Novel Epigenetic Marks for HIV Latency Entry and Reversal The latent HIV reservoirs in human immunodeficiency type 1 virus (HIV) infection poses a major challenge to the eradication of HIV. A better understanding of the molecular mechanisms of HIV transcription is essential for developing proper strategies to attack the latent HIV reservoirs. HIV transcription and latency are fundamentally controlled by epigenetic regulations surrounding the chromatin proximal to HIV promoter. However, our understanding of epigenetic regulation of HIV transcription is still incomplete. This is demonstrated by the fact that an effective reduction of the HIV reservoir has not been achieved in HIV+ patients by the inhibition of histone deacetylase alone or in combination with killing strategies. We recently found that HIV was activated from latency when crotonylation is induced. This was associated with enhanced histone crotonylation and acetylation, but reduced histone methylation at the HIV LTR. When histone crotonylation is inhibited, latency reversal was blocked. Crotonylation induction greatly enhanced latency reversal elicited by the activation of noncanonical NF-κB (ncNF-κB) signaling, which was mediated via the enhancing induction of p100 cleavage into p52, one of the essential steps during ncNF-κB activation. Transcription of HIV appears to be regulated by a network of crotonylation interactome which orchestrates an efficient HIV transcription. These preliminary observations indicate that crotonylation- a novel and previously unrecognized protein modification - is directly involved in HIV transcription. Of interest, the opposite may also hold, and when crotonylation is reversed, HIV may be enforced into latency. Importantly, while crotonylation is controlled by the same enzymes stimulating acetylation to activate gene transcription (e.g., p300), crotonylation and its downstream signaling are regulated by distinct mechanisms. Similarly, although crotonylation is reversed by the same enzymes regulating deacetylation to induce latency (e.g., HDACs), the mechanism of decrotonylation signaling is different from deacetylation. The overall objective of this R21 application is to determine the molecular mechanisms of crotonylation that underlie the direct activation of HIV transcription and how the regulation of protein decrotonylation facilitates HIV into latency. We hypothesize that, distinct from acetylation, protein crotonylation plays a direct role in HIV transcription, and this can be applied to our current efforts to eliminate HIV latency, and perhaps to future efforts to enforce HIV into latency. Our goals will be achieved through 2 specific aims, directed at the following premises: Aim 1: Crotonylation is distinct from acetylation and directly induces HIV transcription by effects on epigenetics and signaling. Aim 2: Decrotonylation suppresses HIV transcription to enforce HIV latency by direct effects at the HIV LTR and on the cellular milieu, which is inde...