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The DNA damage response is mediated by both DNA repair proteins and epigenetic markers. Here, we observe that N 6 -methyladenosine (m 6 A), a mark of the epitranscriptome, was common in RNAs accumulated at UV-damaged chromatin; however, inhibitors of RNA polymerases I and II did not affect the m 6 A RNA level at the irradiated genomic regions. After genome injury, m 6 A RNAs either diffused to the damaged chromatin or appeared at the lesions enzymatically. DNA damage did not change the levels of METTL3 and METTL14 methyltransferases. In a subset of irradiated cells, only the METTL16 enzyme, responsible for m 6 A in non-coding RNAs as well as for splicing regulation, was recruited to microirradiated sites. Importantly, the levels of the studied splicing factors were not changed by UVA light. Overall, if the appearance of m 6 A RNAs at DNA lesions is regulated enzymatically, this process must be mediated via the coregulatory function of METTL-like enzymes. This event is additionally accompanied by radiation-induced depletion of 2,2,7-methylguanosine (m 3 G/TMG) in RNA. Moreover, UV-irradiation also decreases the global cellular level of N 1 - methyladenosine (m 1 A) in RNAs. Based on these results, we prefer a model in which m 6 A RNAs rapidly respond to radiation-induced stress and diffuse to the damaged sites. The level of both (m 1 A) RNAs and m 3 G/TMG in RNAs is reduced as a consequence of DNA damage, recognized by the nucleotide excision repair mechanism.

Main Conclusion: UV-B-induced flavonoid biosynthesis is epigenetically regulated by site-specific demethylation of AaMYB1, AaMYC, and AaWRKY TF-binding sites inAaPAL1promoter-causing overexpression ofAaPALgene inArtemisia annua. The present study was undertaken to understand the epigenetic regulation of flavonoid biosynthesis under the influence of ultraviolet-B radiation using Artemisia annua L. as an experimental model. In-vitro propagated and acclimatized plantlets were treated with UV-B radiation (2.8 W m -2 ; 3 h), which resulted in enhanced accumulation of total flavonoid and phenolics content as well as eleven individual flavonoids measured through HPLC-DAC. Expression of eight genes (phenylanaline ammonia lyase, cinnamate-4-hydroxylase, 4-coumarate: CoA ligase; chalcone synthase, chalcone isomerase, cinnamoyl reductase, flavonoid-3'-hydroxylase, and flavones synthase) from upstream and downstream flavonoid biosynthetic pathways was measured through RT-PCR and RT-Q-PCR and all were variably induced under UV-B irradiation. Among them, AaPAL1 transcript and its protein were most significantly upregulated. Global DNA methylation analysis revealed hypomethylation of genomic DNA in A. annua. Further epigenetic characterization of promoter region of AaPAL1 revealed cytosine demethylation at five sites, which in turn caused epigenetic activation of six transcription factor-binding sites including QELEMENT, EBOXBNNAPA/MYCCONSENSUSAT, MYBCORE, MYBCOREATCYCB1, and GCCCORE. MYB transcription factors are positive regulators of flavonoid biosynthesis. Epigenetic activation of transcription-enhancing cis-regulatory elements in AaPAL1 promoter and subsequent overexpression of AaMYB1 and AaMYC and AaWRKY transcription factors under UV-B irradiation may probably be the reason for higher AaPAL1 expression and hence greater biosynthesis of flavonoids in A. annua L. The present study is the first report that provides mechanistic evidence of epigenetic regulation of flavonoid biosynthesis under UV-B radiation in A. annua L.