Quan Du

In contrast to the well-established genomic 5-methylcytosine (5mC), the existence of N⁶-methyladenine (6 mA) in eukaryotic genomes was discovered only recently. Initial studies found that it was actively regulated in cancer cells, suggesting its involvement in the process of carcinogenesis. However, the contribution of 6 mA in tongue squamous cell carcinoma (TSCC) still remains uncharacterized. In this study, a pan-cancer type analysis was first performed, which revealed enhanced 6 mA metabolism in diverse cancer types. The study was then focused on the regulation of 6 mA metabolism, as well as its effects on TSCC cells. To these aspects, genome 6 mA level was found greatly increased in TSCC tissues and cultured cells. By knocking down 6 mA methylases N6AMT1 and METTL4, the level of genomic 6 mA was decreased in TSCC cells. This led to suppressed colony formation and cell migration. By contrast, knockdown of 6 mA demethylase ALKBH1 resulted in an increased 6 mA level, enhanced colony formation, and cell migration. Further study suggested that regulation of the NF-κB pathway might contribute to the enhanced migration of TSCC cells. Therefore, in the case of TSCC, we have shown that genomic 6 mA modification is involved in the proliferation and migration of cancer cells.

As one of the most abundant DNA methylation form in prokaryotes, N⁶-methyladenine nucleotide (6 mA) was however only recently identified in eukaryotic genomes. To explore the implications of N⁶-adenine methylation in adipogenesis, genomic N⁶-adenine methylation was examined across adipocyte differentiation stages of 3T3-L1 cells. When the N⁶-adenine methylation profiles were analyzed and compared with the levels of gene expression, a positive correlation between the density of promoter 6 mA and gene expression level was uncovered. By means of in vitro methylation and gene knockdown assay, METTL4, a homologue of Drosophila methylase CG14906 and C. elegans methylase DAMT-1, was demonstrated to be a mammalian N⁶-adenine methylase that functions in adipogenesis. Knockdown of Mettl4 led to altered adipocyte differentiation, shown by defective gene regulation and impaired lipid production. We also found that the effects of N⁶-adenine methylation on lipid production involved the regulation of INSR signaling pathway, which promotes glucose up-taking and lipid production in the terminal differentiation stage.

To explore the editing specificity of CRISPR/Cpf1 system, effects of target mutation were systematically examined using a reporter activation assay, with a set of single-nucleotide mutated target site. Consistent with our previous study performed with CRISPR/Cas9, a “core” sequence region that is highly sensitive to target mutation was characterized. The region is of 4-nucleotide long, located from +4 to +7 position of the target site, and positioned within a positively charged central channel when assembled into Cpf1 endonuclease. Single-nucleotide mutation at the core sequence could abolish gene editing mediated by a however active sgRNA. With a great majority of the target sites, a kind of ‘super’ off-target gene editing was observed with both CRISPR/Cpf1 and CRISPR/Cas9. For a given target site, mutation at certain positions led to greatly enhanced off-target gene editing efficacy, even up to 10-fold of that of the fully-matched target. Study further found that these effects were determined by the identity of target nucleotide, rather than the nucleotide of crRNA. This likely suggests that the interactions between target nucleotide and the endonuclease are involved in this process.

Adipocyte differentiation is a coordinated cellular process, which involves a series of dynamic molecular events. Up-regulation of long noncoding RNA slincRAD expression was found to occur in the early differentiation stages of 3T3-L1 cell, prior to the regulation of major transcription factors. By interacting with DNMT1 in S phase, slincRAD guides this essentially epigenetic factor to mediate promoter methylation of a batch of cell cycle-related genes, including cyclin-dependent kinase inhibitor p21. The regulation promotes the growth-arrested cells to re-enter into cell cycle under hormone induction and thereby advances the process of differentiation to clonal expansion stage. The abolishment of the interaction between slincRAD and DNMT1 by slincRAD knockdown results in a defective epigenetic regulation and finally compromised adipogenesis. Collectively, our study characterizes the epigenetic regulation of lncRNA involved in the early stage of adipogenesis.

Lineage-specific cell differentiation is a precise and coordinated biological process. To explore the roles of long noncoding RNA (lncRNA) in this process, the expression of polyA-minus RNAs was comparatively studied during the course of myocyte and adipocyte differentiation. In addition to identifying thousands of novel lncRNAs, distinct lncRNA profiles were revealed during lineage-specific differentiation, showing their active involvement in this process. This study further found that lncRNAs were organized in clusters and are co-regulated, constituting transcription open domains (TODs). In myocyte differentiation of C2C12 cells, loss-of-function screening identified three myogenic lncRNAs. Knockdown of their expression compromised not only the differentiation process, but also the essential signaling pathway. In addition to showing that lncRNAs are actively involved in cell differentiation, our results start to reveal a comprehensive signaling pathway, involving both protein and lncRNA factors.

Targeting specificity is an essential issue in the development of CRISPR-Cas technology. Using a luciferase activation assay, off-target cleavage activity of sgRNA was systematically investigated on single nucleotide-mismatched targets. In addition to confirming that PAM-proximal mismatches are less tolerated than PAM-distal mismatches, our study further identified a “core” sequence that is highly sensitive to target-mismatch. This sequence is of 4-nucleotide long, located at +4 to +7 position upstream of PAM, and positioned in a steric restriction region when assembled into Cas9 endonuclease. Our study also found that, single or multiple target mismatches at this region abolished off-target cleavage mediated by active sgRNAs, thus proposing a principle for gene-specific sgRNA design. Characterization of a mismatch sensitive “core” sequence not only enhances our understanding of how this elegant system functions, but also facilitates our efforts to improve targeting specificity of a sgRNA.