High specificity and tight spatial restriction of self-biotinylation by DNA and RNA G-Quadruplexes complexed in vitro and in vivo with Heme.

Guanine-rich, single-stranded DNAs and RNAs that fold to G-quadruplexes (GQs) are able to complex tightly with heme and display strongly enhanced peroxidase activity. Phenolic compounds are particularly good substrates for these oxidative DNAzymes and ribozymes; we recently showed that the use of biotin-tyramide as substrate can lead to efficient GQ self-biotinylation. Such biotinylated GQs are amenable to polymerase chain reaction amplification and should be useful for a relatively non-perturbative investigation of GQs as well as GQ-heme complexes within living cells. Here, we report that in mixed solutions of GQ and duplex DNA in vitro, GQ biotinylation is specifically >104-fold that of the duplex, even in highly concentrated DNA gels; that a three-quartet GQ is tagged by up to four biotins, whose attachment occurs more or less uniformly along the GQ but doesn't extend significantly into a duplex appended to the GQ. This self-biotinylation can be modulated or even abolished in the presence of strong GQ ligands that compete with heme. Finally, we report strong evidence for the successful use of this methodology for labeling DNA and RNA within live, freshly dissected Drosophila larval salivary glands.

Homeodomain-interacting protein kinase (Hipk) plays roles in nervous system and muscle structure and function.

Homeodomain-interacting protein kinases (Hipks) have been previously associated with cell proliferation and cancer, however, their effects in the nervous system are less well understood. We have used Drosophila melanogaster to evaluate the effects of altered Hipk expression on the nervous system and muscle. Using genetic manipulation of Hipk expression we demonstrate that knockdown and over-expression of Hipk produces early adult lethality, possibly due to the effects on the nervous system and muscle involvement. We find that optimal levels of Hipk are critical for the function of dopaminergic neurons and glial cells in the nervous system, as well as muscle. Furthermore, manipulation of Hipk affects the structure of the larval neuromuscular junction (NMJ) by promoting its growth. Hipk regulates the phosphorylation of the synapse-associated cytoskeletal protein Hu-li tai shao (Hts; adducin in mammals) and modulates the expression of two important protein kinases, Calcium-calmodulin protein kinase II (CaMKII) and Partitioning-defective 1 (PAR-1), all of which may alter neuromuscular structure/function and influence lethality. Hipk also modifies the levels of an important nuclear protein, TBPH, the fly orthologue of TAR DNA-binding protein 43 (TDP-43), which may have relevance for understanding motor neuron diseases.

Role of nuclear factor of activated T-cells 5 in regulating hypertonic-mediated secretin receptor expression in kidney collecting duct cells.

A growing body of evidence suggests that secretin (SCT) is an important element in the osmoregulatory pathway. It is interesting to note that both SCT and its receptor (SCTR) gene are activated upon hyperosmolality in the kidney. However, the precise molecular mechanisms underlying the induction of the SCTR gene expression in response to changes in osmolality have yet to be clarified. Detailed DNA sequence analysis of the promoter regions of the SCTR gene reveals the presence of multiple osmotic response elements (ORE). The ORE is the binding site of a key osmosensitive transactivator, namely, the nuclear factor of activated T-cells 5 (NFAT5). SCTR and NFAT5 are co-expressed in the kidney cortex and medulla collecting duct cells. We therefore hypothesize that NFAT5 is responsible for modulating SCTR expression in hypertonic environments. In this study, we found hypertonicity stimulates the promoter activities and endogenous gene expression of SCTR in mouse kidney cortex collecting duct cells (M1) and inner medulla collecting duct cells (mIMCD3). The overexpression and silencing of NFAT5 further confirmed it to be responsible for the up-regulation of the SCTR gene under hypertonic conditions. A significant increase in the interaction between NFAT5 and the SCTR promoter was also observed following chromatin immunoprecipitation assay. In vivo, osmotic stress up-regulates the SCTR gene in the kidney cortex and medulla of wild-type mice, but does not do so in NFAT5(+/-) animals. Hence, this study provides comprehensive information on how NFAT5 regulates SCTR expression in different osmotic environments.