The Functional Analysis Of A Gene Silencing Regulator ENPT And A Histone Demethylase JMJ16 In Arabidopsis

Epigenetic regulation plays important roles in maintaining genomic stability and regulating gene expression.DNA methylation,histone modification and deposition of histone variants are important for regulating gene silencing in plants and animals.To study the mechanism of gene silencing in plants,we performed a genetic screen using a transgenic line L119 and identified a novel factor involved in regulating gene silencing,named as ENPT(Enhancer of NPT?),and JMJ16(Jumonji domain-containing protein 16).ENPT encodes a conserved protein with unknown functions in plants.In this study,we found that ENPT was expressed in root apex,shoot apical meristem and lateral root primordium,and ENPT protein was localized in the nucleus.ENPT specifically interacted with histone H2A variants,i.e.H2A.Z(HTA11)and H2A.X(HTA5)in vitro and in vivo.Previous studies indicated that H2A.Z at+1 nucleosome near the transcription start sites is required to repress gene expression.To study the relationship between ENPT and H2A.Z,we performed chromatin immunoprecipitation(ChIP)assays using anti-H2A.Z antibodies.Our results showed that the enrichment of H2A.Z was decreased in the upregulated genes in enpt-1.In addition,the transcriptome analysis revealed that the upregulated genes in enpt-1 significantly overlapped with genes enriched by H2A.Z.These results suggested that ENPT could work in concert with H2A.Z to maintain gene silencing.The whole-genome bisulfite sequencing assays showed that CHH methylation of transposons decreased in enpt-1,and 63%of CHH hypo-DMRs in enpt-1 overlapped with CHH hypo-DMRs in h2a.z piel,suggesting that ENPT could maintain CHH methylation of transposons.To investigate how enpt-1 affects CHH methylation,we examined the 24 nt siRNA level in enpt-1by Northern blot and whole-genome small RNA sequencing.The results showed that the level of 24 nt siRNA was decreased in enpt-1,which may explain why CHH methylation was decreased in enpt-1.Therefore,we conclude that ENPT could maintain gene silencing by regulating the enichment of H2A.Z of regulated genes,and played a role in maintainig the level of 24 nt siRNA and CHH methylation.These findings may shed light on the mechanism of how H2A.Z affects gene expression and maintains CHH methylation.JMJ16 is a Jumonji C(JmjC)domain-containing protein belonging to the KDM5/JARID1 family.The in vivo demethylation assays indicated that JMJ16 demethylated H3K4me3/2/1,while did not affect H3K9me3/2/1,H3K27me3/2/1 and H3K36me3/2/1,indicating that JMJ16 is a specific H3K4 demethylase.The ChIP assay was performed to examine the H3K4me3 methylation level in jmjl6-1,and the results showed that H3K4me3 was increased in Pro35S:NPT?,IG1 and soloLTR loci,and the expression levels of these genes were upregulated as shown by RT-qPCR data.These results revealed that JMJ16 could demethylate H3K4me3 at specific loci to maintain gene silencing.In conclusion,this study identified a factor involved in regulating gene silencing,JMJ16,and demonstrated that JMJ16 act as a specific histone H3K4 demethylase to maintain gene silencing.Hence,this study laid the foundation for functional analysis of JMJ16 in further research.