Summary Transcription factors control cell specific gene expression programs through interactions with diverse coactivators and the transcription apparatus. Gene activation may involve DNA loop formation between enhancer-bound transcription factors and the transcription apparatus at the core promoter, but this process is not well understood. We report here that Mediator and Cohesin physically and functionally connect the enhancers and core promoters of active genes in embryonic stem cells. Mediator, a transcriptional coactivator, forms a complex with Cohesin, which can form rings that connect two DNA segments. The Cohesin loading factor Nipbl is associated with Mediator/Cohesin complexes, providing a means to load Cohesin at promoters. DNA looping is observed between the enhancers and promoters occupied by Mediator and Cohesin. Mediator and Cohesin occupy different promoters in different cells, thus generating cell-type specific DNA loops linked to the gene expression program of each cell.

In this chapter, we review the mechanism of action of lithium salts from a chemical perspective. A description on how lithium salts are used to treat mental illnesses, in particular bipolar disorder, and other disease states is provided. Emphasis is not placed on the genetics and the psychopharmacology of the ailments for which lithium salts have proven to be beneficial. Rather we highlight the application of chemical methodologies for the characterization of the cellular targets of lithium salts and their distribution in tissues.

It is important to note that components of the cohesin and mediator complexes, including Smc1a, Nipbl, Rad21 and Med10, were identified in previous RNA interference screens for maintenance of embryonic stem cells [1][2][3] .

G-proteins play a central role in signal transduction by fluctuating between “on” and “off” phases that are determined by a conformational change. cAMP is a secondary messenger whose formation is inhibited or stimulated by activated Giα1 or Gsα subunit. We used tryptophan fluorescence, UV/vis spectrophotometry, and circular dichroism to probe distinct structural features within active and inactive conformations from wild-type and tryptophan mutants of Giα1 and Gsα. For all proteins studied, we found that the active conformations were more stable than the inactive conformations, and upon refolding from higher temperatures, activated wild-type subunits recovered significantly more native structure. We also observed that the wild-type subunits partially regained the ability to bind nucleotide. The increased compactness observed upon activation was consistent with the calculated decrease in solvent accessible surface area for wild-type Giα1. We found that as the temperature increased, Gα subunits, which are known to be rich in α-helices, converted to proteins with increased content of β-sheets and random coil. For active conformations from wild-type and tryptophan mutants of Giα1, melting temperatures indicated that denaturation starts around hydrophobic tryptophan microenvironments and then radiates toward tyrosine residues at the surface, followed by alteration of the secondary structure. For Gsα, however, disruption of secondary structure preceded unfolding around tyrosine residues. In the active conformations, a π-cation interaction between essential arginine and tryptophan residues, which was characterized by a fluorescence-measured red shift and modeled by molecular dynamics, was also shown to be a contributor to the stability of Gα subunits. The folding properties of Gα subunits reported here are discussed in the context of diseases associated to G-proteins.

Guanine nucleotide binding proteins (G‐proteins), in conjunction with their receptors (GPCR), are one of the most prevalent signaling systems and are involved in the regulation of an array of cellular processes. Upon binding of an extracellular stimulus to the GPCR, the activated α‐subunit (Gα) exchanges GDP for GTP, releases both the βγ‐complex and the GPCR, and associates with an effector that further relays the signal. The signal terminates with the hydrolysis of GTP to GDP, causing the α‐subunit to reunite with the βγ‐subunits and the GPCR. G‐protein signaling must be tightly regulated to ensure the appropriate responses to extracellular stimuli. Any misregulation caused by mutations can be detrimental. Gsα and Giα1 are G‐protein subunits that, respectively, stimulate or inhibit adenylyl cyclase and regulate the production of the secondary messenger, cAMP. We studied two mutations observed in cancers of the intestine (R231H mutation in Gsα) and the colon (R208Q mutation in Giα1). Spectroscopic and crystallographic approaches were used to further understand these disease states.Fluorescence spectroscopy, circular dichroism, and UV/Vis spectroscopy showed lower melting temperature in the active conformation of the oncogenic mutants, indicating disruptions in the noncovalent interactions. Using malachite green and fluorescence GTP assays, we found that the kcat values for oncogenic Giα1 were lower than for the WT protein; the trend was the opposite for the WT and oncogenic Gsα proteins. When comparing the x‐ray structure of the R208Q mutant with that of the WT Giα1, the oncogenic point mutations do not show significant differences in the structure of the protein as a whole. However, molecular dynamics simulations modeling the interactions of T181 in Giα1 and T204 in Gsα (key residues in the hydrolysis of GTP) show drastically different interaction energies between mutants and WT proteins. Taken together, these findings suggest that the altered rates of GTP hydrolysis lead to an imbalance of cAMP in tumor cells.Support or Funding InformationNIH R15GM112025This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.