Mechanism of Action of ING4 as a Transcriptional Coactivator of p53

ING4 belongs to a family of proteins that have been implicated in tumor suppression and has been linked to the activation of p53 target genes in a p53- dependent manner. However the mechanism by which it interacts with p53 to activate transcription from these genes is unclear. In this study I use in vitro reconstitutions of cellular processes to biochemically dissect the activity of the ING4 complex, complementing the cell based assays that give a more physiological but less defined window into ING4’s effect on p53 transcriptional activity. Purification of the ING4 complex allowed verification of previously known subunits and identification of BRPF1/2/3 as a core ING4 component. Reconstitution of the ING4 complex allowed the identification of two distinct variants of the ING4 complex which utilized either JADE or BRPF as the central scaffold subunit. Both these variants were required to recapitulate the histone acetyltransferase activity of the endogenous complex. Using these complexes, I demonstrate that the ING4 complex histone modifying activity is not directly affected by p53 but is rather indirectly modulated through p53-dependent p300 activity and enhanced by the presence of the p53-dependent trimethylated H3K4 histone mark. I also establish an ING4 complex-mediated p53-dependent transcription system on chromatin templates and show that the ING4 complex has a direct effect on p53 dependent transcription. Additionally, the same conditions that enhance ING4 complex histone acetylation, namely p300 and trimethylated H3K4, also enhance the ING4 complex’ effects on p53 dependent transcription. Taken together, these results establish ING4 as a transcriptional coactivator of p53 and suggest two mechanisms by which p53 affects ING4 complex histone modification and transcriptional activity

PRMT5-mediated regulation of developmental myelination

Oligodendrocytes (OLs) are the myelin-forming cells of the central nervous system. They are derived from differentiation of oligodendrocyte progenitors through a process requiring cell cycle exit and histone modifications. Here we identify the histone arginine methyl-transferase PRMT5, a molecule catalyzing symmetric methylation of histone H4R3, as critical for developmental myelination. PRMT5 pharmacological inhibition, CRISPR/cas9 targeting, or genetic ablation decrease p53-dependent survival and impair differentiation without affecting proliferation. Conditional ablation of Prmt5 in progenitors results in hypomyelination, reduced survival and differentiation. Decreased histone H4R3 symmetric methylation is followed by increased nuclear acetylation of H4K5, and is rescued by pharmacological inhibition of histone acetyltransferases. Data obtained using purified histones further validate the results obtained in mice and in cultured oligodendrocyte progenitors. Together, these results identify PRMT5 as critical for oligodendrocyte differentiation and developmental myelination by modulating the cross-talk between histone arginine methylation and lysine acetylation

Discovery of a chemical probe for PRDM9

PRDM9 is a PR domain containing protein which trimethylates histone 3 on lysine 4 and 36. Its normal expression is restricted to germ cells and attenuation of its activity results in altered meiotic gene transcription, impairment of double-stranded breaks and pairing between homologous chromosomes. There is growing evidence for a role of aberrant expression of PRDM9 in oncogenesis and genome instability. Here we report the discovery of MRK-740, a potent (IC50: 80 ± 16 nM), selective and cell-active PRDM9 inhibitor (Chemical Probe). MRK-740 binds in the substrate-binding pocket, with unusually extensive interactions with the cofactor S-adenosylmethionine (SAM), conferring SAM-dependent substrate-competitive inhibition. In cells, MRK-740 specifically and directly inhibits H3K4 methylation at endogenous PRDM9 target loci, whereas the closely related inactive control compound, MRK-740-NC, does not. The discovery of MRK-740 as a chemical probe for the PRDM subfamily of methyltransferases highlights the potential for exploiting SAM in targeting SAM-dependent methyltransferases