The Forkhead Transcription Factor Foxi1 Is a Master Regulator of Vacuolar H+-ATPase Proton Pump Subunits in the Inner Ear, Kidney and Epididymis

The vacuolar H+-ATPase dependent transport of protons across cytoplasmic membranes in FORE (forkhead related) cells of endolymphatic epithelium in the inner ear, intercalated cells of collecting ducts in the kidney and in narrow and clear cells of epididymis require expression of several subunits that assemble into a functional multimeric proton pump. We demonstrate that expression of four such subunits A1, B1, E2 and a4 all co-localize with the forkhead transcription factor Foxi1 in a subset of epithelial cells at these three locations. In cells, of such epithelia, that lack Foxi1 we fail to identify any expression of A1, B1, E2 and a4 demonstrating an important role for the transcription factor Foxi1 in regulating subunit availability. Promoter reporter experiments, electrophoretic mobility shift assays (EMSA) and site directed mutagenesis demonstrate that a Foxi1 expression vector can trans-activate an a4-promoter reporter construct in a dose dependent manner. Furthermore, we demonstrate using chromatin immunoprecipitation (ChIP) assays that Foxi1-dependent activation to a large extent depends on cis-elements at position −561/−547 in the a4 promoter. Thus, we provide evidence that Foxi1 is necessary for expression of at least four subunits in three different epithelia and most likely is a major determinant for proper assembly of a functional vacuolar H+-ATPase complex at these locations

Detection of Methoxymethanol as a Photochemistry Product of Condensed Methanol

We report the identification of methoxymethanol (CH3OCH2OH) as a photochemistry product of condensed methanol (CH3OH) based on temperature-programmed desorption studies conducted following photon irradiation at energies below the ionization threshold (9.8 eV) of condensed methanol. The first detection of methoxymethanol in the interstellar medium was reported in 2017 based on data from Bands 6 and 7 from the Atacama Large Millimeter/submillimeter Array (ALMA). The cosmic synthesis of “complex” organic molecules such as methyl formate (HCOOCH3), dimethyl ether (CH3OCH3), acetic acid (CH3COOH), ethylene glycol (HOCH2CH2OH), and glycolaldehyde (HOCH2CHO) has been attributed to UV photolysis of condensed methanol found in interstellar ices. Experiments conducted in 1995 demonstrated that electron-induced radiolysis of methanol cosmic ice analogues yields methoxymethanol. In three recent publications (2016, 2017, and 2018), methoxymethanol was considered as a potential tracer for reactions induced by secondary electrons resulting from the interaction of cosmic rays with interstellar ices. However, the results presented in this study suggest that methoxymethanol can be formed from both radiation chemistry and photochemistry of condensed methanol

Whole-Genome Analysis of Temporal Gene Expression during Foregut Development

We have investigated the cis-regulatory network that mediates temporal gene expression during organogenesis. Previous studies demonstrated that the organ selector gene pha-4/FoxA is critical to establish the onset of transcription of Caenorhabditis elegans foregut (pharynx) genes. Here, we discover additional cis-regulatory elements that function in combination with PHA-4. We use a computational approach to identify candidate cis-regulatory sites for genes activated either early or late during pharyngeal development. Analysis of natural or synthetic promoters reveals that six of these sites function in vivo. The newly discovered temporal elements, together with predicted PHA-4 sites, account for the onset of expression of roughly half of the pharyngeal genes examined. Moreover, combinations of temporal elements and PHA-4 sites can be used in genome-wide searches to predict pharyngeal genes, with more than 85% accuracy for their onset of expression. These findings suggest a regulatory code for temporal gene expression during foregut development and provide a means to predict gene expression patterns based solely on genomic sequence

Foxp2 Regulates Gene Networks Implicated in Neurite Outgrowth in the Developing Brain

Forkhead-box protein P2 is a transcription factor that has been associated with intriguing aspects of cognitive function in humans, non-human mammals, and song-learning birds. Heterozygous mutations of the human FOXP2 gene cause a monogenic speech and language disorder. Reduced functional dosage of the mouse version (Foxp2) causes deficient cortico-striatal synaptic plasticity and impairs motor-skill learning. Moreover, the songbird orthologue appears critically important for vocal learning. Across diverse vertebrate species, this well-conserved transcription factor is highly expressed in the developing and adult central nervous system. Very little is known about the mechanisms regulated by Foxp2 during brain development. We used an integrated functional genomics strategy to robustly define Foxp2-dependent pathways, both direct and indirect targets, in the embryonic brain. Specifically, we performed genome-wide in vivo ChIP–chip screens for Foxp2-binding and thereby identified a set of 264 high-confidence neural targets under strict, empirically derived significance thresholds. The findings, coupled to expression profiling and in situ hybridization of brain tissue from wild-type and mutant mouse embryos, strongly highlighted gene networks linked to neurite development. We followed up our genomics data with functional experiments, showing that Foxp2 impacts on neurite outgrowth in primary neurons and in neuronal cell models. Our data indicate that Foxp2 modulates neuronal network formation, by directly and indirectly regulating mRNAs involved in the development and plasticity of neuronal connections

Genetic and molecular analysis of the osmotically regulatedproU operon of Salmonella typhimurium

When there is an increase in the osmotic strength of the environment of an organism, osmotic stress is encountered. Osmotic stress causes a rapid efflux of water, producing cellular dehydration. Organisms respond to this situation by increasing the intracellular concentrations of certain compounds like proline and glycinebetaine. These compounds act as osmotic balancers serving to prevent cellular dehydration without having an inhibitory effect on intracellular processes. In the enteric bacterium Salmonella typhimurium the increase of these osmotic balancers in response to osmotic stress if mediated by the ProU transport system, which is encoded by genes of the proU operon. As an initial step in understanding the mechanism by which the proU operon is expressed, the proU operon was cloned from S. typhimurium and the nucleotide sequence was determined for the transcriptional control region and part of the first structural gene. The predicted amino acid sequence of the gene product had homology to other proteins that are inner membrane associated components of binding protein-dependent transport systems. Mapping of the proU messenger RNA revealed endpoints that are close enough to sequences weakly resembling consensus bacterial promoters. By deletion analysis, the sequence mediating osmoregulated expression of the proU operon was shown to be contained on a 260 base-pair fragment, 60 nucleotides upstream and 200 nucleotides downstream of the mRNA start point. Strains carrying lacZ expression vectors containing proU fragments with the promoter, but not the DNA between positions +30 and +200 directed a high level constitutive expression of $\beta$-galactosidase, owing to increased levels of mRNA synthesis. This suggests that there is a negative regulatory element for the proU operon, downstream of the promoter, within the first structural gene. It was ruled out that this element is an osmotic stress-dependent transcription terminator. Mutations were identified that resulted in constitutive expression of the proU operon, and each mutation was closely linked to the proU locus and cis-dominant over the wild-type allele. The failure to obtain either recessive or unlinked mutations suggests that transcriptional regulation of the operon is not under the negative control of a specific repressor protein that is dispensable for cell viability