Project Summary Multiple sclerosis (MS) is likely caused by a combination of genetic and environmental factors; however, the mechanisms contributing to these factors remain poorly understood. Epstein-Barr virus (EBV) in particular is a well-established environmental risk factor for MS. We have created a computational algorithm that systematically searches for common molecular mechanisms that might be impacted at multiple MS-associated loci. Using this algorithm, we have discovered that over 40% of MS-associated loci contain MS genetic variants that fall within regions of the human genome occupied by the EBV-encoded EBNA2 protein (44 out of 109, >4.6- fold enrichment, P<10-25). Other top proteins include known EBNA2 human interacting proteins (RBPJ, RELA, SPI1) and proteins recently shown to participate in “EBV super-enhancers”, which enable proliferation and survival of EBV infected B cells. The same MS-associated variants also impact gene expression levels of MS- associated genes in EBV-infected B cell lines. Our hypothesis is that allele-dependent binding of EBNA2 and its co-factors explains the allele-dependent risk at many MS genetic loci. Importantly, this hypothesis links the genetic associations of MS to the known molecular roles played by EBV and Notch signaling. In total, >40 of the known MS associations might be explained by this common mechanism uniting the genetic and the environmental risk components of MS. We propose the following Aims. Aim 1. Genome-wide experimental assessment of allele-dependent EBNA2 and human protein binding to risk alleles at MS loci. We will examine allele-dependent protein binding in MS patient-derived EBV- transformed B cell lines and in T cells using ChIP-seq. We will identify genome-wide allele-dependent co-binding of EBNA2 with its partners using our new, innovative Split Dam ID-seq technique. Resulting data will be used to create an improved model of MS genetic risk mechanism and results will be freely disseminated. Aim 2. Discover specific MS-associated loci where allele-dependent EBNA2 or human protein-DNA interactions result in allele-dependent gene expression. We will assess allele-dependent binding of these proteins at likely causal variants, and allele-dependent gene expression by multiple experimental approaches. Aim 3. Establish causality for intermediate phenotypes at selected MS-associated variants. We will establish the causal effect of these variants by demonstrating the necessity and sufficiency of candidate risk alleles in MS patient-derived cells through a novel dead Cas9 activation system, and by performing genome editing with CRISPR/Cas9 followed by gene expression monitoring. The concept that disease might be influenced by allele-dependent assembly of protein complexes controlled by a virus is highly innovative and has never before been experimentally demonstrated. Results from this proposal would provide strong rationale to develop therapies that interfere with EBNA2 binding, or ...