Publications by Year: 2014

Promoter-proximal pausing of RNA polymerase II (Pol II) is a major checkpoint in transcription. An unbiased search for new human proteins that could regulate paused Pol II at the HSPA1B gene identified TRIM28. In vitro analyses indicated HSF1-dependent attenuation of Pol II pausing upon TRIM28 depletion, whereas in vivo data revealed de novo expression of HSPA1B and other known genes regulated by paused Pol II upon TRIM28 knockdown. These results were supported by genome-wide ChIP-sequencing analyses of Pol II occupancy that revealed a global role for TRIM28 in regulating Pol II pausing and pause release. Furthermore, in vivo and in vitro mechanistic studies suggest that transcription-coupled phosphorylation regulates Pol II pause release by TRIM28. Collectively, our findings identify TRIM28 as a new factor that modulates Pol II pausing and transcriptional elongation at a large number of mammalian genes.

Protease mediated peptide synthesis (PMPS) was first described in the 1930s but remains underexploited today. In most PMPS, the reaction equilibrium is shifted toward synthesis by the aqueous insolubility of product generated. Substrates and proteases are selected by trial and error, yields are modest, and reaction times are slow. Once implemented, however, PMPS reactions can be simple, environmentally benign, and readily scalable to a commercial level. We examined the PMPS of a precursor of the artificial sweetener aspartame, a multiton peptide synthesis catalyzed by the enzyme thermolysin. X-ray structures of thermolysin in complex with aspartame substrates separately, and after PMPS in a crystal, rationalize the reaction's substrate preferences and reveal an unexpected form of substrate inhibition that explains its sluggishness. Structure guided optimization of this and other PMPS reactions could expand the economic viability of commercial peptides beyond current high-potency, low-volume therapeutics, with substantial green chemistry advantages.

The Ets1 transcription factor is a member of the Ets protein family, a group of evolutionarily related DNA-binding transcriptional factors. Ets proteins activate or repress the expression of genes that are involved in various biological processes, including cellular proliferation, differentiation, development, transformation and apoptosis. FOXO1 is a member of the forkhead-box proteins (FOX proteins), which comprise a large family of functionally diverse transcription factors involved in cellular proliferation, transformation and differentiation. The FOXO subgroup of FOX proteins regulates the transcription of genes that control metabolism, cell survival, cellular proliferation, DNA damage responses, stress resistance and longevity. The DNA-binding domains (DBDs) of Ets1 and FOXO1 were crystallized in complex with DNA containing a composite sequence for a noncanonical forkhead binding site (AATAACA) and an ETS site (GGAA), FOX:ETS, by the sitting-drop vapor-diffusion method. The FOX:ETS motif has been shown to be a conserved cis-acting element in several endothelial cell-specific genes, including Vegfr2, Tie2, Mef2c and ve-cadherin. Crystals were grown at 291 K using 30% polyethylene glycol 400, 50 mM Tris pH 8.5, 100 mM KCl, 10 mM MgCl2 as the reservoir solution. The crystals belonged to space group C222(1), with unit-cell parameters a = 68.7, b = 104.9, c = 136.3 Å. Diffraction data were collected to a resolution of 2.2 Å.