Fig. 3: PHF6 deficiency in ILPHF6 cells reduces calcium channel β3 expression and stress-responsive gene induction. | Molecular Psychiatry

Fig. 3: PHF6 deficiency in ILPHF6 cells reduces calcium channel β3 expression and stress-responsive gene induction.

From: Regulation of stress susceptibility by chromatin-binding protein PHF6 in the pituitary intermediate lobe

Fig. 3

a Schematic of CUT&Tag using an anti-PHF6 antibody on the enriched pituitary tissue dissected from 100 wild-type mice under basal conditions and 100 wild-type mice exposed to RS stress. b Heatmaps of genome-wide PHF6 binding in the pituitary IL, generated from normalized PHF6 CUT&Tag reads under basal and RS-stress conditions. PHF6 occupancy is shown within 3 kb upstream of transcription start sites (TSS) and 3 kb downstream of transcription end sites (TES). Right panels display the average PHF6 enrichment under basal and RS-stress conditions. c Pairwise comparison of PHF6 CUT&Tag under basal and RS-stress conditions revealed 2,121 overlapping, 329 basal-only, and 16,203 RS-stress-only annotated genes. d Schematic of SMART-RNA-seq performed on LCM-captured IL tissue from control and Phf6 cKO mice under basal and RS-stress conditions. Representative bright-field image of pituitary section after LCM is shown. LCM, Laser-capture Microdissection. e Volcano plot of the differentially expressed genes (DEGs) in ILPHF6 cells from Phf6 cKO mice and control mice under basal conditions (Ctrl-base, n = 6; Phf6 cKO-base, n = 6), with a pie chart indicating 21 stress-upregulated and 37 stress-downregulated genes. f GO analysis of functional categories enriched among DEGs in Phf6 cKO mice. g Genomic binding profile of PHF6 at the Cacnb3 locus under basal conditions. h Fold change in normalized SMART-RNA-seq counts of Cacnb3 under basal conditions in control (n = 6) and Phf6 cKO mice (n = 6). i Representative smFISH (RNAscope) images showing Cacnb3 mRNA (green) in the pituitary of control and Phf6 cKO mice. White dotted lines indicate the IL. Scale bars, 100 μm. j Left: Volcano plot of the DEGs in ILPHF6 cells from control mice after RS stress (Ctrl-base, n = 6; Ctrl-stress, n = 6), with a pie chart indicating 52 stress-upregulated and 14 stress-downregulated genes. Right: Volcano plot of the DEGs in ILPHF6 cells from Phf6 cKO mice after RS stress (Phf6 cKO-base, n = 6; Phf6 cKO-stress, n = 6), with a pie chart indicating 25 stress-upregulated and 26 stress-downregulated genes. k Heatmap of the quadrant-wise analysis of DEGs in control and Phf6 cKO mice under basal and RS-stress conditions. The heatmap illustrated 66 differentially expressed genes in control mice (RS-stress vs. basal), with cutoffs at FDR (adjusted p-value) < 0.05, and mean of normalized counts ≥ 50. l Fold changes in normalized counts of Fosb, Fosl2, and Nr4a2 from SMART-RNA-seq under RS-stress conditions versus basal conditions in control (n = 6) and Phf6 cKO mice (n = 6).

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