Extended Data Fig. 6: Astrocyte-specific TR deletion (cKO).
From: Divergent transcriptional regulation of astrocyte reactivity across disorders
a. Experimental models: Transgenic mice expressing mGFAP-Cre12 were used for astrocyte-specific deletion (cKO) of Smarca4 or Stat3. Smarca4 was selected as a newly identified TREA-predicted reactivity TR that lacks DNA binding motifs and acts as a chromatin regulator via protein-protein interactions. Stat3 is a well-established astrocyte reactivity TR that acts via DNA-binding motifs12,24 and that was predicted by both TREA and ATAC-seq motif analysis as a reactivity TR in both LPS and SCI. b. Heatmaps show that Smarca4-astro-cKO had minimal effects on overall gene expression or on the expression of highly enriched astrocyte genes under basal conditions in untreated mice, shown as mean FPKM (fragments per kilobase of transcript sequence per million mapped fragments). c. Immunohistochemistry images and graphs (mean + sem) of various staining parameters show that Smarca4-astro-cKO had no visibly detectable or quantifiably significant effects on the appearance or number of astrocytes, neurons or microglia in untreated mice. Unpaired t tests, ns non-significant WT versus Smarca4-cKO, n = 4 mice per group. d. Effects of Smarca4-astro-cKO on RNA-seq reads of Gfap and seven predicted Smarca4-regulated DEGs that are not expressed in WT untreated and are upregulated by LPS in WT but not Smarca4-astro-cKO mice. e. In situ hybridization shows predicted Smarca4-regulated gene, Cnr1, expresed in WT, but not Smarca4-astro-cKO mice after LPS. f. Multichannel immunofluorescence demonstration of Stat3 and Srebf1, or of Smarca4 and Zfp36, in the same reactive astrocytes after SCI. g. Heatmaps compare changes from healthy in differential gene expression (DEG) or differential chromatin accessibility (DAG) across the same genes after LPS or SCI in WT, Smarca4-cKO or Stat3-cKO mice, and graphs show an 86 to 96% congruence between changes in gene expression and changes in chromatin accessibility in both Smarca4- and Stat3-regulated DEGs after LPS or SCI respectively. h. Heatmaps show that Stat3-cKO significantly alters the changes from healthy normally observed after SCI in both differential gene expression (DEG) and differential chromatin accessibility (DAG) across the same 31 genes that lack Stat3-binding motifs. i. Heatmaps show that Stat3-cKO significantly alters the changes from healthy normally observed after SCI in differential gene expression (DEG) of 71 chromatin regulators. j. Multichannel immunofluorescence of Irf9 and Cxcl10 in the same reactive astrocyte after SCI. k. Graphs (mean + sem) show effects of Smarca4-astro-cKO and Stat3-astro-cKO on various microglial histopathological responses to LPS, P values are cKO+LPS versus WT+LPS, one-way ANOVA with Bonferroni’s test, ns nonsignificant; for all graphs WT with no LPS n = 18 mice, all other conditions n = 6 mice. PCA shows composite microglia histopathology score derived from histopathological quantifications in the four graphs. l. Immunohistochemistry and graphs of mean + sem (n > 6) cell counts of the neuronal marker, NeuN, shows that high dose systemic LPS sufficient to cause pronounced microglial activation and behavioural effects did not lead to detectable neuronal loss in either spinal cord or brain. One-way ANOVA with Bonferroni’s test, ns nonsignificant; for both graphs WT with no LPS n = 18 mice, all other conditions n = 4 mice. m. Distance biplot of PCA for the effects of Smarca4-astro-cKO and Stat3-astro-cKO on composite locomotor scores after SCI. The locations of values for each individual locomotor parameter, from either the longitudinal observer scored Open Field (OF) evaluations or ladder walk testing at 28 days after SCI, indicates their contributions to defining the PC space. Graph (mean + sem) shows composite SCI locomotor score derived from PCA of all OF and ladderwalk locomotor parameters recorded over 28 days of recovery; P values are cKO versus WT, one-way ANOVA with Bonferroni’s test, n = 11 mice per group. n. Immunofluorescence images and graph (mean + sem) of staining intensity show effects of Smarca4-astro-cKO and Stat3-astro-cKO on CD68 and Gfap 28 days after SCI. o. Bar graph (mean + sem) shows composite histology scores derived from PCA of Gfap, CD13, MBP and CD68 quantifications at 28 days after SCI; P values are cKO versus WT, one-way ANOVA with Bonferroni’s test, WT n = 8, Smarca4cKO n = 7, Stat3cKO n = 9 mice. Line graph shows correlation analysis of composite locomotor and histological scores. p. DEG-associated functional signalling pathway analysis shows that deletion of either Smarca4 or Stat3 alters many functions normally associated with WT astrocyte reactivity after LPS or SCI. Each immunohistochemical or in situ hybridization evaluation shown (c,e,f,j,l,n) was repeated at least three times with similar results.
