Gene expression examination revealed that Müller cells upregulated the expression of DNA methyltransferases
Dnmt1 and
Dnmt3a after injury and in aging (
Supplementary Fig. S10), suggesting that DNA methylation patterns were dynamically regulated in Müller cells. Moreover, inhibition of DNA methylation perturbs the regeneration activity of Müller cells in zebrafish.
76 To examine the CpG methylation patterns in Müller cells during injury and aging, we performed RRBS sequencing of the genomes of Müller cells isolated from retinas 2 days after NMDA treatment and from 1.5-year-old mice. Searching for DMRs of 200-bp tiles of Müller cell genomes showed that a number of regions exhibited altered DNA methylation levels in response to injury (
Fig. 7A, methylation level change > 25%), while aging had a mild effect on the DNA methylation patterns of Müller cells (
Fig. 7B). DMRs in both injury and aging conditions were largely distributed in the intergenic region, introns and exons, and 8% and 16% were distributed in the promoter regions in the injury condition and aging condition, respectively.
Ascl1 is an essential TF for the regeneration of the zebrafish retina and is quickly upregulated in Müller cells after injury,
50,51 while it fails to do so in mammals (
Fig. 3B).
52 To test whether
Ascl1 expression is sequestered by promoter methylation in Müller cells in mice, we examined the genomic region of
Ascl1. The data showed that the promoter region was hypomethylated in Müller cells in all the conditions tested, similar to the methylation pattern in RPCs (
Fig. 7E). Similarly,
Lin28a and
Lin28b, two other important regeneration regulators for Müller cells,
51 also had similar promoter methylation patterns in Müller cells and RPCs (
Fig. 7E), suggesting that promoter methylation is not the blockade for the expression of
Ascl1, as well as its downstream effectors,
Lin28a and
Lin28b, in Müller cells, consistent with the previously published restriction PCR result.
76 To examine whether
Ascl1 binding to genomic targets might be influenced by DNA methylation, we calculated the CpG methylation levels of genomic
Ascl1-targeting sites in Müller cells under different conditions, as well as in RPCs.
Ascl1-targeting sites were slightly less methylated in Müller cells than in RPCs, while this methylation level was slightly upregulated in injury conditions (
Fig. 7F), suggesting that
Ascl1-targeting sites in Müller cells were not locked by DNA methylation.