Chromatin modifications that enhance DNA accessibility

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Project Summary/Abstract Chromatin modifications that enhance DNA accessibility: Eukaryotic DNA is tightly packaged into nucleosomes, which form structural barriers to transcription, yet RNA polymerases effectively read through chromatinized DNA in cells. Crucial to this apparent paradox are ATP-independent histone chaperones, which include the heterodimeric FACT (FAcilitates Chromatin Transcription) complex and the monomeric proteins lens epithelium-derived growth factor (LEDGF) and hepatoma-derived growth factor 2 (HDGF2). Whereas FACT’s role as a histone chaperone is well-established, LEDGF and HDGF2 were only recently implicated as having histone chaperone activity. Furthermore, LEDGF and HDGF2 have also been implicated in modulating human immunodeficiency virus type 1 (HIV-1) DNA integration into chromatin, with LEDGF playing a dominant role, but the mechanisms remain unclear. We hypothesize that: (i) FACT facilitates chromatin remodeling by preferentially binding to nucleosomes destabilized by post-translational histone modifications, preserving histone-DNA interactions necessary for nucleosome reassembly; (ii) LEDGF and HDGF2 function as reader proteins, working selectively through their preferential binding to regions rich in H3K36me2/3 histone modifications, but have mechanistic similarities to FACT imparted by auxiliary domains; (iii) through their histone chaperone activity, LEDGF and HDGF2 proteins modulate HIV-1 DNA integration into actively transcribed genes characterized by chaperone-destabilized H3K36me2/3-rich chromatin. We propose two aims: Aim 1: Define the differential effects of FACT on the chromatin state To test the hypothesis that FACT acts as a chaperone by preferentially binding to unwound chromatin intermediates and chromatin destabilized by histone modifications, we will apply forces with optical tweezers to determine the equilibrium stability, fluctuational opening rate, and the ability of nucleosomes to reassemble after disruption in the presence of WT, mutant, and truncated FACT for histones modified through destabilizing acylation modifications. The results will reveal the extent to which FACT activity can be regulated by destabilizing histone modifications. Aim 2: Determine the mechanisms by which LEDGF and HDGF2 act as histone chaperones and facilitators of HIV-1 integration. To test the hypotheses that LEDGF and HDGF2 bind H3K36me2/3-containing nucleosomes and mediate nucleosome disassembly and reassembly and that LEDGF and HDGF2 direct HIV-1 DNA integration at H3K36me2/3-enriched loci through their nucleosome chaperone activity at these sites, we will measure the effects of wild type and mutant LEDGF and HDGF2 on nucleosome stability, dynamics and reassembly, as well as their effects on HIV-1 integrase binding. The results will determine the mechanism of LEDGF and HDGF2 nucleosome chaperone activity and the role played by histone methylation in regulating that activity.