Mono-methylation of lysine 27 at histone 3 confers lifelong susceptibility to stress

Abstract

Histone post-translational modifications are critical for mediating persistent alterations in gene expression. By combining unbiased proteomics profiling and genome-wide approaches, we uncovered a role for mono-methylation of lysine 27 at histone H3 (H3K27me1) in the enduring effects of stress. Specifically, mice susceptible to early life stress (ELS) or chronic social defeat stress (CSDS) displayed increased H3K27me1 enrichment in the nucleus accumbens (NAc), a key brain-reward region. Stress-induced H3K27me1 accumulation occurred at genes that control neuronal excitability and was mediated by the VEFS domain of SUZ12, a core subunit of the polycomb repressive complex-2, which controls H3K27 methylation patterns. Viral VEFS expression changed the transcriptional profile of the NAc, led to social, emotional, and cognitive abnormalities, and altered excitability and synaptic transmission of NAc D1-medium spiny neurons. Together, we describe a novel function of H3K27me1 in the brain and demonstrate its role as a "chromatin scar" that mediates lifelong stress susceptibility.

Keywords: H3K27me1; SUZ12; depression; early life stress; histone modifications; medium spiny neurons; nucleus accumbens; polycomb repressive complex 2.

Figure 1.. H3K27 Dynamic Methylation in NAc following CSDS.

(A) Schematic and timeline of CSDS experiment. Susceptible (SUS), control (CON) and resilient (RES) mice. (B) Interaction ratio: One-way ANOVA: F_(2,13)=17.32, p=0.0002. Tukey’s test: ***p<0.001 (n=5-6 mice/group). (C) Heatmap for relative abundance of H3.3K27 modifications. Arrows indicate significance (yellow, increase: ^††p<0.0001; blue, decrease: ^†p<0.001). (D) H3.3K27me1: One-way ANOVA: F_(2,13)=65.21; p<0.0001. Tukey’s test: ****p<0.0001. (E) H3.3K27me1 and interaction ratio negative correlation. (F) H3.3K27me2: One-way ANOVA: F_(2,13)=14.15, p=0.0005. Tukey’s test: Different from CON (**p<0.01) and RES (***p<0.001). (G) H3.3K27me2 and interaction ratio positive correlation. (H) H3K27me1 intensity in MSNs (n=3-4 bilateral sections; 4 mice/group). Left: Coronal section showing H3K27me1 (magenta) in Drd1- (cyan) and Drd2- (yellow) MSNs. Right: Two-way ANOVA: Group: F_(1,12)=17.87; p=0.0012. Sidak’s Group effect: **Different from CON in Drd1-MSNs, p<0.0036. (I) H3K27me2 intensity in MSNs. Left: Coronal section showing H3K27me2 (red) in Drd1- and Drd2-MSNs. Right: Two-way ANOVA: Group: F_(1,12)=4.07; p=0.07; Cell-type: F_(1,12)=0.156; p=0.69, or interaction: F_(1,12)=0.15; p=0.705. Scale bar: 20μm. Data: Mean ± SEM.

Figure 2.. Genome-wide Deposition of H3K27me1 and H3K27me2 in NAc under Control and CSDS Conditions.

(A) Schematic of CUT&RUN-seq experiment (n=4 samples, 4 mice/group pooled tissue). Antibodies validation in Figure S2. (B) Interaction ratio of CON, SUS and RES mice (n=16/group). One-way ANOVA: F_(2,45)=39.49; p<0.0001. Tukey’s test: ****p<0.0001. (C) Number of H3K27me1 and H3K27me2 differentially enriched (yellow) of depleted (blue) peaks in SUS vs CON, RES vs CON and SUS vs RES. (D) Enrichment heatmap for H3K27me1 differential peaks within ±1kb around the start (SS) and end (ES) sites in SUS vs CON. The blue-to-red gradient indicates low-to-high counts within specified regions. Heatmap for H3K27me2 in Figure S2H. (E) Genomic distribution of H3K27me1 differential peaks in SUS vs CON. (F). Gene ontology (GO) molecular functions of H3K27me1-enriched genes. (G) Integrative Genomics Viewer (IGV) tracks of ΔH3K27me1-enriched sites across introns 4, 5, 6 and 9 of the Gabrb2 gene in CON, SUS and RES. (H) Gabrb2 mRNA: One-way ANOVA: F_(2,20)=4.46, p<0.05. Tukey’s test: *p<0.05. Gabrg1 mRNA: One-way ANOVA: F_(2,20)= 3.7, p<0.05. Tukey’s test: *p<0.05. (I) GO molecular functions of H3K27me1-depleted genes. (J) IGV track of ΔH3K27me1-depleted sites within introns 1 and 9 of the Kdm4b gene. (K) Volcano plot of differential peaks in SUS vs RES. (L) H3K27me1 differential peaks distribution in SUS vs RES. (M) GO molecular functions of H3K27me1-enriched genes in SUS vs RES. (N) GO molecular functions of H3K27me1-depleted genes in SUS vs RES. Data: Mean ± SEM.

Figure 3.. VEFS Domain of SUZ12 Induces H3K27me1 in NAc D1-MSNs.

(A) SUZ12: One-Way ANOVA: F_(2,12)=3.62; p<0.05. Tukey’s test: *p<0.05. (B) EZH2: One-Way ANOVA: F_(2,15)=0.18; p=0.83. (C) EED: One-Way ANOVA: F_(2,14)=1.28; p=0.3. One empty well was left between samples (n=5-6/group). (D) Left: Schematic of the SUZ12 protein (yellow). Right: SUZ12 colored to highlight its interacting (VEFS, blue) and localization (ΔVEFS) domains. The VEFS domain determines SUZ12 and EZH2 interaction. Modified with permission from Højfeldt, et al. (2019). Copyright Elsevier. (E) Schematic of VEFS expression viral construct. (F) Timeline of viral infection. (G) Amplification of VEFS or ΔVEFS domains (n=4/group). VEFS: t₍₄₎=3.95, p<0.05; ΔVEFS: t₍₆₎=0.57, p=0.58. (H) H3K27me1 expression in AAV-GFP-injected (top) or AAV-VEFS-injected (bottom) NAc D1-MSNs. Scale bars: 20μm. (I) Percentage of H3K27me1 fluorescence intensity in GFP-positive and GFP-negative neurons. Two-way ANOVA: Virus: F_(1,16)=6.43; p<0.05; Cell-type: F_(1,16)=4.52; p<0.05; Virus by Cell-type: F_(1,16)=4.52; p<0.05. Tukey’s test: *Different from AAV-GFP in GFP+ cells, p<0.05. (J) H3K27me1 abundance in AAV-VEFS: t₍₈₎=2.57; p<0.05 (n=5/group). (K) Enrichment heatmap for H3K27me1 differential peaks within ±1kb around the start (SS) and end (ES) sites in AAV-VEFS vs AAV-GFP (n=6 samples, 2 mice/group pooled tissue). The blue-to-red gradient indicates low-to-high counts within annotated regions. (L) Number of enriched (yellow) or depleted (blue) H3K27me1 peaks in AAV-VEFS vs AAV-GFP. (M) H3K27me1 differential peaks distribution in AAV-VEFS vs AAV-GFP. (N). GO molecular functions of H3K27me1-depleted genes. (O) IGV track of ΔH3K27me1 peaks across intron 8 of the Gabra4 gene. Data: Mean ± SEM.

Figure 4.. VEFS Domain of SUZ12 in NAc D1-MSNs Induces Susceptibility to Sub-Threshold Social Defeat Stress (SbD).

(A) Timeline for viral infection and SbD exposure. (B) Social interaction time. Three-way ANOVA: Virus: F_(1,68)=10.17; p<0.01; Session by Virus: F_(1,68)= 4.226; p<0.05. Tukey’s test: AAV-VEFS-SbD different from AAV-GFP-CON and AAV-GFP-SbD, *p<0.05 (C) Time in corners. Two-way ANOVA: Virus: F_(1,36)=7.22; p=0.01; Group: F_(1,36)=4.093; p<0.05. Sidak’s Virus effect: AAV-VEFS-SbD different from AAV-GFP-SbD, *p<0.05. (D) Percentage of SUS and RES mice per group. (E) Social interaction toward conspecifics. Three-way ANOVA: Social target (ST): F_(1,46)=81.89; p<0.0001; ST by Group: F_(1,22)=13.34; p<0.01, ST by Group by Virus: F_(1,22)=4.57; p<0.05. Tukey’s test: ST different from empty cage (EC) only in AAV-GFP-CON, AAV-VEFS-CON and AAV-GFP-SbD, ****p<0.0001. (F) Time in center. Two-way ANOVA: Virus: F_(1,36)=7.63; p=0.009; Group: F_(1,36)=4.091; p<0.05. Sidak’s Virus effect: AAV-VEFS-SbD different from AAV-GFP-CON, *p<0.05. (G-H) Acquisition of the reversal learning task (RLT). (G) Percentage of correct lever presses: Three-way ANOVA: Session: F_(2,111)=18.32; p<0.0001. Sidak’s Session effect: D6 different from D4, *p<0.05. (H) Sessions to learning: Two-way ANOVA: Non-significant Virus effect: F_(1,35)=0.033; p=0.85; Group: F_(1,35)=0.51; p=0.47, or interactions. (I-J) Reversal phase of the RLT. (I) Percentage of correct lever presses: Three-way ANOVA: Session: F_(4,180)=64.77; p<0.0001; Virus: F_(1,180)=51.36; p<0.0001; Session by Virus: F_(4,180)=3.084; p<0.05, Group by Virus: F_(1,180)=13.64; p<0.001. Tukey’s test: AAV-GFP-SbD different from AAV-GFP-CON in reversal day 4 (R4), *p<0.05. AAV-VEFS-CON different from AAV-GFP-SbD in R3 and R4, **p<0.01; AAV-VEFS-SbD different from AAV-GFP-CON in R5, *p<0.05 and AAV-VEFS-SbD different from AAV-GFP-SbD, during R3, R4 and R5, ****p<0.0001. (J) Sessions to reversal learning: Two-way ANOVA: Virus: F_(1,35)=8.16; p<0.01; Virus by Group: F_(1,35)=5.88; p<0.05. Tukey’s test: AAV-VEFS-SbD different from AAV-GFP-SbD, *p<0.01. Data: Mean ± SEM.

Figure 5.. VEFS Domain of SUZ12 Alters NAc Transcriptional Signatures.

(A) Volcano plot showing differentially expressed genes (DEGs) upon VEFS expression compared to GFP (DEG criteria: Log₂(Fold Change) >∣0.20∣, and p<0.05). Additional comparisons in Figure S10A-D (n=8 mice/group/virus). (B) GO molecular functions in VEFS-injected mice. (C-D) Rank-rank hypergeometric overlap (RRHO) plots showing coordinated transcription of H3K27me1-enriched genes (ΔH3K27me1-genes) between AAV-GFP-SbD vs AAV-GFP-CON and AAV-VEFS-SbD vs AAV-VEFS-CON (C) or between AAV-VEFS-CON vs AAV-GFP-CON and AAV-VEFS-SbD vs AAV-GFP-SbD (D). Arrows within quadrants indicate directionality of transcriptional overlap, and heat denotes strength of the overlap. ΔH3K27me1-genes selected according to the SUS vs CON comparison in Figure 2. (E) Union heatmap of differentially expressed ΔH3K27me1-genes in AAV-VEFS-SbD vs AAV-GFP-CON (bottom-row), ranked from lowest (blue) to highest (yellow) across comparisons. ΔH3K27me1-genes selected from the overlap shown in Figure S11C. Differential criteria: Log₂(Fold Change) >∣0.20∣, and p<0.05). (F) Heatmap of H3K27me1-enriched genes involved in neuronal excitability and synaptic transmission in AAV-GFP-SbD and AAV-VEFS-SbD compared to AAV-GFP-CON. *p<0.05, †p<0.01, pairwise comparisons.

Figure 6.. VEFS Domain of SUZ12 Alters Neuronal Excitability and Synaptic Transmission in NAc D1-MSNs.

(A) Schematic of electrophysiology recordings in D1-MSNs from AAV-GFP- and AAV-VEFS-injected mice (n=41-46 neurons/6–8 mice/virus). (B) Current-evoked spikes. Two-way ANOVA: Virus: F_(1,85)=6.86; p<0.01, Current: F_(30,2550)=127.4; p<0.0001, Virus by Current: F_(30,2550)=1.60; p=0.02. Sidak’s Virus effect: *Different from AAV-GFP; p<0.05. (C) Number of spikes at 200 pA current: t₍₈₅₎= 2.713; p<0.01. (D) Representatives of voltage traces. (E) Threshold potential: t₍₈₅₎=2.95; p<0.01. (F) Resting membrane potential: t₍₈₅₎=0.52; p=0.59. (G) Percentage of D1-MSNs exhibiting spontaneous firing. (H) Miniature inhibitory postsynaptic currents (mIPSCs) traces. (I) Cumulative fraction of mIPSC amplitude. Inset: Cells average: t₍₃₆₎=2.059; p<0.05. (J) Cumulative fraction of mIPSC interevent intervals (IEIs). Inset: Cells average: t₍₃₆₎=2.59; p<0.05. (K) Miniature excitatory postsynaptic currents (mEPSCs) traces. (L) Cumulative fraction of mEPSC amplitude. Inset: Cells average: t₍₄₉₎=1.602; p=0.11. (M) Cumulative fraction of mEPSC IEIs. Inset: Cells average: t₍₄₅₎=3.48; p<0.01. Data: Mean ± SEM.

Figure 7.. Persistent H3K27me1 Increase in the NAc after ELS.

(A) Timeline of the ELS experiment. (B) H3K27me1 relative abundance. Two-way ANOVA: ELS: F_(1,8)=59.11; p<0.0001; Age: F_(1,8)=21.7; p<0.01; ELS by Age: F_(1,8)=9.349; p<0.05. Tukey’s test: ELS different from Std at PND21 (*p<0.05) and Adult (***p<0.001). Std in Adult different from Std at PND21, **p<0.01. (C) H3K27me2 relative abundance. Two-way ANOVA: ELS: F_(1,8)=7.675; p<0.05. Sidak’s ELS effect: ELS different from Std group at PND21, *p<0.05. (D) “Double-hit” paradigm: pups exposed to ELS and SbD in adulthood (n=8/group). (E) Social interaction. Two-way ANOVA: ELS: F_(1,28)=10.19, p<0.01; SbD: F_(1,28)=8.78, p<0.01; ELS by SbD: F_(1,28)=1.94, p=0.17. Sidak’s ELS effect: ELS-SbD different from Std-CON (***p<0.001), and ELS-CON (*p<0.05). (F) Number of differential enriched (yellow) of depleted (blue) H3K27me1 peaks in ELS-CON, Std-SbD, and ELS-SbD relative to Std-CON. (G) Enrichment heatmap for H3K27me1 differential peaks within ±1kb around the start (SS) and end (ES) sites in ELS-SbD vs Std-CON. Gradient blue-to-red color indicates low-to-high counts. (H) Average H3K27me1 density within differential peaks and their ±1kb flanking regions. (I) Volcano plot of ELS-CON vs ELS-Std differential peaks. (J) Volcano plot of ELS-CON vs ELS-Std differential peaks. (K) GO molecular functions in H3K27me1-enriched genes in ELS-SbD vs Std-CON. (L) IGV tracks of ΔH3K27me1 peaks across introns 1 and 3 of the Kcnn2 gene. (M) Enrichment heatmap for H3K27me1 in ELS-CON and ELS-SbD comparisons within H3K27me1 differential peaks in SUS vs CON. (N) Venn diagram showing H3K27me1 peak overlap in SUS vs CON and ELS-SbD vs Std-CON comparisons. (O) GO molecular functions of shared H3K27me1-enriched genes across stress models. Data: Mean ± SEM.