This paper identifies the N-acetylglucosamine transferase OGT as binding partner for TET2/3 proteins. Their genome-wide chromatin binding and the characterization of the Set1/COMPASS complex as OGT target implies coordinated gene regulation.

The two tandem bromodomains of the BET proteins enable chromatin binding to facilitate transcription. Drugs that inhibit both bromodomains equally have shown efficacy in certain malignant and inflammatory conditions. To explore the individual functional contributions of the first (BD1) and second (BD2) bromodomains in biology and therapy, we developed selective BD1 and BD2 inhibitors. We found that steady-state gene expression primarily requires BD1 whereas the rapid increase of gene expression induced by inflammatory stimuli requires both BD1 and BD2 of all BET proteins. BD1 inhibitors phenocopied the effects of pan-BET inhibitors in cancer models whereas BD2 inhibitors were predominantly effective in models of inflammatory and autoimmune disease. These insights into the differential requirement of BD1 and BD2 for the maintenance and induction of gene expression may guide future BET targeted therapies.

Wnt growth factors mediate cell fate determination during embryogenesis and in the renewal of tissues in the adult. Wnts act by stabilizing cellular levels of the transcriptional coactivator beta-catenin, which forms complexes with sequence-specific DNA-binding Tcf/Lef transcription factors. In the absence of nuclear beta-catenin, Tcf/Lefs act as transcriptional repressors by binding to Groucho/TLE proteins. The molecular basis of the switch from transcriptional repression to activation during Wnt signaling has not been clear, in particular whether factors other than beta-catenin are required to disrupt the interaction between Groucho/TLE and Tcf/Lef. Using highly purified proteins, we demonstrate that beta-catenin displaces Groucho/TLE from Tcf/Lef by binding to a previously unidentified second, low-affinity binding site on Lef-1 that includes sequences just N-terminal to the DNA-binding domain, and that overlaps the Groucho/TLE-binding site.

Dynamic interactions between proteins comprise a key mechanism for temporal control of cellular function and thus hold promise for development of novel drug therapies. It remains technically challenging, however, to quantitatively characterize these interactions within the biologically relevant context of living cells. Although, bioluminescence resonance energy transfer (BRET) has often been used for this purpose, its general applicability has been hindered by limited sensitivity and dynamic range. We have addressed this by combining an extremely bright luciferase (Nanoluc) with a means for tagging intracellular proteins with a long-wavelength fluorophore (HaloTag). The small size (19 kDa), high emission intensity, and relatively narrow spectrum (460 nm peak intensity) make Nanoluc luciferase well suited as an energy donor. By selecting an efficient red-emitting fluorophore (635 nm peak intensity) for attachment onto the HaloTag, an overall spectral separation exceeding 175 nm was achieved. This combination of greater light intensity with improved spectral resolution results in substantially increased detection sensitivity and dynamic range over current BRET technologies. Enhanced performance is demonstrated using several established model systems, as well as the ability to image BRET in individual cells. The capabilities are further exhibited in a novel assay developed for analyzing the interactions of bromodomain proteins with chromatin in living cells.

SUMMARY The transcription factor HIF1A is a key mediator of the cellular response to hypoxia. Despite the importance of HIF1A in homeostasis and various pathologies, little is known about how it regulates RNA polymerase II (RNAPII). We report here that HIF1A employs a specific variant of the Mediator complex to stimulate RNAPII elongation. The Mediator-associated kinase CDK8, but not the paralog CDK19, is required for induction of many HIF1A target genes. HIF1A induces binding of CDK8-Mediator and the Super Elongation Complex (SEC), containing AFF4 and CDK9, to alleviate RNAPII pausing. CDK8 is dispensable for HIF1A chromatin binding and histone acetylation, but it is essential for binding of SEC and RNAPII elongation. Global analysis of active RNAPII reveals that hypoxia-inducible genes are paused and active prior to their induction. Our results provide a mechanistic link between HIF1A and CDK8, two potent oncogenes, in the cellular response to hypoxia.