5-Fluorouracil dose escalation generated desensitized colorectal cancer cells with reduced expression of protein methyltransferases and no epithelial-to-mesenchymal transition potential

Background:Colorectal cancer(CRC)is one of the most frequently diagnosed cancers.In many cases,the poor prognosis of advanced CRC is associated with resistance to treatment with chemotherapeutic drugs such as 5-Fluorouracil(5-FU).The epithelial-to-mesenchymal transition(EMT)and dysregulation in protein methylation are two mechanisms associated with chemoresistance in many cancers.This study looked into the effect of 5-FU dose escalation on EMT and protein methylation in CRC.Materials and Methods:HCT-116,Caco-2,and DLD-1 CRC cell lines were exposed to dose escalation treatment of 5-FU.The motility and invasive potentials of the cells before and after treatment with 5-FU were investigated through wound healing and invasion assays.This was followed by aWestern blot which analyzed the protein expressions of the epithelial marker E-cadherin,mesenchymal marker vimentin,and the EMT transcription factor(EMTTF),the snail family transcriptional repressor 1(Snail)in the parental and desensitized cells.Western blotting was also conducted to study the protein expressions of the protein methyltransferases(PMTs),Euchromatic histone lysine methyltransferase 2(EHMT2/G9A),protein arginine methyltransferase(PRMT5),and SET domain containing 7/9(SETD7/9)along with the global lysine and arginine methylation profiles.Results:The dose escalation method generated 5-FU desensitized CRC cells with distinct morphological features and increased tolerance to high doses of 5-FU.The 5-FU desensitized cells experienced a decrease in migration and invasion when compared to the parental cells.This was reflected in the observed reduction in E-cadherin,vimentin,and Snail in the desensitized cell lines.Additionally,the protein expressions of EHMT2/G9A,PRMT5,and SETD7/9 also decreased in the desensitized cells and global protein lysine and arginine methylation became dysregulated with 5-FU treatment.Conclusion:This study showed that continuous,dose-escalation treatment of 5-FU in CRC cells generated 5-FU desensitized cancer cells that seemed to be less aggressive than parental cells.

Mutual regulation between microRNA-373 and methyl-CpGbinding domain protein 2 in hilar cholangiocarcinoma

AIM:To investigate the reciprocal modulation between microRNA(miRNA) and DNA methylation via exploring the correlation between miR-373 and methyl-CpGbinding domain protein(MBD)2.METHODS:MiR-373 expression was examined using the TaqMan miRNA assay.Methylation of miR-373 was investigated using methylation-specific polymerase chain reaction,and recruitment of methyl binding proteins was studied using the chromatin immunoprecipitation assay.Mutation analysis was conducted using the QuikChange Site-Directed Mutagenesis kit.The activity of miR-373 gene promoter constructs and targeting at MBD2-three prime untranslated region(3'UTR) by miR-373 were evaluated by a dual-luciferase reporter gene assay.RESULTS:In hilar cholangiocarcinoma,miR-373 decreased and was closely associated with poor cell differentiation,advanced clinical stage,and shorter survival.The promoter-associated CpG island of miR-373 gene was hypermethylated and inhibited expression of miR-373.MBD2 was up-regulated and enriched at the promoter-associated CpG island of miR-373.Methylation-mediated suppression of miR-373 required MBD2 enrichment at the promoter-associated CpG island,and miR-373 negatively regulated MBD2 expression through targeting the 3'UTR.CONCLUSION:MiR-373 behaves as a direct transcriptional target and negative regulator of MBD2 activity through a feedback loop of CpG island methylation.

OsPRMT6a-mediated arginine methylation of OsJAZ1 regulates jasmonate signaling and spikelet development in rice

Although both protein arginine methylation(PRMT)and jasmonate(JA)signaling are crucial for regulating plant development,the relationship between these processes in the control of spikelet development remains unclear.In this study,we used the CRISPR/Cas9 technology to generate two OsPRMT6a loss-of-function mutants that exhibit various abnormal spikelet structures.Interestingly,we found that OsPRMT6a can methylate arginine residues in JA signal repressors OsJAZ1 and OsJAZ7.We showed that arginine methylation of OsJAZ1 enhances the binding affinity of OsJAZ1 with the JA receptors OsCOI1a and OsCOI1b in the presence of JAs,thereby promoting the ubiquitination of OsJAZ1 by the SCF^(OsCOI1a/OsCOI1b) complex and degradation via the 26S proteasome.This process ultimately releases OsMYC2,a core transcriptional regulator in the JA signaling pathway,to activate or repress JA-responsive genes,thereby maintaining normal plant(spikelet)development.However,in the osprmt6a-1 mutant,reduced arginine methylation of OsJAZ1 impaires the interaction between OsJAZ1 and OsCOI1a/OsCOI1b in the presence of JAs.As a result,OsJAZ1 proteins become more stable,repressing JA responses,thus causing the formation of abnormal spikelet structures.Moreover,we discovered that JA signaling reduces the OsPRMT6a mRNA level in an OsMYC2-dependent manner,thereby establishing a negative feedback loop to balance JA signaling.We further found that OsPRMT6a-mediated arginine methylation of OsJAZ1 likely serves as a switch to tune JA signaling to maintain normal spikelet development under harsh environmental conditions such as high temperatures.Collectively,our study establishes a direct molecular link between arginine methylation and JA signaling in rice.

AtPRMT5 Regulates Shoot Regeneration through Mediating Histone H4R3 Dimethylation on KRPs and Pre-mRNA Splicing of RKP in Arabidopsis

Protein arginine methylation plays important roles in diverse biological processes, but its role in regulating shoot regeneration remains elusive. In this study, we characterized the function of the protein arginine methyltransferase AtPRMT5 during de novo shoot regeneration in Arabidopsis. AtPRMT5 encodes a type II protein arginine methyltransferase that methylates proteins, including histories and RNA splicing factors. The frequency of shoot regeneration and the number of shoots per callus were decreased in the atprmt5 mutant compared with those in the wild type. Chromatin immunoprecipitation analysis revealed that AtPRMT5 targets KIP-RELATED PROTEINs （KRPs）, which encode the cyclin-dependent kinase inhibitors that repress the cell cycle. During shoot regeneration, the KRP transcript level increased in the atprmt5 mutant, which resulted from reduced histone H4R3 methylation in the KRP promoter. Overexpression of KRP significantly reduced the frequency of shoot regeneration and shoot number per callus. Furthermore, abnormal pre-mRNA splicing in the gene RELATED TO KPC1 （RKP）, which encodes an ubiquitin E3 ligase, was detected in the atprmt5 mutant. RKP functions in regulating KRP protein degradation, and mutation in RKP inhibited shoot regeneration. Thus, AtPRMT5 regulated shoot regeneration through histone modification-mediated KRP transcription and RKP pre-mRNA splicing. Our findings provide new insights into the function of protein arginine methylation in de novo shoot regeneration.

Cellular functions of MLL/SET-family histone H3 lysine 4 methyltransferase components

The MLL/SET family of histone H3 lysine 4 methyltransferases form enzyme complexes with core subunits ASH2L, WDR5, RbBP5, and DPY-30 （often abbreviated WRAD）, and are responsible for global histone H3 iysine 4 methylation, a hallmark of actively transcribed chromatin in mammalian cells. Accordingly, the function of these proteins is required for a wide variety of processes including stem cell differentiation, cell growth and division, body segmentation, and hematopoiesis. While most work on MLL-WRAD has focused on the function this core complex in histone methylation, recent studies indicate that MLL-WRAD proteins interact with a variety of other proteins and IncRNAs and can localize to cellular organelles beyond the nucleus. In this review, we focus on the recently described activities and interacting partners of MLL-WRAD both inside and outside the nucleus.