Protein acetylation is a key post-translational modification that regulates diverse biological activities in eukaryotes. Here we report bioorthogonal chemical reporters that enable direct in-gel fluorescent visualization and proteome-wide identification of acetylated proteins via Cu(I)-catalyzed azide-alkyne cycloaddition, often termed "click chemistry". We demonstrate that two alkynyl-acetyl-CoA analogues, 4-pentynoyl-CoA and 5-hexynoyl-CoA, function as efficient substrates of lysine acetyltransferase p300 and serve as sensitive reagents for monitoring p300-catalyzed protein acetylation in vitro. In addition, we demonstrate that three alkynylacetate analogues, sodium 3-butynoate, sodium 4-pentynoate, and sodium 5-hexynoate, can be metabolically incorporated onto cellular proteins through biosynthetic mechanisms for profiling of acetylated proteins in diverse cell types. Mass spectrometry analysis of the enriched 4-pentynoate-labeled proteins revealed many reported as well as new candidate acetylated proteins from Jurkat T cells and specific sites of lysine acetylation. The bioorthogonal chemical reporters described here should serve as powerful tools for investigating protein acetylation.

JAGGED1 is a member of the Delta/Serrate/Lag-2 (DSL) family of proteins that are cell-bound ligands for Notch receptors. Initiation of Notch signaling occurs through a series of proteolytic events upon the binding of Notch to a DSL protein presented on neighboring cells. Whether DSL proteins themselves are capable of initiating an intrinsic signaling mechanism within the cell they are expressed is not known. Aberrant misexpression of JAGGED1 and DELTA1 has been documented in several human tumors; however, the mechanism by which misexpression of JAGGED1 contributes to oncogenesis has not been elucidated. We report that expression of human JAGGED1 transforms RKE cells in culture, therefore providing a model system to elucidate the function of DSL proteins. JAGGED1-mediated transformation occurs in a dose-dependent manner and requires a PDZ-ligand at the C terminus. Mutation of the PDZ-ligand did not affect the ability of JAGGED1 to initiate Notch signaling in neighboring cells. However, the PDZ-ligand is required for changes in the expression of JAGGED1 target genes and transcriptional activation of luciferase reporter constructs. Our data indicate the existence of a novel PDZ-dependent signaling pathway intrinsic to JAGGED1. We propose a bi-directional signaling model such that DSL proteins may have two distinct functions: to initiate Notch signaling in a neighboring cell and to initiate a PDZ-dependent signaling mechanism in the DSL-expressing cell. Moreover, we conclude that this intrinsic signaling mechanism of JAGGED1 may partly provide a link between aberrant misexpression of JAGGED1 and tumorigenesis.

c-Myc is frequently overexpressed in tumors and plays an important role in the regulation of cancer metabolism. Hypoxia-inducible factor-1 (HIF1), the master regulator of the hypoxic response, enhances tumorigenesis and influences metabolism via upregulation of the glycolytic pathway and suppression of mitochondrial respiration. Together, deregulated Myc and HIF1 cooperate to lend metabolic advantages to proliferating cancer cells and contribute to the Warburg Effect. Here we show that overexpression of Myc significantly stabilizes the alpha subunit of HIF1 (HIF1alpha) under normoxic conditions and enhances HIF1alpha accumulation under hypoxic conditions in cells. Post-transcriptional regulation of HIF1α by Myc led to the induction of HIF1α gene targets. Normoxic HIF1α protein expression was also dependent on Myc. Functionally; HIF1α expression was required for Myc-induced anchorage-independent growth and cell proliferation. Myc-dependent stabilization of HIF1α involved either disruption of binding to the VHL complex or post-translational protein modifications. Taken together, our findings uncover a previously uncharacterized regulatory relationship between Myc and HIF1 that has important implications for cancer metabolism and development.

SUMMARY Many genes are regulated at the level of a Pol II that is recruited to a nucleosome-free region upstream of the +1 nucleosome. How the Mediator coactivator complex, which functions at multiple steps, affects transcription through the promoter proximal region, including this nucleosome, remains largely unaddressed. We have established a fully defined in vitro assay system to delineate mechanisms for Pol II transit across the +1 nucleosome. Our results reveal cooperative functions of multiple cofactors, particularly of Mediator and elongation factor SII, in transcribing into this nucleosome. This is achieved, in part, through an unusual activity of SII that alters the intrinsic catalytic properties of promoter-proximal Pol II and, in concert with the Mediator, leads to enhancement in transcription of nucleosomal DNA. Our data provide additional mechanistic bases for Mediator function after recruitment of Pol II and, potentially, for regulation of genes controlled via nucleosome-mediated promoter-proximal pausing.

Forkhead factors are important regulators of animal development and homeostasis. They are among the earliest to bind quiescent genes, which they activate in conjunction with other transcription factors. Many liver-specific genes are under the control of FoxA2, a liver-enriched forkhead protein. Here we confirmed by chromatin immunoprecipitation that FoxA2 is one of the factors bound to the promoter-proximal enhancer of the gene encoding apolipoprotein AI (a component of high density lipoprotein) and that it functions in synergy with the nuclear receptor hepatocyte nuclear factor-4␣. Furthermore, toward identifying additional cofactors that could potentially regulate FoxA2 activity, we identified DNA-dependent protein kinase (DNA-PK) as a FoxA2-associated factor upon affinity purification of epitope-tagged FoxA2. We show that FoxA2, found to be a phosphoprotein in vivo, is also an efficient substrate for DNA-PK, which targets serine 283. This residue is contained within a conserved serine-glutamine phosphorylation signal for DNA-PK, located within the C-terminal third of the polypeptide, just distal to its winged-helix DNA binding domain. We establish that this residue is critical for FoxA2 function because FoxA2 bearing a mutation at this site is severely compromised in its ability to activate a reporter gene under the control of its cognate DNA-binding site (apoAI site B). Complementary experiments rule out that this mutation compromises the ability of FoxA2 to either translocate to the nucleus or to bind site B. We therefore conclude that DNA-PK-dependent phosphorylation of FoxA2 plays a critical role in its transcriptional activation function per se.