Fig. 3: Kdm4a regulates Trpm7 expression via RNA Pol II pausing at exonal region.
From: Kdm4a is an activity downregulated barrier to generate engrams for memory separation
a Differentially expressed genes were selected according to the conditions of p < 0.05 and |log2(fold change)| > 1. Two-sided test, Benjamini-Hochberg multiple test correction. b Knockdown of Kdm4a in DG GCs resulted in upregulation of the transcription level of the cation channel Trpm7. shCtrl n = 3, shKdm4a n = 3. Two-tailed unpaired t-test, Trpm7: t4 = 3.299, * p = 0.0300; Kdm4a: t4 = 3.055, * p = 0.0379; EYFP: t4 = 0.3798, p = 0.7234. Data are presented as mean ± s.e.m. c Knockdown of Kdm4a in DG GCs increased the level of histone H3K36me3 modification. shKdm4a n = 3, shCtrl n = 3. Two-tailed unpaired t-test, H3K9me3: t4 = 0.6265, p = 0.5649; H3K36me3: t4 = 3.473, * p = 0.0255. Data are presented as mean ± s.e.m. d (Left) Knockdown of Kdm4a resulted in a significant increase in the level of histone H3K36me3 modification on the Trpm7 exonic regions. shKdm4a n = 3, shCtrl n = 3. Two-way ANOVA followed by Sidak test, F1,4 = 16.0, * p = 0.0161. (Right) Knockdown of Kdm4a did not significantly increase the level of histone H3K36me3 modification in the Trpm7 intronic regions. shKdm4a n = 3, shCtrl n = 3. Two-way ANOVA followed by Sidak test, F1,4 = 1.367, p = 0.3072. e (Left) Knockdown of Kdm4a did not affect the level of histone H3K9me3 modification on the Trpm7 gene loci. shKdm4a n = 3, shCtrl n = 3. Two-way ANOVA followed by Sidak test, F1,4 = 0.2911, p = 0.6182. Data are presented as mean. (Right) Knockdown of Kdm4a leads to an increase in the level of histone H3K36me3 modification on the Trpm7 gene. shKdm4a n = 3, shCtrl n = 3. Two-way ANOVA followed by Sidak test, F1,4 = 10.44, * p = 0.0319. f Mass spectrometry analysis of KDM4A PUP-IT proximately labeled proteins. (Left) Schematic diagram of the PUP-IT proximity labeling system. (Right) Plot showing enriched proteins (z-score > 2.0) that were proximal to the KDM4A in living cells. g (Left) The Co-IP results of KDM4A and YTHDC2 indicate that KDM4A interacts with YTHDC2. Co-IP experiments were repeated for 3 times. Data are presented as mean ± s.e.m. h Schematic representation of in vitro nuclear real-time transcription assay (or nuclear run-on assay, NRO). i, NRO-qPCR results for nascent RNAs. (Up) NRO-qPCR primers designed for the mouse Trpm7 gene loci. Primers P1 to P4 target 5’UTR, Intron 3-4, Exon 12 and Exon 19 on the Trpm7 gene loci respectively. (Down) Knockdown of Kdm4a increased the transcription velocity of nascent RNA in the Trpm7 E12 region. n = 3, unpaired two-tailed t-test, P1: t4 = 0.6641, p = 0.0.4680; P2: t4 = 0.1909, p = 0.8579; P3: t4 = 3.345, * p = 0.0287; P4: t4 = 0.09449, p = 0.9293. Knockdown of Ythdc2 reduces the transcription velocity of nascent RNA in the Trpm7 E12 region. n = 3, unpaired two-tailed t-test, P1: t4 = 0.1281, p = 0.9043; P2: t4 = 1.287, p = 0.2677; P3: t4 = 3.065, * p = 0.0375; P4: t4 = 1.64, p = 0.1763. Kdm4a/Ythdc2 double knockdown has no significant effect on the transcription velocity of nascent RNA in the Trpm7 E12 region. n = 3, unpaired two-tailed t-test, P1: t4 = 0.3208, p = 0.7644; P2: t4 = 0.03464, p = 0.974; P3: t4 = 0.4775, p = 0.658; P4: t4 = 1.353, p = 0.2474. Data are presented as mean ± s.e.m. j Schematic diagram of RNA immunoprecipitation (RIP) experiments. n = 3 per group, Two-way ANOVA test followed by Sidak test, *** p = 0.0001, **** p < 0.0001. Ythdc2-RIP-qPCR results show that neuronal Trpm7 mRNA E12 can bind Ythdc2 protein. n = 3, Two-way ANOVA followed by Sidak test, * p = 0.0309, *** p = 0.0001. Data are presented as mean ± s.e.m. k Illustration of the Exon 12 sequence of Trpm7 and its predicted m6A sites. n = 4 per group, Two-way ANOVA followed by Sidak test, *** p = 0.0002. Overexpression of Ythdc2 can significantly increase the mRNA expression level of Rluc. Trpm7 E12 Mutant results in disruption of Ythdc2 binding to RNA. n = 4, Two-way ANOVA followed by Sidak test, * p = 0.0395. Data are presented as mean ± s.e.m. l, ChIP-qPCR assay for Flag-Kdm4a at the exonic and intronic regions of gene Trpm7 in WT or Ythdc2 KD cells. Exon12: n = 3, two-tailed unpaired t-test, t4 = 3.313, * p = 0.0296; Intron 3-4: n = 3, t4 = 0.9444, ns p = 0.3984. Data are presented as mean ± s.e.m. m Schematics of the split luciferase complementation assay. n, Schematic illustrations of the fused protein variants. o, Relative luminescence intensity of each pair of interacting partners. n = 4 per group, One-way ANOVA followed by Bonferroni test, F12,39 = 17.6, p < 0.0001. * p = 0.0366, **** p < 0.0001. Data are presented as mean ± s.e.m. p Schematic diagram of Ythdc2 binds to m6A sites in the exonic region of Trpm7 mRNA to recruit the transcription repressor Kdm4a to clear the histone H3K36me3 methylation modifications, thereby reducing the transcription velocity of the exon region of the Trpm7 gene.
