Mediator Acts Upstream of the Transcriptional Activator Gal4

We show that Mediator, a protein originally isolated on the basis of its ability to respond to transcriptional activators, and thought to be regulated by an activator, can also be the master that controls the activator

Chromatin-Dependent Cooperativity between Constitutive and Inducible Activation Domains in CREB

The cyclic AMP (cAMP)-responsive factor CREB induces target gene expression via constitutive (Q2) and inducible (KID, for kinase-inducible domain) activation domains that function synergistically in response to cellular signals. KID stimulates transcription via a phospho (Ser133)-dependent interaction with the coactivator paralogs CREB binding protein and p300, whereas Q2 recruits the TFIID complex via a direct association with hTAF(II)130. Here we investigate the mechanism underlying cooperativity between the Q2 domain and KID in CREB by in vitro transcription assay with naked DNA and chromatin templates containing the cAMP-responsive somatostatin promoter. The Q2 domain was highly active on a naked DNA template, and Ser133 phosphorylation had no additional effect on transcriptional initiation in crude extracts. Q2 activity was repressed on a chromatin template, however, and this repression was relieved by the phospho (Ser133) KID-dependent recruitment of p300 histone acetyltransferase activity to the promoter. In chromatin immunoprecipitation assays of NIH 3T3 cells, cAMP-dependent recruitment of p300 to the somatostatin promoter stimulated acetylation of histone H4. Correspondingly, overexpression of hTAFII130 potentiated CREB activity in cells exposed to cAMP, but had no effect on reporter gene expression in unstimulated cells. We propose that cooperativity between the KID and Q2 domains proceeds via a chromatin-dependent mechanism in which recruitment of p300 facilitates subsequent interaction of CREB with TFIID

The mammalian-membrane two-hybrid assay (MaMTH) for probing membrane-protein interactions in human cells

Cell signaling, one of the key processes involved in human health and disease, is coordinated by numerous membrane protein-protein interactions (PPIs) that change in response to stimuli. Currently, there is a lack of assays that can detect these changes in stimuli- and disease-related contexts. Here, we present a novel split-ubiquitin-method for the detection of integral membrane PPIs in human cells, termed Mammalian-Membrane-Two-Hybrid (MaMTH). We highlight the strength of this technology by showing that it detects stimuli (hormone/agonist)- and phosphorylationdependent PPIs. Importantly, it can detect changes in PPIs conferred by mutations such as those in oncogenic ErbB-receptor variants or by treatment with drugs like the tyrosine-kinase inhibitor erlotinib. Using MaMTH as a screening assay, we identified CRKII as an interactor of oncogenic EGFRL858R, promoting persistent activation of aberrant signaling. In conclusion, our study illustrates that MaMTH is a powerful tool for investigating dynamic interactomes of human integral membrane proteins.The work was supported by grants from the Ontario Genomics Institute (303547), Canadian Institutes of Health Research (Catalyst - NHG99091; ppp-125785), Canadian Foundation for Innovation (IOF-LOF), Natural Sciences and Engineering Research Council of Canada (RGPIN 372393-12), Canadian Cystic Fibrosis Foundation (300348), Canadian Cancer Society (2010-700406), Novartis, UNiversity Health Network (GL2-01-018), FWF-Erwin Schrödinger Fellowship progra