Protein lysine methyltransferases G9a and GLP modulate the transcriptional repression of a variety of genes via dimethylation of Lys9 on histone H3 (H3K9me2) as well as dimethylation of non-histone targets. Here we report the discovery of UNC0638, an inhibitor of G9a and GLP with excellent potency and selectivity over a wide range of epigenetic and non-epigenetic targets. UNC0638 treatment of a variety of cell lines resulted in lower global H3K9me2 levels, equivalent to levels observed for small hairpin RNA knockdown of G9a and GLP with the functional potency of UNC0638 being well separated from its toxicity. UNC0638 markedly reduced the clonogenicity of MCF7 cells, reduced the abundance of H3K9me2 marks at promoters of known G9a-regulated endogenous genes and disproportionately affected several genomic loci encoding microRNAs. In mouse embryonic stem cells, UNC0638 reactivated G9a-silenced genes and a retroviral reporter gene in a concentration-dependent manner without promoting differentiation.

SIRT6 is a member of the evolutionarily conserved sirtuin family of NAD ؉ -dependent protein deacetylases and functions in genomic stability and transcriptional control of glucose metabolism. Early reports suggested that SIRT6 performs ADPribosylation, whereas more recent studies have suggested that SIRT6 functions mainly as a histone deacetylase. Thus, the molecular functions of SIRT6 remain uncertain. Here, we perform biochemical, kinetic, and structural studies to provide new mechanistic insight into the functions of SIRT6. Utilizing three different assays, we provide biochemical and kinetic evidence that SIRT6-dependent histone deacetylation produces Oacetyl-ADP-ribose but at a rate ϳ1,000 times slower than other highly active sirtuins. To understand the molecular basis for such low deacetylase activity, we solved the first crystal structures of this class IV sirtuin in complex with ADP-ribose and the non-hydrolyzable analog of O-acetyl-ADP-ribose, 2 -N-acetyl-ADP-ribose. The structures revealed unique features of human SIRT6, including a splayed zinc-binding domain and the absence of a helix bundle that in other sirtuin structures connects the zinc-binding motif and Rossmann fold domain. SIRT6 also lacks the conserved, highly flexible, NAD ؉ -binding loop and instead contains a stable single helix. These differences led us to hypothesize that SIRT6, unlike all other studied sirtuins, would be able to bind NAD ؉ in the absence of an acetylated substrate. Indeed, we found that SIRT6 binds NAD ؉ with relatively high affinity (K d ‫؍‬ 27 ؎ 1 M) in the absence of an acetylated substrate. Isothermal titration calorimetry and tryptophan fluorescence binding assays suggested that ADP-ribose and NAD ؉ induce different structural perturbations and that NADH does not bind to SIRT6. Collectively, these new insights imply a unique activating mechanism and/or the possibility that SIRT6 could act as an NAD ؉ metabolite sensor.Sirtuins comprise an ancient and diverse family of nicotinamide adenine dinucleotide (NAD ϩ )-dependent protein deacetylases that are evolutionarily conserved from bacteria to eukaryotes (1). Unlike other classes of histone deacetylases, which utilize an active site Zn 2ϩ and involve direct attack of a water molecule on acetylated lysines, sirtuins transfer the acetyl group from the lysine side chain of a protein or peptide substrate to the co-factor NAD ϩ , generating nicotinamide, 2Ј-Oacetyl-ADP-ribose (OAADPr) 3 (2, 3), and a deacetylated substrate. This unique requirement of NAD ϩ suggests that sirtuins might act as sensors of the cellular metabolic state (4), relaying changes in cellular metabolism to reverse acetylation-mediated pathways, which include transcription, cell cycle progression, genome maintenance, apoptosis, and organism longevity. The founding member of the sirtuin family, yeast Sir2 (silent information regulator 2), has emerged as an important regulator in extending the life span of Saccharomyces cerevisiae (5). In other organisms, such as Caenorhabditis elegans and Drosophila me...

The CEBPA gene is mutated in 9% of patients with acute myeloid leukemia (AML). Selective expression of a short 30 kDa C/EBPα translational isoform, termed p30, represents the most common type of CEBPA mutations in AML. The molecular mechanisms underlying p30-mediated transformation remain incompletely understood. We show that C/EBPα p30, but not the normal p42 isoform, preferentially interacts with Wdr5, a key component of SET/MLL histone-methyltransferase complexes. Accordingly, p30-bound genomic regions were enriched for MLL-dependent H3K4me3 marks. The p30-dependent increase in self-renewal and inhibition of myeloid differentiation required Wdr5, as its down-regulation inhibited proliferation and restored differentiation in p30-dependent AML models. OICR-9429 is a novel small-molecule antagonist of the Wdr5-MLL interaction. This compound selectively inhibited proliferation and induced differentiation in p30-expressing human AML cells. Our data reveal the mechanism of p30-dependent transformation and establish the essential p30-cofactor Wdr5 as a therapeutic target in CEBPA-mutant AML.