The Transcription Factor Rfx3 Regulates β-Cell Differentiation, Function, and Glucokinase Expression

OBJECTIVE: Pancreatic islets of perinatal mice lacking the transcription factor Rfx3 exhibit a marked reduction in insulin-producing beta-cells. The objective of this work was to unravel the cellular and molecular mechanisms underlying this deficiency. RESEARCH DESIGN AND METHODS: Immunofluorescence studies and quantitative RT-PCR experiments were used to study the emergence of insulin-positive cells, the expression of transcription factors implicated in the differentiation of beta-cells from endocrine progenitors, and the expression of mature beta-cell markers during development in Rfx3(-/-) and pancreas-specific Rfx3-knockout mice. RNA interference experiments were performed to document the consequences of downregulating Rfx3 expression in Min6 beta-cells. Quantitative chromatin immunoprecipitation (ChIP), ChIP sequencing, and bandshift experiments were used to identify Rfx3 target genes. RESULTS: Reduced development of insulin-positive cells in Rfx3(-/-) mice was not due to deficiencies in endocrine progenitors or beta-lineage specification, but reflected the accumulation of insulin-positive beta-cell precursors and defective beta-cells exhibiting reduced insulin, Glut-2, and Gck expression. Similar incompletely differentiated beta-cells developed in pancreas-specific Rfx3-deficient embryos. Defective beta-cells lacking Glut-2 and Gck expression dominate in Rfx3-deficent adults, leading to glucose intolerance. Attenuated Glut-2 and glucokinase expression, and impaired glucose-stimulated insulin secretion, were also induced by RNA interference-mediated inhibition of Rfx3 expression in Min6 cells. Finally, Rfx3 was found to bind in Min6 cells and human islets to two well-known regulatory sequences, Pal-1 and Pal-2, in the neuroendocrine promoter of the glucokinase gene. CONCLUSIONS: Our results show that Rfx3 is required for the differentiation and function of mature beta-cells and regulates the beta-cell promoter of the glucokinase gene

NLRC5 deficiency impairs MHC class I-dependent lymphocyte killing by cytotoxic T cells

Purpose/Objective: Hepatitis C virus (HCV) establishes a chronic infection in approximately 80% of infected individuals and is a leading cause of liver disease. Knowledge gaps exist with regard to the mechanisms of viral replication within hepatocytes and disease progression during chronic infection. The present study aimed to explore the regulation of gene expression during chronic HCV infection in liver biopsy samples. Materials and methods: RNA was isolated from liver biopsies from patients who were chronically infected with HCV. Biopsies from noninfected patients with other liver diseases were used as controls to focus on HCV-associated changes in hepatic gene expression. This excluded altered gene expression patterns resulting from either inflammation or fibrosis. We applied high-throughput RNA-Seq technology to provide a more complete overview of the interactions between HCV and the host. Differential gene expression was analysed using DESeq and edgeR and gene enrichment analysis was performed with GSEA and Gestalt. Results: Expression profiling identified 181 genes that were differentially regulated (P < 0.05 after adjusting for multiple testing) between infected and uninfected biopsies. These genes fell into distinct regulatory pathways including immune response, antigen processing and interferon*stimulated genes (ISGs). Of note a significant enrichment of the IFIT and PARP gene families was observed as well as upregulation of multiple genes involved in the ISGylation pathway such as ISG15, UBE2L6 and HERC5. Validation of these gene targets was carried out in liver biopsies and tissue culture cells. Functional analysis of ISG15 indicated that depleting mRNA levels resulted in increased HCV RNA abundance. On-going studies are underway to explore the role of IFIT and PARP family members during chronic HCV. Conclusions: High-throughput sequencing has shed new light on the pathways that may regulate HCV replication and associated pathology during chronic infection. Future studies will address how HCV is able to persist in the presence of a stimulated IFN response within the liver