HISTONE MODIFICATIONS GUIDING HIV INTEGRATION PROJECT SUMMARY / ABSTRACT Human immunodeficiency virus (HIV-1) is the causative agent of acquired immunodeficiency syndrome (AIDS) with ~36.7 million people currently infected worldwide. Integration of the viral genome establishes an irreversible insertion of the proviral sequence into the host chromatin. The integrated genome ensures effective HIV gene expression and ultimately virus production or the establishment of latency where the provirus remains dormant for an extended time. Retroviral integration is mediated by the viral integrase (IN) protein that is bound to the direct-repeated ends of the viral DNA genome, along with additional cellular and viral co-factors that direct integration to host chromatin sites. The overarching goal of this proposal is to understand how viral integration is targeted to chromatin by histone post translational modifications (PTMs), and how cellular factors that tightly control the localization of these PTMs influence target site choice. LEDGF/p75 is a key cellular transcription co-activator that binds to HIV-1 IN via a C-terminal integrase binding domain (IBD). The N-terminus of LEDGF/p75 contains a PWWP domain that is expected to recognize histone methylated lysine PTMs. Bimodal tethering of the HIV-1 integration complex (intasome) to a transcription-related histone PTM has been proposed to account for the observed ~76% of chromosomal integrations that occur in actively transcribed genes. LEDGF/p75 purportedly targets HIV-1 to nucleosomes containing trimethylation of histone H3 lysine 36 residues (H3K36me3). However, H3K36me3 is most often found at the 3’ ends of transcribed genes, whereas HIV-1 integrates more frequently toward the 5’ ends of genes. Bioinformatic correlations of HIV-1 integration sites with histone PTMs is limited by the data available in the Encyclopedia of DNA Elements (ENCODE) database. Notably absent from ENCODE are genomic maps of the H3K36me2 PTM in any cell type. We performed a ChIP-Seq analysis of H3K36me2 and determined that it was commonly found near the 5’ transcription start site of genes. Moreover, we found that HIV-1 integration sites correlate better with the location of H3K36me2 than H3K36me3. We propose to examine the role of H3K36me2 and H3K36me3 with two Specific Aims. Aim 1 will expand the current understanding of H3K36 methylation and its connection to HIV-1 integration efficiency and site selection in vivo. Aim 2 will probe the influence of H3K36me2 and H3K36me3 on HIV-1 integration in vitro. Our approaches will include technologically advanced mass spectrometry, integration site mapping, and single molecule fluorescence microscopy. The results of these studies will clearly determine the role of H3K36me2 and H3K36me3 during HIV-1 integration.