Satb2 determines miRNA expression and long-term memory in the adult central nervous system
- Medical University of Innsbruck, Austria
- University of Innsbruck, Austria
- Technical University Braunschweig, Germany
- AG Neuroinflammation and Neurodegeneration (NIND), Germany
- German Center for Neurodegenerative Diseases, Germany
Figures
Figure 1 with 2 supplements
Characterization of Satb2 conditional mutants.
(A) Satb2 is mainly expressed in the adult forebrain. Immunostaining for Satb2 of sagittal brain sections from adult mice. Scale bar: 1000 μm. (B) Immunobloting analysis of the Satb2 protein level …
Figure 1—figure supplement 1
Satb2 is expressed in the cortex and hippocampus of both Satb2 conditional mutants and littermate controls at postnatal day 15.
Immunostaining for Satb2 of coronal brain sections from juvenile, postnatal day 15 Satb2 cKO mice (cKO) and Satb2flox/flox littermate controls (Ctrl). Nuclei were counterstained with DAPI. …
Figure 1—figure supplement 2
Postnatal Satb2 deletion does not cause alterations in body weight, gross brain morphology and cortical layer-specific marker expression.
(A) Satb2 cKO mice (cKO) are visually indistinguishable from Satb2flox/flox mice (Ctrl) and have similar body weights to their control littermates (Ctrl, n = 12; cKO, n = 17; Student's t test, t27 = …
Figure 2 with 1 supplement
Satb2 is required for long-term memory formation.
(A) In a contextual fear conditioning paradigm, Satb2 cKO mice (cKO), showed (i) similar levels of freezing to Satb2flox/flox mice (Ctrl) during the fear-acquisition phase (cKO, n = 7; Ctrl, n = 6; …
Figure 2—figure supplement 1
Satb2 cKO mice show normal responses to electric foot shock.
Flinch-Jump test revealed similar general responses to electric foot shock between Satb2 cKO mice (n = 5) and control littermates (n = 5), Student’s t-test, flinch: t8 = 0, p = 1.0, run/jump: t8 = …
Figure 3
Late-LTP maintenance is impaired in Satb2 conditional mutants.
(A) Schaffer collateral-CA1 late-LTP was significantly impaired in Satb2 cKO mice (Student's t test, t28 = 4.92, p < 0.0001 for the interval 180–185 min post-theta burst stimulation, TBS). Shown are …
Figure 4 with 2 supplements
Synaptic activity and BDNF up-regulate Satb2 in primary hippocampal neurons.
(A) Increased synaptic activity up-regulates Satb2 protein depending on calcium influx through L-type VGCC. Representative Western blot (top) and quantification (bottom) of the Satb2 protein level …
Figure 4—figure supplement 1
BDNF-induced Satb2 expression requires gene transcription.
Representative image of Western blot (top) and quantification (bottom) of Satb2 protein levels 24 hr after stimulation with BDNF in the presence or absence of actinomycin D, (n = 4, ANOVA followed …
Figure 4—figure supplement 2
Time-course analysis of Satb2 expression after BDNF treatment.
(A) Representative image of Western blot (top) and quantification (bottom) showing that Satb2 protein is significantly induced 6 hr and peaks at around 12 hr after BDNF application, (n = 4; ANOVA …
Figure 5 with 4 supplements
Satb2 binding sites are enriched on active gene promoters including miRNA promoters.
(A) Pie-chart illustrating the genomic annotation of Satb2 binding sites. (B) Heatmap of Satb2 binding to TSS (±3 Kb) regions. (C) Average profiles of H4K16ac, H3K27ac, H3K4me1, H3K4me2, H3K4me3 and …
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Figure 5—source data 1
Major GO terms and KEGG pathways, revealed by ChIP-Enrich bioinformatics tool, found to be enriched among the genes having Satb2 peaks within their promoters.
- https://doi.org/10.7554/eLife.17361.012
Figure 5—figure supplement 1
ChIP-qPCR validation of Satb2 enrichment at various identified target regions using chromatin from AAV-Satb2-V5-transduced and non-transduced primary hippocampal neurons.
Each bar represents the ratio of precipitated DNA (bound) to the total input DNA (% Input). Each target region was tested in at least two independent chromatin samples derived from non-transduced …
Figure 5—figure supplement 2
Satb2 binding sites are enriched on active gene promoters and do not correlate with the Polycomb-associated H3K27me3 repressive mark (PcR).
(A) Heat maps depicting H3K4me3 (GSM530197), H3K4me2 (GSM1544912), H3K4me1 (GSM1544908), H3K27Ac (GSM1629392), H4K16Ac (GSM1629377), PolII (GSM1544942), and Satb2 tag densities (read counts/million …
Figure 5—figure supplement 3
Satb2 is deposited at CpGs.
Average tag density profiles (ChIP/Input) and heat maps depicting Satb2 ChIP-seq tag density at CpG islands localized at proximal promoters (A), gene body (B), intergenic regions (C), and miRNA …
Figure 5—figure supplement 4
ChIP-qPCR validation of Satb2 targets in vivo.
ChIP-qPCR analysis of chromatin derived from CA1 hippocampal tissue of control and Satb2 cKO mice immunoprecipitated with an antibody to Satb2 at several target sites identified by ChIP-seq in …
Figure 6 with 3 supplements
Satb2 regulates miRNA expression in CA1 hippocampal area.
(A) Pie-chart showing the percentage of differentially expressed miRNAs (up-and down-regulated, Figure 6—source data 1) in the CA1 region of Satb2 cKO mice vs. littermate controls as assessed by …
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Figure 6—source data 1
Differentially expressed genes between Satb2 cKO mice and Satb2flox/flox littermate controls in the CA1 region as assessed by RNA-seq.
Shown are the genomic coordinates of the identified Satb2 binding sites and the distance to the TSS of the corresponding gene.
- https://doi.org/10.7554/eLife.17361.018
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Figure 6—source data 2
No major significant differences in splicing were observed between the CA1 region of Satb2 cKO mice (n = 9) and littermate controls (n = 9).
Only one gene showed differential exon usage at FDR corrected p-value significance level 0.01 and 12 genes at FDR 0.1 (based on DEXSeq R-package with default parameters).
- https://doi.org/10.7554/eLife.17361.019
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Figure 6—source data 3
List of differentially expressed miRNAs between Satb2 cKO mice and littermate controls (Satb2flox/floxmice) in the CA1 region as assessed by small RNA-seq analysis (base mean above 10 counts, 1.5-fold change, and adjusted p < 0.05).
- https://doi.org/10.7554/eLife.17361.020
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Figure 6—source data 4
List of differentially expressed miRNAs in the CA1 region of Satb2 cKO mice vs. littermate controls that contain Satb2 peak(s) within their promoter.
- https://doi.org/10.7554/eLife.17361.021
Figure 6—figure supplement 1
Validation of the differential expression of selected genes by qPCR.
qPCR quantification of selected gene mRNA levels in the CA1 field of Satb2 cKO mice compared to the control group (Oxr1: Ctrl, n = 11; cKO, n = 11; Ghsr: Ctrl, n = 10; cKO, n = 10; Adra2a: Ctrl, n = …
Figure 6—figure supplement 2
Validation of the differential expression of selected miRNAs by qPCR.
qPCR quantification of miRNA levels in the CA1 field of Satb2 cKO mice compared to the control group (miR-124: Ctrl, n = 9; cKO, n = 9; miR-125b: Ctrl, n = 9; cKO, n = 9; miR-22: Ctrl, n = 7; cKO, n …
Figure 6—figure supplement 3
Satb2 binds to miRNA’s promoters.
IGV genome browser images revealing overlapping between Satb2 peaks and predicted promoters (depicted as red rectangles) of selected miRNAs: miR124a (a), miR125a (b), mir132 (c), mir182 (d), found …
Figure 7
Hippocampal Satb2 re-expression rescues Arc levels and long-term fear memory deficits.
(A) Representative Western blot (left) and quantification (right) of Arc protein level in the CA1 field of Satb2 cKO mice and littermate controls. β3-tubulin was used as a loading control (Ctrl, n = …
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Figure 7—source data 1
List of miRNAs predicted to target mouse Arc 3’UTR by the bioinformatics tools TargetScan, PITA and miRanda.
The miRNAs identified as up-regulated in the CA1 region of Satb2 cKO vs. control mice are shown in red.
- https://doi.org/10.7554/eLife.17361.026
Author response image 1
(A) Mean intensity of Egr1 in each layer was normalized to corresponding layer 4 for each mouse to obtain relative Egr1 expression. Dark-rearing leads to a significant reduction in relative Egr1 …
Additional files
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Supplementary file 1
List of primers used in RT-qPCR and ChIP-qPCR analyses.
- https://doi.org/10.7554/eLife.17361.027
