Understanding the transcriptional changes that are engaged in stress resilience may reveal novel antidepressant targets. Here we use gene co-expression analysis of RNA-sequencing data from brains of resilient mice to identify a gene network that is unique to resilience. Zfp189, which encodes a previously unstudied zinc finger protein, is the highest-ranked key driver gene in the network, and overexpression of Zfp189 in prefrontal cortical neurons preferentially activates this network and promotes behavioral resilience. The transcription factor CREB is a predicted upstream regulator of this network and binds to the Zfp189 promoter. To probe CREB–Zfp189 interactions, we employ CRISPR-mediated locus-specific transcriptional reprogramming to direct CREB or G9a (a repressive histone methyltransferase) to the Zfp189 promoter in prefrontal cortex neurons. Induction of Zfp189 with site-specific CREB is pro-resilient, whereas suppressing Zfp189 expression with G9a increases susceptibility. These findings reveal an essential role for Zfp189 and CREB–Zfp189 interactions in mediating a central transcriptional network of resilience.
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The RNA-seq data reported in the paper are deposited in GEO with the accession number GSE118317. Other data that support the findings of this study are available from the corresponding author upon request.
Scripts and code utilized in the analysis of study data are available from the corresponding author upon request.
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This work was supported by National Institutes of Health grants F30MH110073 (Z.S.L.), T32GM007280 (Z.S.L.), T32MH096678 (Z.S.L.), K99DA045795 (P.J.H), U01AG046170 (B.Z.), R01AG057907 (B.Z.), RF1AG057440 (B.Z.), P50MH096890 and R01MH051399 (E.J.N.), and by the Hope for Depression Research Foundation.
The authors declare no competing interests.
Peer review information: Nature Neuroscience thanks Cornelius Gross and the other, anonymous, reviewers for their contribution to the peer review of this work.
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Integrated supplementary information
a) Resilient module enrichment for known and predicted protein-protein interactions (PPIs). Presence of color indicates statistical significance (FDR q < 0.05) with intensity of color scaled according to -log10(p-value). b-c) Module preservation analysis in RNA-seq data from post-mortem brain from (b) human controls and (c) MDD patients. Dotted line represents significance threshold (Bonferroni corrected p < 0.05). d) Resilient module preservation in human controls and MDD. Presence of color indicates statistical significance (Bonferroni p < 0.05) with intensity of color scaled according to -log10(p-value). e) Change in resilient module preservation between human controls and MDD. Modules showing more preservation in controls (difference between control and MDD -log 10 p-value > 1) are shown in blue. Modules showing more preservation in MDD (difference between control and MDD -log 10 p-value < -1) are shown in red. Modules not significantly (Bonferroni corrected p < 0.05) preserved or preserved similarly (difference in |-log 10 (p-value)| < 1) are not assigned a color.
a) Pink module genes overlaid with genes upregulated (yellow) and downregulated (blue) in the differentially expressed gene (DEG) comparison resilient vs. control in PFC (p < 0.05, FC > 1.3). b) Connections between pink module key driver genes. c) Correspondence between differential expression for the top 10 key driver genes and DEG enrichment for the pink module across phenotypes and brain areas. Presence of color indicates statistical significance (p q < 0.05) with intensity of color scaled according to -log10(p-value).
a) RNAscope highlighting robust Zfp189 mRNA (red) expression in mouse PFC neurons (green, NeuN mRNA). Scale bar is 5 μm. Roughly 80% of Zfp189+ cells are NeuN+. Repeated with similar results in five animals. B) IHC showing colocalization of HSV transgene expression (GFP) and neurons (NeuN) in the mouse PFC. 20x magnification; Scale bar is 50 μm. Repeated with similar results in four animals.
a) Injection of HSV-Zfp189 in PFC increases Zfp189 mRNA relative to HSV-GFP (t=2.718, p = 0.024, n = 10). b) Exposure to CSDS reduces OFT exploration in HSV-GFP mice (χ2(3) = 10.903, p = 0.012, n = 8-14, Mann Whitney post hoc p = 0.031), but not HSV-Zfp189 mice. However, unstressed mice show baseline differences (n = 8,11,10,14). c) Defeat reduces locomotion regardless of whether Zfp189 is overexpressed (F1,41 = 30.26, p < 0.001, n = 9,12,10,14). d) Null effects of Zfp189 overexpression in FST (χ2(3) = 0.564, p = 0.905, n = 9,11,10,14). e-g) Null effects in (h) OFT (t = 0.049, p = 0.962), (i) locomotor behavior (t = 0.077, p = 0.450), and (j) FST (t = 0.142, p = 0.888) for previously susceptible mice injected with Zfp189 in PFC (n = 12,13). *p < 0.05, **p < 0.01, ***p < 0.001. Bar graphs show mean ± SEM.
a) DEGs (p < 0.05, FC > log2|0.2|) in PFC in Zfp189 overexpressing unstressed controls. b) Module-wide enrichment for HSV-Zfp189 overexpression in PFC in unstressed mice. c-d) Gene Ontology Biological Process (GOBP) pathways affected Zfp189 overexpression in PFC in (c) previously susceptible mice and (d) unstressed controls (DEG threshold = p < 0.05, FC > log2|0.2|). *FDR q < 0.05.
Cells infected by an HSV vector (expressing mCherry) overlap 100% with cells infected by a previously injected AAV vector (expressing GFP) in the mouse PFC. Scale bar is 50 μm. Repeated with similar results in three animals.
a) Effect of CREB knockout (KO) and Zfp189 overexpression in splash test (interaction F1,40 = 0.067, p = 0.797, n = 10,11,11,12). b) Effect of CREB KO and Zfp189 overexpression in FST (interaction F1,35 = 0.121, p = 0.730, n = 10,11,10,8).
a) Targeting dCas9 with no functional domain to the Zfp189 promoter does not affect Zfp189 expression (U = 4.0, p = 0.343, n = 4). b) Targeting dCas9 fused to phosphomimetic CREBS133D increases Zfp189 (U = 1.0, p = 0.009, n = 5,6). c) Localizing dCas9 fused to a phospho-null CREBS133A does not affect Zfp189 expression (t=0.9868, p = 0.362, n = 4).
a-b) GOBP pathways affected by dCas9-CREBS133D combined with Zfp189-sgRNA in PFC in (a) mice exposed to social defeat and (b) unstressed controls. (DEG threshold = p < 0.05, FC > log2|0.2|, Asterisk = FDR q < 0.05) c) Overlap for PFC DEGs (p < 0.05, log2FC > |0.2|) resulting from Zfp189-targeted dCas9-CREBS133D compared to NT-dCas9-CREBS133D in unstressed controls. d) Pink module genes differentially expressed in unstressed controls. e) On and off-target CRISPR gene regulation in RNA-seq datasets. Expression of the targeted Zfp189 gene is significantly up-regulated (p < 0.05, FC > log2|0.2|) in both defeated and un-stressed mice. The other 49 genes analyzed were those in closest proximity to other genomic regions with closest homology to the Zfp189-sgRNA used. No genomic region contains fewer than 3 mismatches with this sgRNA. Only one of these predicted off-target sites is affected by dCas9-CREBS133D plus Zfp189-sgRNA, but this regulation was only seen in defeated mice, suggesting that this is not an off-target effect of the CRISPR tools, but rather an effect of the defeat per se.
Pink module genes and key drivers.
Human cohort demographics.
Upstream motifs of module genes.
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Nature Reviews Neuroscience (2019)