The DACH1 Gene Transcriptional Activation and Protein Degradation Mediated by Transactivator Tas of Prototype Foamy Virus.

Foamy viruses are members of the Retroviridae family's Spumaretrovirinae subfamily. They induce cell vacuolation and exhibit a foamy pathogenic impact after infecting cells. DACH1 (dachshund family transcription factor 1) is a crucial cytokine linked to tumor development, and is associated with the growth of many different malignant tumor cells. Additionally, DACH1 suppresses pancreatic cell proliferation and is involved in diabetes insulin signaling. Prototype foamy viruses (PFVs) were used for the investigation of the regulatory mechanism of FVs on cellular DACH1 expression. The results show that DACH1 expression in PFV-infected cells was inconsistent at both the transcriptional and protein levels. At the transcriptional level, DACH1 was significantly activated by PFV transactivator Tas, and dual-luciferase reporter gene tests, EMSA, and ChIP assays found a Tas response element of 21 nucleotides in the DACH1 promoter. PFV and Tas did not boost the levels of DACH1 protein in a manner consistent with the high levels of DACH1 transcription expression. It was noted that Tas increased the expression of the Ser/Thr protein phosphatase PPM1E, causing PPM1E-mediated post-translational SUMOylation alterations of DACH1 to prompt DACH1 to degrade. The reason for DACH1 protein degradation is that DACH1 inhibits PFV replication. To sum up, these findings show that PFV upregulated the transcription of DACH1, while urging its protein into PPM1E-mediated SUMOylation, to eliminate the adverse effect of DACH1 overexpression of host cells on viral replication and promote virus survival.

Comprehensive Analysis and Functional Characteristics of Differential Expression of N6-Methyladenosine Methylation Modification in the Whole Transcriptome of Rheumatoid Arthritis.

N6-methyladenosine (m6A) modification is the most prevalent chemical modification in eukaryotic mRNA and is associated with the development of various immune diseases. However, the role of m6A methylation in rheumatoid arthritis (RA) development is unclear. We preliminarily explored the role of m6A methylation-related mRNAs in RA for its clinical application. The discovery of m6A methylation-modifying genes in this study may provide a fresh perspective on the development of drugs for RA treatment. High-throughput sequencing combined with methylated RNA immunoprecipitation (MeRIP-seq) and RNA sequencing were used to assess whole-transcriptome m6A modifications in the synovium of patients with RA. The relationship between m6A-modified target genes and RA inflammation and macrophages was determined. The expression of the m6A-modified significant transcript-enriched inflammatory signaling pathway was assessed through animal experiments. Differentially expressed m6A genes were correlated with macrophage activation involved in immune response, vascular endothelium, MAPK signaling pathway, PI3K - Akt signaling pathway, and other inflammatory processes. Furthermore, combined analysis with m6A-seq and RNA-seq revealed 120 genes with significant changes in both m6A modification and mRNA expression. We selected the top 3 candidate mRNAs that were upregulated and downregulated simultaneously. The expression of phosphatase and tensin homolog deleted on chromosome ten (PTEN) mRNA and protein in RA patients was lower than that in healthy control (HC). SHC-binding protein 1 (SHCBP1) and neurexophilin-3 (NXPH3) mRNA expressions were increased in RA patients. The expression of M1 macrophages was increased in RA patients. RA markers are such as rheumatoid factor (RF) and peptide containing citrulline (CCP). Further animal experiments showed that the expression of synovial MAPK, PI3K, and Akt1 proteins in the RA model was increased, and the PTEN, p-PTEN protein expression was decreased. PI3K, Akt1, PTEN, and p-PTEN were correlated to RA joint inflammation. This study revealed a unique pattern of differential m6A methylation modifications in RA and concluded that m6A modification is related to the occurrence of RA synovial inflammation.

An Accurate and Comprehensive Clinical Sequencing Assay for Cancer Targeted and Immunotherapies.

BACKGROUND: Incorporation of next-generation sequencing (NGS) technology into clinical utility in targeted and immunotherapies requires stringent validation, including the assessment of tumor mutational burden (TMB) and microsatellite instability (MSI) status by NGS as important biomarkers for response to immune checkpoint inhibitors. MATERIALS AND METHODS: We designed an NGS assay, Cancer Sequencing YS panel (CSYS), and applied algorithms to detect five classes of genomic alterations and two genomic features of TMB and MSI. RESULTS: By stringent validation, CSYS exhibited high sensitivity and predictive positive value of 99.7% and 99.9%, respectively, for single nucleotide variation; 100% and 99.9%, respectively, for short insertion and deletion (indel); and 95.5% and 100%, respectively, for copy number alteration (CNA). Moreover, CSYS achieved 100% specificity for both long indel (50-3,000 bp insertion and deletion) and gene rearrangement. Overall, we used 33 cell lines and 208 clinical samples to validate CSYS's NGS performance, and genomic alterations in clinical samples were also confirmed by fluorescence in situ hybridization, immunohistochemistry, and polymerase chain reaction (PCR). Importantly, the landscape of TMB across different cancers of Chinese patients (n = 3,309) was studied. TMB by CSYS exhibited a high correlation (Pearson correlation coefficient r = 0.98) with TMB by whole exome sequencing (WES). MSI measurement showed 98% accuracy and was confirmed by PCR. Application of CSYS in a clinical setting showed an unexpectedly high occurrence of long indel (6.3%) in a cohort of tumors from Chinese patients with cancer (n = 3,309), including TP53, RB1, FLT3, BRCA2, and other cancer driver genes with clinical impact. CONCLUSION: CSYS proves to be clinically applicable and useful in disclosing genomic alterations relevant to cancer target therapies and revealing biomarkers for immune checkpoint inhibitors. IMPLICATIONS FOR PRACTICE: The study describes a specially designed sequencing panel assay to detect genomic alterations and features of 450 cancer genes, including its overall workflow and rigorous clinical and analytical validations. The distribution of pan-cancer tumor mutational burden, microsatellite instability, gene rearrangement, and long insertion and deletion mutations was assessed for the first time by this assay in a broad array of Chinese patients with cancer. The Cancer Sequencing YS panel and its validation study could serve as a blueprint for developing next-generation sequencing-based assays, particularly for the purpose of clinical application.