Defining neddylation pathways and glinking histone ubiquitination to histone demethylation

Covalent modification of proteins can be used to regulate enzymes' activities or their protein-protein interactions. As a rapid and generally reversible mode of regulation, it is used to quickly change the program of cells and can have a wide range of biological consequences.; Ubiquitin-like proteins modify target proteins, altering their activities or causing them to be slated for degradation. These modifications are used to efficiently regulate key events in the cell. To explore the set of proteins modified by a small ubiquitin-like protein, I have developed a proteomic approach. Affinity purification of an epitope-tagged Nedd8 allowed the identification of the majority of proteins known to be involved with the neddylation pathway. This purification not only isolated the known targets of neddylation, but the assembly of enzymes and complexes known to regulate neddylation and deneddylation, including the COP9 signalosome, Nub1, and enzymes in the neddylation cascade. This purification scheme can be applied to other small ubiquitin-like proteins, especially those with limited protein targets such as the SUMOs (1, 2 and 3), Isg15, or FAT10.; Histones can be acetylated, methylated and ubiquitylated, among other modifications. These modifications result in changes in gene regulation of nearby genes and are employed to enact diverse biological events including cell differentiation and development, as well as cell cycle and response to external stimuli. Here, through our studies on a Pcgf6/JARID1d complex, we examine the interplay of histone ubiquitination and methylation. We define a novel histone demethylase complex that can ubiquitylate histones.