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.

Grant Number: 1R21DA062828-01
NIH Institute/Center: NIH
Principal Investigator: Bassem Al-Sady