Regulation of the epigenome through RNA modifications.

Chemical modifications of nucleotides expand the complexity and functional properties of genomes and transcriptomes. A handful of modifications in DNA bases are part of the epigenome, wherein DNA methylation regulates chromatin structure, transcription, and co-transcriptional RNA processing. In contrast, more than 150 chemical modifications of RNA constitute the epitranscriptome. Ribonucleoside modifications comprise a diverse repertoire of chemical groups, including methylation, acetylation, deamination, isomerization, and oxidation. Such RNA modifications regulate all steps of RNA metabolism, including folding, processing, stability, transport, translation, and RNA's intermolecular interactions. Initially thought to influence all aspects of the post-transcriptional regulation of gene expression exclusively, recent findings uncovered a crosstalk between the epitranscriptome and the epigenome. In other words, RNA modifications feedback to the epigenome to transcriptionally regulate gene expression. The epitranscriptome achieves this feat by directly or indirectly affecting chromatin structure and nuclear organization. This review highlights how chemical modifications in chromatin-associated RNAs (caRNAs) and messenger RNAs (mRNAs) encoding factors involved in transcription, chromatin structure, histone modifications, and nuclear organization affect gene expression transcriptionally.

DNA Methylation of Human Choline Kinase Alpha Promoter-Associated CpG Islands in MCF-7 Cells.

Choline kinase (CK) is the enzyme catalyzing the first reaction in CDP-choline pathway for the biosynthesis of phosphatidylcholine. Higher expression of the α isozyme of CK has been implicated in carcinogenesis, and inhibition or downregulation of CKα (CHKA) is a promising anticancer approach. This study aimed to investigate the regulation of CKα expression by DNA methylation of the CpG islands found on the promoter of this gene in MCF-7 cells. Four CpG islands have been predicted in the 2000 bp promoter region of ckα (chka) gene. Six CpG island deletion mutants were constructed using PCR site-directed mutagenesis method and cloned into pGL4.10 vectors for promoter activity assays. Deletion of CpG4C region located between -225 and -56 significantly increased the promoter activity by 4-fold, indicating the presence of important repressive transcription factor binding site. The promoter activity of methylated full-length promoter was significantly lower than the methylated CpG4C deletion mutant by 16-fold. The results show that DNA methylation of CpG4C promotes the binding of the transcription factor that suppresses the promoter activity. Electrophoretic mobility shift assay analysis showed that cytosine methylation at MZF1 binding site in CpG4C increased the binding of putative MZF1 in nuclear extract. In conclusion, the results suggest that DNA methylation decreased the promoter activity by promoting the binding of putative MZF1 transcription factor at CpG4C region of the ckα gene promoter.

MicroRNA-367-3p induces apoptosis and suppresses migration of MCF-7 cells by downregulating the expression of human choline kinase α.

Choline kinase (ChK) catalyzes the first step in the CDP-choline pathway for the synthesis of phosphatidylcholine. The α isoform of this enzyme is overexpressed in various types of cancer and its inhibition or downregulation has been applied as an anticancer strategy. In spite of increasing attention being paid to ChK expression, as well as its activity and inhibition in cancer, there are only limited studies available on the regulation of ChK, including its regulation by microRNAs (miRNAs/miRs). The dysregulation of gene expression by miRNAs is a common cause for carcinogenesis. In the present study, miR-367-3p was predicted to target the 3'-untranslated region (UTR) of the ChK α (chka) mRNA transcript. The binding of miR-367-3p to the 3'-UTR of chka was validated by a luciferase assay. The effects of the miR-367-3p mimic on chka gene and protein expression levels were determined by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. miR-367-3p significantly downregulated the expression of chka to ~60% of the negative control. Cells transfected with miR-367-3p exhibited higher levels of apoptosis and a lower cell migration compared with the control. To the best of our knowledge, the present study provided the first experimental evidence of the regulation of chka expression by miR-367-3p. The pro-apoptotic and suppressive effects of miR-367-3p on cell migration were similar to the anticancer effects resulting from the inhibition of ChK enzyme activity or the knockdown of chka gene expression by small interfering RNA. Therefore, these findings may potentially lead to the use of miR-367-3p in anticancer strategies that target ChK.

Keratin 5/14­mediated cell differentiation and transformation are regulated by TAp63 and Notch­1 in oral squamous cell carcinoma­derived cells.

Keratins 5/14 (K5/14) are intermediate filament proteins expressed in the basal layer of stratified epithelial cells and are known targets of p63. Previous research in our laboratory showed that upon K5/14 downregulation in oral squamous cell carcinoma (OSCC)­derived cells, there was an increase in intracellular Notch­1 levels and differentiation markers such as involucrin, keratin 1 and a decrease in tumorigenic potential in vivo. However, the molecules involved in the K14 regulated cell differentiation and transformation are not known to date. In order to understand the possible role of TAp63, we downregulated TAp63 in a K14­knockdown background. We observed that there was a decrease in the expression of Notch­1. Expression levels of differentiation markers such as involucrin, K1, loricrin and filaggrin were also decreased. Furthermore, TAp63 downregulation led to an increase in invasion, migration and in vivo tumorigenic potential of these cells. We observed a decrease in ß­catenin signaling in K14­downregulated cells. Notably, when TAp63 was downregulated in K14­knockdown cells, there was increase in non­phospho ß­catenin levels. Hence, this study indicates that TAp63 plays an important role in K14­downregulated cells possibly by regulating the Notch­1 expression. K14 regulates the expression of TAp63 which in turn regulates expression of Notch­1. The present study is a step forward in our quest to understand the functional significance of molecules that regulate the process of differentiation and tumorigenesis in stratified epithelial cells.

Multimodal approach to explore the pathogenicity of BARD1, ARG 658 CYS, and ILE 738 VAL mutants.

BARD1-BRCA1 complex plays an important role in DNA damage repair, apoptosis, chromatin remodeling, and other important processes required for cell survival. BRCA1 and BARD1 heterodimer possess E3 ligase activity and is involved in genome maintenance, by functioning in surveillance for DNA damage, thereby regulating multiple pathways including tumor suppression. BRCT domains are evolutionary conserved domains present in different proteins such as BRCA1, BARD1, XRCC, and MDC1 regulating damage response and cell-cycle control through protein-protein interactions. Nonetheless, the role of BARD1BRCT in the recruitment of DNA repair mechanism and structural integrity with BRCA1 complex is still implicit. To explicate the role of BARD1BRCT in the DNA repair mechanism, in silico, in vitro, and biophysical approach were applied to characterize BARD1 BRCT wild-type and Arg658Cys and Ile738Val mutants. However, no drastic secondary and tertiary structural changes in the mutant proteins were observed. Thermal and chemical denaturation studies revealed that mutants Arg658Cys and Ile738Val have a decrease in Tm and ∆G than the wild type. In silico studies of BARD1 BRCT (568-777) and mutant protein indicate loss in structural compactness on the Ile738Val mutant. Comparative studies of wild-type and mutants will thus be helpful in understanding the basic role of BARD1BRCT in DNA damage repair.