Remote Memory and Cortical Synaptic Plasticity Require Neuronal CCCTC-Binding Factor (CTCF)

Abstract

The molecular mechanism of long-term memory has been extensively studied in the context of the hippocampus-dependent recent memory examined within several days. However, months-old remote memory maintained in the cortex for long-term has not been investigated much at the molecular level yet. Various epigenetic mechanisms are known to be important for long-term memory, but how the 3D chromatin architecture and its regulator molecules contribute to neuronal plasticity and systems consolidation is still largely unknown. CCCTC-binding factor (CTCF) is an 11-zinc finger protein well known for its role as a genome architecture molecule. Male conditional knock-out mice in which CTCF is lost in excitatory neurons during adulthood showed normal recent memory in the contextual fear conditioning and spatial water maze tasks. However, they showed remarkable impairments in remote memory in both tasks. Underlying the remote memory-specific phenotypes, we observed that female CTCF conditional knock-out mice exhibit disrupted cortical LTP, but not hippocampal LTP. Similarly, we observed that CTCF deletion in inhibitory neurons caused partial impairment of remote memory. Through RNA sequencing, we observed that CTCF knockdown in cortical neuron culture caused altered expression of genes that are highly involved in cell adhesion, synaptic plasticity, and memory. These results suggest that remote memory storage in the cortex requires CTCF-mediated gene regulation in neurons, whereas recent memory formation in the hippocampus does not.SIGNIFICANCE STATEMENT CCCTC-binding factor (CTCF) is a well-known 3D genome architectural protein that regulates gene expression. Here, we use two different CTCF conditional knock-out mouse lines and reveal, for the first time, that CTCF is critically involved in the regulation of remote memory. We also show that CTCF is necessary for appropriate expression of genes, many of which we found to be involved in the learning- and memory-related processes. Our study provides behavioral and physiological evidence for the involvement of CTCF-mediated gene regulation in the remote long-term memory and elucidates our understanding of systems consolidation mechanisms.

Keywords: 3D genome architecture; CTCF; cortical plasticity; remote memory; systems consolidation.

Figure 1.

CTCF cKO mice have reduced expression of CTCF. A, B, Immunohistochemistry analysis showed that CTCF cKO mice have reduced CTCF protein expression in the ACC and hippocampus. Blue represents DAPI. Red represents NeuN. Green represents CTCF. C, D, qRT-PCR analysis confirmed that CTCF mRNA level is reduced in CTCF cKO mice (WT, n = 4; cKO, n = 3; C, WT, 1 ± 0.0471; cKO, 0.5094 ± 0.025; unpaired t test; p = 0.0004; D, WT, 0.9674 ± 0.076; cKO, 0.6512 ± 0.102; unpaired t test; p = 0.0257). *p < 0.05, ***p < 0.001.

Figure 2.

CTCF cKO mice display impaired cortex-dependent memory. A, In the CFC test, CTCF cKO mice showed intact recent fear memory but impaired remote fear memory (WT, n = 8; cKO, n = 9; two-way repeated-measures ANOVA; effect of interaction, F_(1,15) = 9.831, p = 0.0068; Bonferroni post hoc test for day 29, p < 0.01). B, During the training phase of the MWM, CTCF cKO mice showed longer escape latency on days 4–6 (WT, n = 14; cKO, n = 11; two-way repeated-measures ANOVA, effect of interaction, F_(5,115) = 4.072, p = 0.0019; Bonferroni post hoc tests, day 4, p < 0.01; day 5, p < 0.01; day 6, p < 0.05). C–E, In the recent memory probe test of the MWM test, CTCF cKO mice performed comparably with WT but showed slower swimming speed (C, two-way ANOVA, effect of interaction of genotype and quadrant, F_(3,92) = 1.246, p = 0.2976; D, WT, 3.071 ± 0.5593; cKO, 1.818 ± 0.5191; unpaired t test; p = 0.1225; E, WT, 20.89 ± 0.7795; cKO, 18.04 ± 1.063; unpaired t test; p = 0.0370). TQ, Target quadrant; OQ, opposite quadrant; AQ, adjacent quadrant. F–H, When the same mice were tested 3 weeks after training, CTCF cKO mice displayed loss of spatial memory with less number of platform crossing and still showed slower swimming speed (F, two-way ANOVA, effect of interaction of genotype and quadrant, F_(3,92) = 3.551, p = 0.0175; G, WT, 5.429 ± 0.7317; cKO, 2.364 ± 0.4724; unpaired t test; p = 0.0031; H, WT, 21.17 ± 0.6098; cKO, 17.16 ± 0.6548; unpaired t test; p = 0.0002). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3.

CTCF cKO mice exhibit impaired cortical synaptic plasticity. A, Hippocampal E-LTP was normal in CTCF cKO mice (WT, n = 13; cKO, n = 8; average of fEPSP slopes for the last 5 min; WT, 140.8 ± 5.8%; cKO, 136.8 ± 8.6%; unpaired t test; p = 0.6947). B, CTCF cKO mice did not exhibit any impairment in the theta burst stimulation-induced hippocampal L-LTP, and the potentiation level was maintained for 3 h at a comparable level with WT (WT, n = 5; cKO, n = 4; average fEPSP slopes for the last 10 min; WT, 144.0 ± 10.4%; cKO, 138.1 ± 16.7%; unpaired t test; p = 0.7635). C, Input–output curve was normal in CTCF cKO mice (WT, n = 11; cKO, n = 8; repeated-measures two-way ANOVA, effect of genotype, F_(1,17) = 0.0929; p = 0.7641). D, CTCF cKO mice displayed normal cortical E-LTP (WT, n = 16; cKO, n = 12; average of fEPSP slopes for the last 4 min; WT, 122.6 ± 2.5%; cKO, 120.6 ± 3.2%; unpaired t test; p = 0.6190). E, CTCF cKO mice showed a significant deficit in cortical L-LTP with a substantially decreased potentiation level after induction (WT, n = 10; cKO, n = 6; average fEPSP slopes for the last 8 min; WT, 141.7 ± 8.1%; cKO, 116.4 ± 6.9%; unpaired t test; p = 0.0498). F, Cortical basal transmission was attenuated in CTCF cKO mice (WT, n = 8; cKO, n = 8; repeated-measures two-way ANOVA, effect of genotype, F_(1,14) = 7.364; p = 0.0168). *p < 0.05.

Figure 4.

CTCF HT mice show a partial deficit in remote memory. A, CFC recent memory and remote memory were normal in CTCF HT mice (WT, n = 15; HT, n = 16; two-way ANOVA; effect of interaction, F_(1,58) = 0.0631; p = 0.8026). B–D, CTCF HT mice performed comparably with WT in the recent memory test of the MWM (WT, n = 7; HT, n = 8; B, two-way ANOVA; effect of interaction of genotype and quadrant, F_(3,52) = 0.4176; p = 0.7411; C, WT, 36.7 ± 2.126; cKO, 36.24 ± 2.155; unpaired t test; p = 0.8835; D, WT, 2.429 ± 0.6117; HT, 2.375 ± 0.4978; unpaired t test; p = 0.9463). E–G, Three weeks later, the CTCF HT mice showed a significant memory deficit in the MWM probe test. Quadrant duration, distance from platform, and number of platform crossings were all impaired (WT, n = 7; HT, n = 8; E, two-way ANOVA; effect of interaction of genotype and quadrant, F_(3,52) = 4.882; p = 0.046; Bonferroni post hoc test for TQ (target quadrant). p = 0.0124; F, WT, 29.23 ± 1.985; HT, 34.55 ± 1.372; unpaired t test; p = 0.0423; G, WT, 4 ± 1.750; HT, 1.750 ± 0.4532; unpaired t test; p = 0.0442). H, Basal transmission was normal in ACC of CTCF HT mice (WT, n = 10; HT, n = 11; two-way ANOVA; effect of interaction, F_(9,171) = 0.2952; p = 0.9753). I, E-LTP was intact in ACC of CTCF HT mice (WT, n = 7; HT, n = 6; average fEPSP slopes for the last 4 min; WT, 132.1 ± 3.252%; HT, 135.1 ± 5.005%; unpaired t test; p = 0.6123). J, L-LTP was also normal in ACC of CTCF HT mice (WT, n = 6; HT, n = 4; average fEPSP slopes for the last 8 min; WT, 164.2 ± 10.12%; HT, 159.8 ± 9.665%; unpaired t test; p = 0.7700). *p < 0.05.

Figure 5.

RNA-seq data from CTCF KD cortical culture reveal differentially expressed genes. A, Volcano plot showed that more genes were downregulated than upregulated in the DEG list. B–E, IPA showed connections of DEGs for four functions: memory, learning, LTP, and synaptic transmission. They were all predicted to be inhibited.

Figure 6.

qRT-PCR of 5 DEGs shows differential results in ACC and hippocampus. A–E, qRT-PCR of ACC tissues from CTCF cKO mice confirmed the RNA-seq data of gene downregulation (WT, n = 4; cKO, n = 3; A, normalized expression of RhoU mRNA; WT, 1 ± 0.04013%; cKO, 0.7669 ± 0.05816%; unpaired t test; p = 0.0187; B, normalized expression of Drd1 mRNA; WT, 1 ± 0.07316%; cKO, 0.6552 ± 0.1332%; unpaired t test; p = 0.0584; C, normalized expression of Pcdhα4 mRNA; WT, 1 ± 0.06878%; cKO, 0.4386 ± 0.07655%; unpaired t test; p = 0.0029; D, normalized expression of Pcdhβ13 mRNA; WT, 1 ± 0.02351%; cKO, 0.3466 ± 0.02203%; unpaired t test; p < 0.0001; E, normalized expression of PcdhγA12 mRNA; WT, 1 ± 0.04239%; cKO, 0.4336 ± 0.02648%; unpaired t test; p = 0.0001). F–J, qRT-PCR of hippocampal tissues from CTCF cKO mice showed a different pattern of gene expression (WT, n = 4; cKO, n = 3; F, normalized expression of RhoU mRNA; WT, 1 ± 0.05991%; cKO, 0.95 ± 0.07146%; unpaired t test; p = 0.6132; G, normalized expression of Drd1 mRNA; WT, 1 ± 0.05873%; cKO, 0.7701 ± 0.114%; unpaired t test; p = 0.1090; H, normalized expression of Pcdhα4 mRNA; WT, 1 ± 0.3823%; cKO, 0.593 ± 0.3689%; unpaired t test; p = 0.4907; I, normalized expression of Pcdhβ13 mRNA; WT, 1 ± 0.07615%; cKO, 0.6268 ± 0.06089%; unpaired t test; p = 0.0155; J, normalized expression of PcdhγA12 mRNA; WT, 1 ± 0.0923%; cKO, 0.6785 ± 0.0057%; unpaired t test; p = 0.0322). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.