Project Summary/Abstract TNF1 is a cytokine critically important in innate host defense. Altered levels of TNFa are deleterious to the host and expression is tightly regulated at multiple levels. We propose that its regulation also includes effects from long ncRNAs. GCF2 is a transcriptional repressor that binds chromatin and localizes to the region of the TNF1 promoter where sense and antisense ncRNAs are found. In K562 cells, a model of hematopoietic stem cells which do not express TNF1, the DNA is highly methylated, chromatin marks of repression are evident and GCF2 is found bound to the -300 region of the promoter. Over-expression of GCF2 in a cell line competent for expression, led to significantly reduced expression of TNF1. The -300 region of the promoter, where GCF2 is found, is the region of maximal histone modifications and is also marked by binding of EZH2 and Suz 12, two polycomb proteins associated with transcriptional repression. . We identified low levels of sense and antisense transcripts throughout the upstream region of TNF1 and these transcripts were predicted to form highly folded structures. Depletion of antisense transcripts by phosphorothioate oligonucleotides led to de- repression of TNF1 expression in K562 cells. The promoter RNA was closely associated with the DNA because the S9.6 antibody, which recognizes RNA:DNA hybrid structures, bound to the TNF1 upstream region in a pattern that paralleled the binding pattern of GCF2. We hypothesize that GCF2 binds chromatin at sites of antisense RNA with high secondary structure and participates in RNA-mediated transcriptional repression. Additional data supporting this hypothesis include (1) GCF2 acts as a repressor in every transient transfection assay reported, (2) GCF2 binds argonautes 1 and 2, (3) Chromatin RNA-IP with an antibody to GCF2 recovered TNF1 upstream RNA. In Aim 1, we will better define the roles of the upstream sequences by quantitation of the transcripts and mapping from cells that are repressed, competent or actively expressing TNF1. To demonstrate their effect on TNF1 mRNA, we will deplete the upstream transcripts with phosphorothioate oligonucleotides. In Aim 2, we will define the binding characteristics of GCF2. We will deplete the upstream sequences and observe the effect on GCF2 binding to the promoter. To assess the other targets of GCF2, we will perform a GCF2 ChIP-seq analysis. In Aim 3, we will determine whether argonaute, EZH2, or Suz 12 are required for GCF2 binding to the upstream region. Aim 3 will also define protein-protein interactions and directly assess the effect on transcription. The data from these aims will define the character of the upstream transcripts and their role in the regulation of TNF1. These studies will advance our understanding of RNA-mediated regulation of gene expression and, specifically, the complex regulation of TNF1 expression. PUBLIC HEALTH RELEVANCE: Project Narrative Previous studies from my laboratory have revealed evidence of a novel form of regulation of TNF1. The studies described in this application are directed at understanding this new RNA-mediated form of transcriptional regulation. The long term goal is to be able to regulate TNF1 expression in disease states characterized by over-production such as Crohn Disease and rheumatoid arthritis.