Fig. 4

Identification of the GR (gene ID: Nr3c1) as a putative key regulator of glial transcriptomic changes in diabetic mice on basis of chromatin accessibility and RNA expression profiles. A Chromatin accessibility was assessed by ATAC-seq on purified Müller cells from 6-month-old mice of both genotypes (2 biological replicates per genotype). Data were subjected to transcription factor binding motif analysis. Left, Density plot showing the distribution and colour code of bins comprising an equal number of peaks corresponding to fold changes in chromatin accessibility between db/db and control mice. Numbers represent the log2 fold change range (db/db vs control) and the corresponding number of peaks per bin. Right, Cluster analysis and heat map of transcription factor (TF) binding motifs significantly enriched when comparing chromatin accessibility of Müller cells from db/db mice with their counterparts from wild-type animals. Bins in shades of green indicate decreased accessibility, while shades of brown indicate increased accessibility. Motifs enriched in the grey bin are those with unchanged chromatin density between genotypes. B TF clusters possibly involved in regulatory changes associated with DR progression as identified by oPOSSUM-3 analysis. Genes/transcripts identified by RNA seq to be significantly up- or down-regulated in Müller cells of 6-months-old diabetic mice were submitted to oPOSSUM-3 analysis [56]. Only TFs that were expressed at transcript level in Müller cells are included. TFs identified in both ATAC-seq and oPPOSSUM-3 analysis are highlighted in green. C Expression levels of TFs highlighted in green in B and of Otx1 and Otx2 as two examples of the TFs that were prominent in A by a high-level reduction in the accessibility of their DNA-binding domains. Data are from the RNA-seq dataset performed on MACS-sorted cells (see Fig. 3). Bars represent mean ± SEM from 3 biological replicates per genotypes (one dot per replicate). Unpaired t-test: *p < 0.05. Mg microglia, vc vascular cells, Mc Müller cells, n neurons