Fig 1: CDYL protein is downregulated by neuronal activity in vivo. a Illustration of the stimulatory hM3Dq DREADD system. A schematic diagram showed the stimulatory hM3Dq DREADD that is activated by clozapine-N-oxide (CNO). b Western blot analysis of indicated proteins in hippocampus tissues obtained from mice injected vehicle or CNO for 5 days (n = 4 mice in each group). c Quantification of the western blotting in b by normalizing the levels of the indicated proteins to that of ß-actin. n = 4, *p < 0.05, **p < 0.01, paired two-tailed Student’s t-test. Data were represented as mean ± s.e.m. d Immunohistochemistry (IHC) images showing the expression of CDYL protein in DG, CA1, striatum and neocortex area of the rat brain. Scale bar, 5 mm (left), 0.2 mm (right). e Western blotting and quantification showing CDYL protein was decreased upon exposure of rats to enriched enviroment (EE). n = 5, *p < 0.05, paired two-tailed Student’s t-test. f Western blotting and quantification showing intracranial injection of NMDA (10 µM) into rat hippocampus DG area significantly decreased CDYL protein level. n = 4,*p < 0.05, paired two-tailed Student’s t-test. g Western blotting and quantification showing CDYL expression levels were gradually reduced after status epilepticus in rat temporal lobe epilepsy model. n = 3, **p < 0.01, ***p < 0.001, one-way ANOVA with Bonferroni’s multiple-comparisons test. Data marked asterisks in the figure are significantly different from control. h IHC results showing CDYL protein was decreased in the 28 days after status epilepticus in rat temporal lobe epilepsy model (left). Scale bar, 0.1 mm (upper), 0.2 mm (bottom). The reduction of CDYL expression was confirmed by western blotting and quantification (right). n = 3, **p < 0.01, paired two-tailed Student’s t-test. Data were represented as mean ± s.e.m.
Fig 2: Genome-wide identification of CDYL target genes in mouse hippocampus. a Genomic distribution of CDYL binding regions determined by ChIP-seq analysis. b Consensus CDYL binding motif is nearly identical to the REST binding motif. Motif screening was performed using MEME suite. c Classification of the genes identified in ChIP-seq experiments with GO analysis. d Verification of the ChIP-seq results in mice hippocampus tissues by ChIP-qPCR. Results are represented as fold change over GAPDH negative control. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, paired two-tailed Student’s t-test. e, f Quantitative real-time RT-PCR analysis to measure the mRNA levels of indicated genes. Hippocampus tissues were obtained from mice infected with lentiviruses containing CDYL shRNA or nonsilencing shRNA for 14 days (e), or from adult WT or CDYL-TG littermates (f). Total RNAs were prepared and mRNA levels of the indicated genes were examined by real-time RT-PCR. The levels of mRNA were normalized against that of GAPDH. *p < 0.05, **p < 0.01, paired two-tailed Student’s t-test. Data were represented as mean ± s.e.m.
Fig 3: CDYL represses SCN8A transcription through an intronic element. a, b Knockdown of CDYL increased SCN8A expression. Hippocampal DG tissues were obtained from mice (a) or rats (b) infected with lentiviruses containing CDYL-shRNA or nonsilencing-shRNA for 14 days. Western blotting was used for mice experiments (a). For rats (b), the mRNA and protein levels of SCN8A and CDYL were examined by real-time RT PCR (left) and western blot analysis (middle) and quantified (right). n = 3, *p < 0.05, paired two-tailed Student’s t-test. c SCN8A is downregulated in transgenic mice over-expressing CDYL. Hippocampus tissues were obtained from adult WT or CDYL-TG littermates. Western blot analysis and quantification were performed. n = 4,*p < 0.05, **p < 0.01, paired two-tailed Student’s t-test. d Mouse hippocampus tissues were obtained from WT and CDYL-TG littermates. ChIP experiments were performed using the indicated antibodies. Real-time PCR assays were performed for the measurement. n = 3, *p < 0.05, paired two-tailed Student’s t-test. e Somatic knockout of CDYL using CRISPR/Cas9 technology. Western blotting and RT-PCR assays were performed to confirm the elimination of CDYL in SH-SY5Y cells. Puromycin-resistant single cell-derived colonies were analyzed by western blotting and clone #1 and #6 were used for the subsequent functional studies. **p < 0.01, paired two-tailed Student’s t-test. f CDYL binds to a regulatory element within SCN8A intron. qChIP assays were performed in SH-SY5Y cells with primer pairs specific to indicated regions (left). Normal rabbit IgG or antibody specific to CDYL was used to immunoprecipitate the protein-DNA complex (middle). Conventional semi-quantitative ChIP assays performed at the indicated regions (right). **p < 0.01, paired two-tailed Student’s t-test. g Depletion of CDYL resulted in decreased regional enrichment of H3K27me3 and increased regional enrichment of H3K27ac. CDYL-KO SH-SY5Y cell lysates were collected, and ChIP-qPCR experiments were performed using the indicated antibodies. n = 3, *p < 0.05, **p < 0.01, paired two-tailed Student’s t-test. h CDYL interacts with REST in vivo. Whole cell lysates from SH-SY5Y cells were prepared, and immunoprecipitated (IP) was performed with anti-CDYL followed by immunoblotted with antibodies against the indicated proteins. i Knockdown of REST or EZH2 in SY5Y cells increased SCN8A expression. The protein and mRNA levels were examined by Western blot analysis and real-time RT PCR. *p < 0.05, **p < 0.01, paired two-tailed Student’s t-test. Data were represented as mean ± s.e.m.
Fig 4: CDYL inhibits seizure susceptibility and latent period duration in temporal lobe epilepsy. a Examples of in vivo EEG recordings obtained from CDYL-shRNA-infected and nonsilencing-shRNA-infected rats. b Graph depicting the time taken to reach status epilepticus after kainic acid (KA) administration from CDYL-shRNA- infected and nonsilencing-shRNA-infected rats. n = 5, **p < 0.01, paired two-tailed Student’s t-test. c Graph depicting the seizure progression in CDYL-shRNA-infected and nonsilencing- shRNA-infected rats, illustrated as mean maximum seizure class reached by 15, 30, 45, 60, 75, 90 min after KA administration. *p < 0.05, unpaired two-tailed Student’s t-test. d Incidence of maximum seizure class reached during the course of the experiments in (c). e Graph depicting the seizure progression in WT and TG mice, illustrated as mean maximum seizure class reached by 15, 30, 45, 60, 75, 90 min after KA administration. *p < 0.05, paired two-tailed Student’s t-test. f Incidence of maximum seizure class reached during the course of the experiments in (e). g Graph showing time taken to reach first spontaneous seizure after administration of KA from CDYL-shRNA-infected and nonsilencing-shRNA- infected rats. n = 5, **p < 0.01, paired two-tailed Student’s t-test. h Graph depicting the seizure progression in shRNA-Control, shRNA-CDYL, and shRNA-CDYL plus shRNA-SCN8A infected mice, illustrated as mean maximum seizure class reached by 15, 30, 45, 60, 75, 90 min after KA administration. n = 12, *p < 0.05, one-way ANOVA with Bonferroni’s multiple-comparison test. i Incidence of maximum seizure class reached during the course of the experiments in (h). j Western blot analysis of SCN8A and CDYL in the hippocampus from control individuals or individuals with TLE. n = 10. k Quantification of the results in H by normalizing the protein levels of SCN8A and CDYL to that of ß-actin in control and TLE groups (n = 10). *p < 0.05, paired two-tailed Student’s t-test. Data were represented as mean ± s.e.m.
Fig 5: Over expression of CDYL in vivo significantly reduced neuronal intrinsic excitability. a Western blotting and real-time RT PCR analysis showed CDYL protein and mRNA levels of the progeny from two founders (TG1: #8 and TG2: #24) of transgenic mice over-expressing CDYL. Western blot of FLAG and CDYL in the hippocampus from adult wild-type (WT) and TG littermates (left). Quantification of CDYL mRNA levels in hippocampus obtained from WT and TG littermates (right). n = 3, **p < 0.01, paired two-tailed Student’s t-test. Because of the similar CDYL expression levels in these transgenic mice, which also behaves similarly (Supplemental Fig. 4a–c), we generally used “TG” to represent transgenic mice from both founders in the following functional experiments for simplicity. b Representative traces obtained from DG neurons from WT and TG mice in response to a series of 400 ms current stepping from -200 to +400 pA with increments of 50 pA. For comparison reasons, the recordings were obtained at the fixed potential of -80 mV. Graphs demonstrate average numbers of action potentials obtained in response to varying depolarizing current pulses when the soma was held at -80 mV. *p < 0.05, one-way ANOVA with Bonferroni’s multiple-comparison test. c Representative traces of DG neurons from WT and TG mice in response to 300 pA positive current injection (left). The avaraged delay of first action potential initiation in WT and TG neurons (right). n = 25, *p < 0.05, unpaired two-tailed Student’s t-test. d Typical spikes (left), associated phase plane plots (middle), and the average spike threshold (right) obtained from DG neurons of WT and TG mice. *p < 0.05, unpaired two-tailed Student’s t-test. Data were represented as mean ± s.e.m.
Supplier Page from MilliporeSigma for Anti-CDYL antibody produced in rabbit