Biochemically counteracting maladaptive functions of G9a/GLP in addiction - ABSTRACT The development of addictive behaviors to stimulants and opiates requires changes in the reward center of the brain, in particular, the Nucleus Accumbens. Animal studies and examination of postmortem human cocaine users have indicated a decrease in some gene repressive-epigenetic modifiers, such as the histone methyl transferases G9a and its paralog G9a-like protein (GLP), which methylates histone 3 (H3) lysine 9 (K9). Decreases in these repressive modifiers and concomitant increases in gene-activating chromatin marks are thought to induce the expression of genes involved in neuroplasticity in the Nucleus Accumbens, facilitating the development of maladaptive addiction behavior. Animal models of cocaine addiction indicate that G9a is involved in the addiction process. However, while its involvement is well documented, whether G9a acts adaptively or maladaptively, remains unresolved and depends on the method of G9a manipulation (conditional versus local untargeted knockout) and addiction model (contingent and non-contingent). Two challenges exist in identifying the G9a/GLP molecular function in addition and then targeting it therapeutically: 1. G9a and GLP have a wide range of functions. Because G9a and GLP are obligate dimers and can form three dimers (G9a, GLP homodimers, and G9a-GLP heterodimer), it is unclear whether each dimer has a different function in addiction, potentially yielding opposing results in different studies. Further, beyond H3K9 methylation, G9a and GLP have nonhistone targets and are part of multiple corepressor complexes. 2. Due to G9a/GLP's gene-regulatory roles in many tissues, all the various inhibitors developed against this methyltransferase remain in preclinical development, given their significant toxicity. The central aim of this proposal is to develop ways to target G9a/GLP activity that is not reliant on catalytic site inhibition. This proposal has two central deliverables: 1. We will identify surfaces that enable the specific manipulation of any one G9a/GLP dimer and its activity on chromatin for future small molecule therapy, 2. The identification of these surfaces allows querying in animal models how each dimer, chromatin-bound or not, contributes to addiction phenotypes. We accomplish these deliverables by leveraging our significant biochemical expertise on G9a and GLP. Specifically, we will determine the molecular mechanism and structure of the G9a-GLP complex on a substrate and reaction intermediate nucleosome. Additionally, we will define the molecular surface that weakens specifically one of the possible dimers. We accomplish this by cryo-electron microscopy structure determination, crosslinking mass spectrometry, and biochemical characterization of G9a/GLP mutants. Further, to initially document the contribution of the chromatin- bound complex or specific dimers, we will examine the transcriptomic and epigenomic impacts of mutants in neural progenitor cells. This proposal does not directly develop a treatment approach for addiction. Instead, we recognize that more insight into the mechanism of G9a/GLP in stimulant addiction is required for the development of such treatments, and our lab is uniquely positioned to elucidate them.
