Article Figures & Data
Figures
- Figure 1.
H2Bubi is reduced in the aged hippocampus. A, We compared baseline levels of H2Bubi in the dorsal CA1 region of the hippocampus in young adult (3 m.o), middle-aged (12 m.o), and aged (24 m.o.) male rats (N = 6–8 per group). H2Bubi levels were significantly reduced in the hippocampus of aged rats compared with young adult and middle-aged. B, Aged (24 m.o.) male rats were trained to contextual fear conditioning and killed 1 h later. H2Bubi levels in fear-conditioned animals were similar to naive controls (N = 6 per group). *p < 0.05.
- Figure 2.
Upregulation of the H2Bubi ligase Rnf20 in the aged hippocampus improves contextual fear memory. A, Aged (22 m.o) male rats (N = 7–8 per group) were stereotaxically injected with a CRISPR-dCcas9-VPR plus Rnf20 gRNA to upregulate expression of the E3 ligase RNF20 responsible for monoubiquitinating histone H2B. B, During training we saw no significant differences in performance. C, However, rats that received the CRISPR-dCas9–mediated upregulation of Rnf20 showed markedly better memory retention during testing than the control group. *p < 0.05.
- Figure 3.
Upregulation of H2Bubi ligase Rnf20 in aged hippocampus improves the learning-related transcriptome. Using whole genome RNA sequencing, we compared differential gene expression in the CA1 region of the dorsal hippocampus between aged fear-conditioned-control and fear-conditioned Rnf20-gRNA + dCas9-VPR groups relative to control-injected naive animals (N = 5 per group). CA1 tissue was collected 1 h after training. A, Volcano plot of DEGs for the trained-control group relative to naive-control animals. B, Volcano plot of DEGs of the trained Rnf20-gRNA + dCas9-VPR group relative to naive-control animals. C, There were 18 common upregulated DEGs between the control-trained and trained-Rnf20-siRNA groups. However, there were 51 DEGs unique to the trained Rnf20-gRNA + dCas9-VPR, 40 being upregulated and 11 downregulated. D, Pathway analysis identifying differences between trained Rnf20-gRNA + dCas9-VPR and trained-control groups showing the increases in genes in different pathways. E, Heat map displaying the expression levels of DEGs across conditions. Please see Extended Data Figure 3-1 for extended data.
Tables
- Table 1.
Differentially expressed genes in the aged hippocampus following fear conditioning
Rnf20 trained Both Control trained Upregulated Genes Acer2 Ddit4 - Apold1 Dusp1 - Arc Egr1 - Arrdc2 Egr2 - Arrdc3 Fos - Cfap46 Junb - Clec18a Lfng - Col8a1 Nfkbia - Dnah1 Nr4a1 - Dnah11 Olig2 - Dnah6 Per1 - Dusp5 Plekhf1 - Egr4 Prokr2 - Elovl7 Sgk1 - Errfi1 Slc2a1 - Fam43a Tmem119 - Fgf1 Tob2 - Foxj1 Cdkn1a - Fstl1 - - Lama3 - - LOC108352943 - - LOC120093683 - - Map2k3 - - Meis2 - - Msx1 - - Nfil3 - - Nt5e - - Pdk4 - - Plat - - Rgs22 - - Rsrp1 - - Sik1 - - Spag6l - - Spred1 - - Sulf1 - - Tinagl1 - - Tmem212 - - Tsc22d3 - - Ttc21a - - Zmynd10 - - Gpd1 Downregulated Genes Ddit4l - LOC100362987 LOC120097130 - Tbrg4 LOC103690059 - Ryr2 Mex3b - - LOC102551114 - - LOC102547703 - - Spry2 - - Adcy1 - - Erf - - Usp2 - - Kcnj2 - -
Figure 3-1
Upregulation of Rnf20 increases H2B monoubiquitination at LOC120093683 in the aged hippocampus. Using chromatin immunoprecipitation analysis, we compared H2B monoubiquitination (H2Bubi) levels at LOC120093683 in the CA1 region of the dorsal hippocampus between aged fear conditioned-control and fear conditioned Rnf20-gRNA+dCas9-VPR groups relative to control-injected naïve animals (N = 5 per group). CA1 tissue was collected 1 hour after training. Upregulation of Rnf20 increased H2Bubi after training. *P < 0.05 from Naïve. #P = 0.0519 from Control Trained. Download Figure 3-1, TIF file.
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