Selective Inhibition of p300 HAT Blocks Cell Cycle Progression, Induces Cellular Senescence, and Inhibits the DNA Damage Response in Melanoma Cells

Epigenetic events, including covalent post-translational modifications of histones, have been demonstrated to have critical roles in tumor development and progression. The transcriptional coactivator p300/CBP possesses both histone acetyltransferase (HAT) activity and scaffolding properties that directly influence the transcriptional activation of targeted genes. We have used a potent and specific inhibitor of p300/CBP HAT activity, C646, in order to evaluate the functional contributions of p300/CBP HAT to tumor development and progression. Here we report that C646 inhibits the growth of human melanoma and other tumor cells and promotes cellular senescence. Global assessment of the p300 HAT transcriptome in human melanoma identified functional roles in promoting cell cycle progression, chromatin assembly, and activation of DNA repair pathways through direct transcriptional regulatory mechanisms. In addition, C646 is shown to promote sensitivity to DNA damaging agents, leading to the enhanced apoptosis of melanoma cells after combination treatment with cisplatin. Together, our data suggest that p300 HAT activity mediates critical growth regulatory pathways in tumor cells and may serve as a potential therapeutic target for melanoma and other malignancies by promoting cellular responses to DNA damaging agents that are currently ineffective against specific cancers

The transcriptional coactivator MAML1 regulates p300 autoacetylation and HAT activity

MAML1 is a transcriptional coregulator originally identified as a Notch coactivator. MAML1 is also reported to interact with other coregulator proteins, such as CDK8 and p300, to modulate the activity of Notch. We, and others, previously showed that MAML1 recruits p300 to Notch-regulated genes through direct interactions with the DNA–CSL–Notch complex and p300. MAML1 interacts with the C/H3 domain of p300, and the p300–MAML1 complex specifically acetylates lysines of histone H3 and H4 tails in chromatin in vitro. In this report, we show that MAML1 potentiates p300 autoacetylation and p300 transcriptional activation. MAML1 directly enhances p300 HAT activity, and this coincides with the translocation of MAML1, p300 and acetylated histones to nuclear bodies

Protein Lysine Acetylation by p300/CBP

Protein Lysine Acetylation by p300/CB

A screening-based platform for the assessment of cellular respiration in Caenorhabditis elegans

Mitochondrial dysfunction is at the core of many diseases ranging from inherited metabolic diseases to common conditions that are associated with aging. Although associations between aging and mitochondrial function have been identified using mammalian models, much of the mechanistic insight has emerged from Caenorhabditis elegans. Mitochondrial respiration is recognized as an indicator of mitochondrial health. The Seahorse XF96 respirometer represents the state-of-the-art platform for assessing respiration in cells, and we adapted the technique for applications involving C. elegans. Here we provide a detailed protocol to optimize and measure respiration in C. elegans with the XF96 respirometer, including the interpretation of parameters and results. The protocol takes similar to 2 d to complete, excluding the time spent culturing C. elegans, and it includes (i) the preparation of C. elegans samples, (ii) selection and loading of compounds to be injected, (iii) preparation and execution of a run with the XF96 respirometer and (iv) postexperimental data analysis, including normalization. In addition, we compare our XF96 application with other existing techniques, including the eight-well Seahorse XFp. The main benefits of the XF96 include the limited number of worms required and the high throughput capacity due to the 96-well format