NuA4 is an essential histone H4/H2A acetyltransferase complex that interacts with activators and stimulates transcription in vitro. We have identified three novel NuA4 subunits: Act3/Arp4, an actin-related protein implicated in epigenetic control of transcription, Act1, and Epl1, a protein homologous to Drosophila Enhancer of Polycomb. Act3/Arp4 binds nucleosomes in vitro and is required for NuA4 integrity in vivo. Mutations in ACT3 and acetyltransferase-encoding ESA1 cause gene-specific transcription defects. Accordingly, NuA4 is localized in precise loci within the nucleus and does not overlap with the silent chromatin marker Sir3. These data along with the known epigenetic roles of Act3/Arp4 and homologs of Epl1 and Esa1 strongly support an essential role for chromatin structure modification by NuA4 in transcription regulation in vivo.

Growing experimental evidence indicates that, in addition to the physical virion components, the non-structural proteins of hepatitis C virus (HCV) are intimately involved in orchestrating morphogenesis. Since it is dispensable for HCV RNA replication, the non-structural viral protein NS2 is suggested to play a central role in HCV particle assembly. However, despite genetic evidences, we have almost no understanding about NS2 protein-protein interactions and their role in the production of infectious particles. Here, we used co-immunoprecipitation and/or fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy analyses to study the interactions between NS2 and the viroporin p7 and the HCV glycoprotein E2. In addition, we used alanine scanning insertion mutagenesis as well as other mutations in the context of an infectious virus to investigate the functional role of NS2 in HCV assembly. Finally, the subcellular localization of NS2 and several mutants was analyzed by confocal microscopy. Our data demonstrate molecular interactions between NS2 and p7 and E2. Furthermore, we show that, in the context of an infectious virus, NS2 accumulates over time in endoplasmic reticulum-derived dotted structures and colocalizes with both the envelope glycoproteins and components of the replication complex in close proximity to the HCV core protein and lipid droplets, a location that has been shown to be essential for virus assembly. We show that NS2 transmembrane region is crucial for both E2 interaction and subcellular localization. Moreover, specific mutations in core, envelope proteins, p7 and NS5A reported to abolish viral assembly changed the subcellular localization of NS2 protein. Together, these observations indicate that NS2 protein attracts the envelope proteins at the assembly site and it crosstalks with non-structural proteins for virus assembly.

We have discovered and developed a series of molecules (thiazole benzenesulfonamides). HA15, the lead compound of this series, displayed anti-cancerous activity on all melanoma cells tested, including cells isolated from patients and cells that developed resistance to BRAF inhibitors. Our molecule displayed activity against other liquid and solid tumors. HA15 also exhibited strong efficacy in xenograft mouse models with melanoma cells either sensitive or resistant to BRAF inhibitors. Transcriptomic, proteomic, and biochemical studies identified the chaperone BiP/GRP78/HSPA5 as the specific target of HA15 and demonstrated that the interaction increases ER stress, leading to melanoma cell death by concomitant induction of autophagic and apoptotic mechanisms.

For gene regulation, some transcriptional activators bind periodically to promoters with either a fast (approximately 1 minute) or a slow (approximately 15 to 90 minutes) cycle. It is uncertain whether the fast cycle occurs on natural promoters, and the function of either cycle in transcription remains unclear. We report that fast and slow cycling can occur simultaneously on an endogenous yeast promoter and that slow cycling in this system reflects an oscillation in the fraction of accessible promoters rather than the recruitment and release of stably bound transcriptional activators. This observation, combined with single-cell measurements of messenger RNA (mRNA) production, argues that fast cycling initiates transcription and that slow cycling regulates the quantity of mRNA produced. These findings counter the prevailing view that slow cycling initiates transcription.