Regulatory Elements Outside Established Pou5f1 Gene Boundaries Are Required for Multilineage Differentiation of Embryonic Stem Cells.

The transcription factor Oct4 can rightfully be considered a pivotal element in maintaining pluripotency. In addition, its ability to function as a pioneer factor enables the reprogramming of somatic cells back into a pluripotent state. To better understand the regulation of the Oct4-encoding gene (Pou5f1), the main genetic elements that regulate its expression in different states of pluripotency ought to be identified. While some elements have been well characterized for their ability to drive Pou5f1 expression, others have yet to be determined. In this work, we show that translocation of the Pou5f1 gene fragment purported to span all essential cis-elements, including the well-known distal and proximal enhancers (DE and PE), into the Rosa26 locus impairs the self-renewal of mouse embryonic stem cells (ESCs) in the naïve pluripotency state, as well as their further advancement through the formative and primed pluripotency states, inducing overall differentiation failure. These results suggest that regulatory elements located outside the previously determined Pou5f1 boundaries are critical for the proper spatiotemporal regulation of this gene during development, indicating the need for their better characterization.

Membrane Permeability of Modified Butyltriphenylphosphonium Cations.

The alkyltriphenylphosphonium (TPP) group is the most widely used vector targeted to mitochondria. Previously, the length of the alkyl linker was varied as well as structural modifications in the TPP phenyl rings to obtain the optimal therapeutic effect of a pharmacophore conjugated with a lipophilic cation. In the present work, we synthesized butyltriphenylphosphonium cations halogenated and methylated in phenyl rings (C4TPP-X) and measured electrical current through a planar lipid bilayer in the presence of C4TPP-X. The permeability of C4TPP-X varied in the range of 6 orders of magnitude and correlates well with the previously measured translocation rate constant for dodecyltriphenylphosphonium analogues. The partition coefficient of the butyltriphenylphosphonium analogues obtained by calculating the difference in the free energy of cation solvation in water and octane using quantum chemical methods correlates well with the permeability values. Using an ion-selective electrode, a lower degree of accumulation of analogues with halogenated phenyl groups was found on isolated mitochondria of rat liver, which is in agreement with their permeability decrease. Our results indicate the translocation of the butyltriphenylphosphonium cations across the hydrophobic membrane core as rate-limiting stage in the permeability process rather than their binding/release to/from the membrane.

Lipophilic ion aromaticity is not important for permeability across lipid membranes.

To clarify the contribution of charge delocalization in a lipophilic ion to the efficacy of its permeation through a lipid membrane, we compared the behavior of alkyl derivatives of triphenylphosphonium, tricyclohexylphosphonium and trihexylphosphonium both in natural and artificial membranes. Exploring accumulation of the lipophilic cations in response to inside-negative membrane potential generation in mitochondria by using an ion-selective electrode revealed similar mitochondrial uptake of butyltricyclohexylphosphonium (C4TCHP) and butyltriphenylphosphonium (C4TPP). Fluorescence correlation spectroscopy also demonstrated similar membrane potential-dependent accumulation of fluorescein derivatives of tricyclohexyldecylphosphonium and decyltriphenylphosphonium in mitochondria. The rate constant of lipophilic cation translocation across the bilayer lipid membrane (BLM), measured by the current relaxation method, moderately increased in the following sequence: trihexyltetradecylphosphonium ([P6,6,6,14]) < triphenyltetradecylphosphonium (C14TPP) < tricyclohexyldodecylphosphonium (C12TCHP). In line with these results, measurements of the BLM stationary conductance indicated that membrane permeability for C4TCHP is 2.5 times higher than that for C4TPP. Values of the difference in the free energy of ion solvation in water and octane calculated using the density functional theory and the polarizable continuum solvent model were similar for methyltriphenylphosphonium, tricyclohexylmethylphosphonium and trihexylmethylphosphonium. Our results prove that both cyclic and aromatic moieties are not necessary for lipophilic ions to effectively permeate through lipid membranes.