeutics to reduce sun-inflicted carcinogenesis.



Biology, Molecular