PROJECT SUMMARY The long-term goal of our proposed research is to elucidate the molecular mechanisms underlying human gene regulation by histone H4 modification with the small ubiquitin-like modifier-3 (SUMO-3) protein. Histone sumoylation is a dramatic post-translational modification that occurs on the lysine12 side-chain in the unstructured N-terminal tail of histone H4 (abbreviated as suH4). Early correlative studies in yeast and human cells associated H4 tail sumoylation with reduced levels of histone H3 acetylation, and the repression of gene transcription. However, the biochemical mechanisms by which histone sumoylation may undertake gene repression remain unknown. Several human diseases, including aggressive cancers of the blood and brain, are directly linked to the untimely activation of gene transcription. Hence, by elucidating the molecular mechanisms underlying transcription repression by histone sumoylation, we will identify new pathways to predictably control gene function. Toward our goals, we developed chemical strategies to access site-specifically sumoylated, methylated and acetylated histones. These histones were used in biophysical and biochemical assays to reveal the direct effect of suH4 on chromatin structure and its biochemical relationship with other modified histones. Our results, revealed that suH4 stimulates the demethylase and deacetylase activities of a key gene-silencing protein complex responsible for silencing neuronal genes in non-neuronal cells. Furthermore, when incorporated in place of H4, suH4 strongly inhibits transcription from chromatinized templates. These exciting preliminary results set the stage for our proposed specific aims: 1. To investigate the mechanism of biochemical crosstalk between histone sumoylation, methylation and acetylation. 2. To investigate the mechanism of transcription inhibition by sumoylated H4. 3. To study gene regulation by sumoylated H4 in human cells. Success in these aims will elucidate the mechanistic underpinnings of suH4-mediated gene repression in cells, and identify its biochemical crosstalk with two histone modifications that are misregulated in several human diseases. Understanding suH4's contribution to the function of multiple chromatin-regulating protein complexes will uncover new targetable pathways to control misregulated genes.