Epigenetic dysregulation of Oxtr in Tet1-deficient mice has implications for neuropsychiatric disorders

Abstract

OXTR modulates a variety of behaviors in mammals, including social memory and recognition. Genetic and epigenetic dysregulation of OXTR has been suggested to be implicated in neuropsychiatric disorders, including autism spectrum disorder (ASD). While the involvement of DNA methylation is suggested, the mechanism underlying epigenetic regulation of OXTR is largely unknown. This has hampered the experimental design and interpretation of the results of epigenetic studies of OXTR in neuropsychiatric disorders. From the generation and characterization of a new line of Tet1 mutant mice - by deleting the largest coding exon 4 (Tet1Δe4) - we discovered for the first time to our knowledge that Oxtr has an array of mRNA isoforms and a complex transcriptional regulation. Select isoforms of Oxtr are significantly reduced in the brain of Tet1Δe4-/- mice. Accordingly, CpG islands of Oxtr are hypermethylated during early development and persist into adulthood. Consistent with the reduced express of OXTR, Tet1Δe4-/- mice display impaired maternal care, social behavior, and synaptic responses to oxytocin stimulation. Our findings elucidate a mechanism mediated by TET1 protein in regulating Oxtr expression by preventing DNA hypermethylation of Oxtr. The discovery of epigenetic dysregulation of Oxtr in TET1-deficient mouse brain supports the necessity of a reassessment of existing findings and a value of future studies of OXTR in neuropsychiatric disorders.

Keywords: Epigenetics; Genetics; Neuroscience; Psychiatric diseases.

Figure 1. Generation and characterization of Tet1 mutant (Tet1^Δe4) ESCs and mice.

(A) Gene targeting strategy for generating Tet1^e4f and Tet1^Δe4 mice. LoxP sites (red arrowheads), FRT sites (blue diamonds), and primers for RT-PCR (blue arrows) and genotyping (green arrows) are indicated. Mutations of other published Tet1 mutant mice are diagramed (Δe11-13, Δe5, Gene-trap; refs. –44). (B) Left panel, DNA Southern blot confirmation of Tet1^e4f (+/f) embryonic stem cells (ESCs). Right panel, RT-PCR of Tet1^Δe4–/– hippocampus. The primer pair of CR1 and CF1(246 bp) amplified the WT mRNA, and the primer pair of CR1 and CF2 (182 bp) detected the exon 4–deleted mutant mRNA. (C) Western blot analysis with a TET1 antibody confirmed deficiency of TET1 protein from Tet1^Δe4–/– (–/–) hippocampus. β-Tubulin used as loading control and fill uncut gel is shown. (D) Generation of homozygous Tet1^Δe4–/– ESCs through sequential targeting. The upper right panel shows the Southern blot confirmation of double-targeted ESCs (f/f), and the lower right panel shows PCR genotyping confirmation of exon4 deletion (–/–) after electroporation with Cre plasmid (Supplemental Figure 1). (E) Tet2 but not Tet3 was upregulated in Tet1^Δe4–/– ESCs. (**P < 0.005, 2-tailed t test, n = 3–5 for +/+ and –/–). (F) Tet2 and Tet3 were not differentially expressed in Tet1^Δe4–/– hippocampus of adult age. (G) Tet1^Δe4–/– mice have reduced weight at weaning (3w) and adulthood (n = 4–12/group at weaning and n = 8–19/group at adult age). *P < 0.05; **P < 0.005, ***P < 0.0005, 2-tailed t test. All data are presented as mean ± SEM.

Figure 2. Tet1 deficiency results in significantly reduced expression of key neural genes, including Npas4 and Oxtr, in brain.

(A) A total of 186 genes were downregulated and 34 were upregulated in Tet1^Δe4–/– hippocampus from RNA-seq analysis using FDR < 0.05 (n = 3/group). (B) Overall, the dysregulated genes in Tet1^Δe4–/– hippocampus did not have a significant overlap with known neuronal activity–regulated genes reported in literate (533 genes). Dysregulated activity–dependent genes such as Npas4 were identified from this analysis. (C) Npas4 was downregulated in hippocampus from Tet1^Δe4–/– mice (n = 6/group; *P = 0.017, 2-tailed t test). (D) Gene Ontology (cellular component classification) analysis using DAVID revealed an enrichment of extracellular dysregulated genes in Tet1^Δe4–/– mice (Benjamini corrected P values indicated). (E) Oxtr was downregulated in hippocampus from Tet1^Δe4–/– mice but not in Tet1^Δe4–/– ESCs. Oxtr_ABEFG primers used. (**P < 0.005, 2-tailed t test; n = 5–6 for each genotype). (F) Significant overlap of ECS-treated Tet1^Δe4–/– hippocampal dysregulated genes with dysregulated genes from neural progenitor cells (NPCs) of Tet1^Δe11–13 (odds ratio [OR] = 7.66, P = 0.003) and untreated hippocampus of Tet1^Δe5 mice (OR = 39.07, P = 2.3 × 10^–57). All data are presented as mean ± SEM.

Figure 3. Tet1^Δe4–/– mice show hypermethylation of the Npas4 and Oxtr CpG islands during early development and complex transcriptional dysregulation of Oxtr.

(A) Diagram of Npas4 promoter (coding regions are shaded), associated CpG island (green bar), and bisulfite-sequencing region (black bar). Npas4 was hypermethylated in hippocampus of Tet1^Δe4–/– mice (n = 3/group; P = 0.004, 2-tailed t test). Blue squares represent unmethylated CpG dinucleotides, red squares represent methylated CpGs, and white squares were undetermined due to the ambivalent sequence reads and the same for other figures. (B) Diagram of Oxtr gene structure (coding regions are shaded), CpG island (green bar), and bisulfite-sequencing regions (BS, black bars). The genomic coordinate of BS1–BS3 in mouse mm9 assembly are as follows: BS1, Chr6:112440814-112441327; BS2, Chr6:112440387-112440815; BS3, Chr6: 112439019-112439542. The human CpG island spans 2319 bp (hg19:Chr3:8808962-8811280) extending to the more 5′ region of OXTR as indicated by a dotted green line. Mouse CpG island is 859 bp (mm9:Chr6:112439019-112439877). Human promoter MT2 region (9) and the region harboring the CG site that is likely to be equivalent to the human –934 CG site (14) are indicated as arrow. BS2 and BS3 were hypermethylated but not BS1 in hippocampus of adult Tet1^Δe4–/– mice (n = 3/group; BS2, P = 0.0015; BS3, P = 0.0000015; 2-tailed t test). BS3 showed intermediate levels of hypermethylation in Tet1^Δe4+/– mice (n = 3–4/group; P = 0.0019, 2-tailed t test). (C) Quantification of DNA methylation of BS1, BS2, and BS3 in hippocampus of Tet1^+/+ and Tet1^Δe4–/–. (D) The hMDeIP shows the comparable level of 5hmC in BS3 between Tet1^+/+ and Tet1^Δe4–/– (n = 5/group; **P < 0.005; ***P < 0.0005; 2-tailed t test).(E) Oxtr BS3 was not hypermethylated in Tet1^Δe4–/– ESCs. (F) Oxtr BS3 was hypermethylated in cerebellum (CB), cortex (CX), and olfactory bulb (OB) of adult Tet1^Δe4–/– adult mice. (G) Oxtr BS3 was hypermethylated in E14.5 cerebellum of Tet1^Δe4–/– mice. (H) Oxtr BS3 was hypermethylated in tissues of heart and lung from the other 2 germ layers (Meso, mesoderm; Endo, endoderm). (I) Whole-genome bisulfite sequencing of neocortex from adult brain of Tet1^Δe4–/– mice revealed Tet1-DMRs are significantly enriched in intragenic CpG islands (CGI) (n = 3 for +/+ and –/–; P = 2.65 × 10^–38, Fisher’s exact test).

Figure 4. Identification of Oxtr mRNA isoforms and reduced expression of Oxtr isoforms in Tet1^Δe4–/– mice.

(A) Oxtr mRNA isoforms (A–H) identified by 5′ RACE and confirmed by RT-PCR and Sanger sequencing (predicted coding regions are shaded). qPCR primers indicated (multicolored arrows and supplemental method). The new sequences for individual isoforms have been deposited in GeneBank with accession no. KU686795-KU686801. (B) qPCR data revealed downregulation of isoform B, but not A or H, in the hippocampus of Tet1^Δe4–/– mice (n = 3–4/group, *P < 0.05, 2-tailed t test). (C) The histone modification of Oxtr in mouse brain. The enrichment of histone H3K4me3 and H3K27me3 in BS1–BS3 regions of Oxtr was revealed from ENCODE project. A second putative regulatory element was identified within exon 3 (large black bar) of Oxtr, which overlaps a CpG island (green bar) (mm9, http://genome.ucsc.edu/). Selection of Oxtr regions for bisulfite sequencing (BS1–BS3) and ChIP-PCR are indicated. ENCODE ChIP data shown is from E14.5 whole brain produced in the laboratory of Bing Ren (H3K4me3, GEO GSM1000095; H3K27me3, GEO GSM1000143). The similar pattern is also observed in ChIP-seq from 8-week cerebellum (http://genome.ucsc.edu) (Supplemental Figure 6). (D) The enrichment of H3K4me3, H3K27me3, and H3K4me1 in DMRs of Tet1^Δe4–/– mice. The hypermethylated DMRs (200 bp bin) in Tet1^Δe4–/– cortex was selected firstly by Fisher test with a threshold of P < 0.05. The 2000 bp genomic windows containing at least 4 hypermethylated DMRs (200 bp) in Tet1^Δe4–/– cortex were selected out as DMRs for histone enrichment analysis. Random control regions with same bin size covered by the same methylome were chosen as controls. The ENCODE ChIP-seq data from WT adult mouse neocortex was used for the analysis. P values (calculated by t test) indicate a significant differential histone enrichment between DMRs and control region. (E–G) ChIP-qPCR revealed altered histone modifications at the bivalent promoter region of and GARDP control (E) and Oxtr (F and G) in the cerebrum of Tet1^Δe4–/– mice. H3K4me3 (active mark), H3K27me3 (repressive mark), and IgG (isotype negative control) were assessed in 2 regions overlapping coding exon 3 and exon 1 in the 5′ UTR. Both H3K4me4 and H3K27me3 were reduced in BS3 hypermethylated region of Tet1^Δe4–/– mice (n = 2/group and 3 replicates for each group).

Figure 5. Tet1^Δe4–/– mice are hypoactive and display impaired social and maternal care behaviors.

(A) Tet1^Δe4–/– mice were hypoactive in the open-field exploration as indicated by a reduced distance traveled (P = 0.0005, 2-tailed t test) and reduced center time (P = 0.04, 2-tailed t test). n = 21 (–/–) and 30(+/+). (B) Tet1^Δe4–/– female mice display significantly increased stationary reactivity (P = 0.017, 2-tailed t test) and threatening postures in the resident intruder test (P = 0.001, 2-tailed t test). Males show a significant increase in threatening postures (P = 0.02, 2-tailed t test). n =13 (male–/–) and 8 (female–/–); n = 17 (male +/+) and 13 (female+/+). (C) Tet1^Δe4–/– mice showed delay in pup retrieval (genotype x day: F[2,30] = 7.244, P = 0.003, Tukey’s multiple comparison’s test: Tet1^Δe4–/– day 1 vs. day 3, P = 0.222; Tet1^+/+ day 1 vs. day 3, P = 0.026) (left panel), and reduced overall crouching time (effect of genotype: F[1,15] = 5.357, P = 0.035) (middle panel). Both genotypes show an increase in crouching over time (effect of day: F[2,30] = 3.534, P = 0.042); however, post-hoc analysis were not significant for any group. n = 7 (–/–) and 10 (+/+). Tet1^Δe4–/– mice showed reduced overall crouching time (effect of genotype: F(1,15) = 5.357, P = 0.035) (middle panel). Tet1^Δe4–/– mice showed increased aggressive interactions in the first 15 minutes of the first day of virgin pup retrieval (P = 0.008, 2-tailed t test) (right panel) RMANOVA with Tukey’s post-hoc correction. (D) Tet1^Δe4–/– mice showed a trend to reduced preference in training, short-term, and long-term memory in the object exploration (effect of genotype: F[1,48] = 3.877, P = 0.055; effect of time: F[2,96] = 0.035, P = 0.053). Train, training phase; STM, short-term memory; LTM, long-term memory. n = 21 (–/–) and 29(+/+). RMANOVA.

Figure 6. Tet1^Δe4–/– mice display impaired response to OXTR agonist stimulation but normal synaptic plasticity in the hippocampus.

(A) The frequency of spontaneous inhibitory current (sIPSC) at baseline and after TGOT stimulation in Tet1^+/+ (n = 8 cells) and Tet1^Δe4–/– (n = 10 cells) mice. The mean frequency at baseline for Tet1^Δe4–/– (4.16 ± 0.67) was borderline lower than that of Tet1^+/+ (2.47 ± 0.48) (P = 0.051, 2-tailed t test). Both Tet1^e4+/+ and Tet1^Δe4–/– cells showed the significant increased frequency in response to TGOT stimulation (Wilcoxon signed ranks test; P = 0.008 for +/+ and P = 0.01 for –/–). (B) The amplitude of spontaneous inhibitory current (sIPSC) at baseline and after TGOT stimulation in Tet1^e4+/+ (n = 8 cells) and Tet1^Δe4–/– (n = 10 cells) mice. The amplitude of sIPSC at baseline for Tet1^Δe4–/– neurons (26.56 ± 6.78) was comparable with that of Tet1^+/+ mice (20.36 ± 2.19). Tet1^+/+ but not Tet1^Δe4–/– cells showed the significant increased amplitude in response to TGOT stimulation (Wilcoxon signed ranks test; P = 0.006 for +/+ and P = 0.65 for –/–). (C) Baseline synaptic transmission not different in hippocampal CA1 of Tet1^Δe4–/– mice. (n = 8 [–/–] slices from 5 mice; n = 11 [+/+] slices from 6 mice of 6–8 weeks old). (D) Paired pulse facilitation (PPF) not different in hippocampal CA1 of Tet1^Δe4–/– mice indicating normal presynaptic function. (n = 8 [–/–] slices from 5 mice; n = 11 [+/+] slices from 6 mice of 6–8 weeks old). (E) Fiber volley not different in hippocampal CA1 of Tet1^Δe4–/– mice indicating normal presynaptic function (n = 8 [–/–] slices from 5 mice; n = 11 [+/+] slices from 6 mice of 6–8 weeks old). (F) LTP in CA1 of Tet1^Δe4–/– was not different from Tet1^+/+ (+/+, 11 slices from 6 mice; LTP, 156% ± 6 %; –/–, 8 slice from 5 mice; LTP, 153% ± 12 %; 2-tailed t test). Arrow indicates the time of stimulation (HFS, 100 Hz, 1 second).