Mapping lysine methyltransferase substrate selectivity reveals gaps in the proteome-wide curation of lysine methylomes.

Promoter-proximal pausing by RNA Pol II is a rate-determining step in gene transcription that is hypothesized to be a prominent point at which regulatory factors act. The pausing factor NELF is known to induce and stabilize pausing, but not all kinds of pausing are NELF-mediated. Here, we find that NELF-depleted Drosophila melanogaster cells functionally recapitulate the NELF-independent pausing we previously observed in fission yeast (which lack NELF). Critically, only NELF-mediated pausing establishes a strict requirement for Cdk9 kinase activity for the release of paused Pol II into productive elongation. Upon inhibition of Cdk9, cells with NELF efficiently shutdown gene transcription, while in NELF-depleted cells, defective, non-productive transcription continues unabated. By introducing a strict checkpoint for Cdk9, the evolution of NELF was likely critical to enable increased regulation of Cdk9 in higher eukaryotes, as Cdk9 availability can be restricted to limit gene transcription without inducing wasteful, non-productive transcription.

Transcription of DNA into RNA is a fundamental, universal, and highly regulated process in all living organisms. RNA Polymerase II (Pol II), is the enzyme that catalyzes this reaction and it is highly regulated by the pausing factors DRB Sensitivity Inducing Factor (DSIF) and Negative Elongation Factor (NELF), and elongation factors, including Positive Transcription Elongation Factor b (P‐TEFb) and Polymerase Associated Factor 1 (PAF1). However, most previous studies of these factors have only provided snapshots of factor binding or snapshots of mRNA production following their depletion. While valuable, these studies provide limited information about the time frames in which Pol II and key transcription factors interact with each other and with the DNA. Using live‐cell imaging of amplified and interphase‐extended Drosophila polytene chromosomes with endogenously tagged transcription factors and Pol II, we can analyze the real time behavior of these molecules at single genetic loci and at high resolution. In particular, heat shock genes, like heat shock protein 70 (Hsp70), have been shown to be a robust system for studying transcription because of their inducible nature; they are able to transition quickly from a paused to a highly active state. Previous work from our lab utilizing this system suggested the formation of a transcription compartment during prolonged bouts of transcription. We propose that transcription compartments form around Hsp70 loci during an extended heat shock due to the activity of Poly ADP Ribose Polymerase (PARP) polymerizing large PAR chains that trap Pol II and other transcription factors, restricting their movement and preventing free diffusion with other molecules in the nucleus. We are tagging proteins of interest with photo‐activatable fluorescent proteins to minimize background by selectively activating a population of molecules only in the small 3‐D volume of specific loci. Utilizing this system we have begun measuring the dynamics of Pol II at specific loci and are working toward measuring the dynamics of key transcription regulating factors and further characterizing PARP‐mediated transcription compartments.

System xc− is a heterodimeric plasma membrane transporter involved in the exchange of intracellular glutamate for extracellular cystine. As such, this transporter plays a critical role in the production of the antioxidant glutathione. Previous studies in our lab have demonstrated that there is an increase in cell surface expression within ten minutes of exposure to H2O2 in confluent U138MG human glioma cells. The study described herein sought to begin to characterize the mechanism by which H2O2 regulates the trafficking of xCT. We hypothesized that Akt signaling is necessary for H2O2‐mediated trafficking of of xCT. A significant increase in Akt phosphorylation was observed in U138MG cells following ten‐minute exposure to 3 mM H2O2 compared to vehicle‐treated cells using western blot analysis. Treatment with the Akt inhibitor 10‐DEBC (2.5μM) for 30 minutes prior to and during H2O2 exposure resulted in a decrease in H2O2‐induced phosphorylation of Akt at Ser473. Similar inhibition of Akt phosphorylation at Thr308 was observed following treatment of cells with 1.0μM API‐2. Next, we used simultaneous treatment of cultured glioma cells with both inhibitors in the presence of H2O2 to determine if such treatment led to a reduction in the trafficking of endogenously expressed xCT to the plasma membrane. Our preliminary data suggests that Akt activation is necessary for H2O2‐induced trafficking xCT to the plasma membrane in cultured glioma cells. To determine if the regulation of xCT cell surface expression is not limited to human glioma cells where xCT is oven overexpressed, we also studied the role Akt plays in the trafficking of recombinant, transiently‐expressed xCT in COS‐7 cells. COS‐7 cells transfected with myc‐tagged xCT, 4F2HC and a constitutively active form of Akt showed higher levels of xCT localized to the membrane compared with cells transfected with a dominant negative form of Akt. Collectively, these data suggest that Akt is an important regulator of xCT cell surface expression.Support or Funding InformationThis work was supported by NSF‐RUI 0843564.

System xc− exchanges intracellular glutamate for extracellular cystine across the membrane of many cell types, including astrocytes. Its activity directly regulates the synthesis of the antioxidant glutathione and the extracellular concentration of glutamate in some areas of the brain. Dysregulation of the transporter can lead to excessive glutamate release and excitotoxic cell death or the depletion of glutathione stores and the development of oxidative stress. We recently demonstrated that oxidants acutely upregulate System xc− activity by triggering the rapid redistribution of the transporter from intracellular compartments to the cell surface. Our current work suggests that the trafficking of the transporter may be regulated by ubiquitination and that oxidant exposure directly influences the ubiquitination of the transporter. Since increased ubiquitination tends to decrease the cell surface expression of many membrane transporters, we sought to test the hypothesis that System xc− is ubiquitinated and that the ubiquitination status of the transporter regulates both its cell surface expression and activity. We have used a mutagenesis approach to disrupt putative ubiquitination sites and a putative ubiquitin ligase binding site within a myc‐tagged System xc− construct so that we can understand the role ubiquitination plays in regulating the cell surface expression of System xc−. There are seven highly conserved lysine residues within xCT that are located on the cytoplasmic side of the membrane. These residues are located at positions 4, 37, 41, 43, 422, 472, and 473. We have created mutant forms of this construct containing single or multiple lysine to arginine mutations so that we could assess the effect of these mutations on cell surface expression of System xc‐. Using biotinylation assays and immunocytochemical analysis, we have demonstrated that mutation of the N‐terminal lysine residues increases the cell surface expression of the transporter. We are currently assessing the ubiquitination status of these mutant transporters to determine if the changes in ubiquitination of the transporter are associated with changes in the cell surface expression and activity of the transporter. In addition, we have identified a putative ubiquitin ligase binding site in the C‐terminus of the transporter. Disruption of this binding site also leads to an increase in cell surface expression of the transporter. Collectively, these data suggest that System xc− is regulated by changes in its ubiquitination status such that factors which lead to diminished ubiquitination will allow for increased cell surface expression of the transporter.Support or Funding InformationThis work was supported by RUI 0843564.