An atlas of combinatorial transcriptional regulation in mouse and man.

Combinatorial interactions among transcription factors are critical to directing tissue-specific gene expression. To build a global atlas of these combinations, we have screened for physical interactions among the majority of human and mouse DNA-binding transcription factors (TFs). The complete networks contain 762 human and 877 mouse interactions. Analysis of the networks reveals that highly connected TFs are broadly expressed across tissues, and that roughly half of the measured interactions are conserved between mouse and human. The data highlight the importance of TF combinations for determining cell fate, and they lead to the identification of a SMAD3/FLI1 complex expressed during development of immunity. The availability of large TF combinatorial networks in both human and mouse will provide many opportunities to study gene regulation, tissue differentiation, and mammalian evolution.

Exciton Primer-mediated SNP detection in SmartAmp2 reactions.

Most commonly used intercalating fluorescent dyes in DNA detection are lacking any sequence specificity, whereas so-called Exciton Primers can overcome this limitation by functioning as "sequence-specific dyes." After hybridization to complementary sequences, the fluorescence of Exciton Primers provides sequence-specific signals for real-time monitoring of amplification reactions. Applied to the SmartAmp2 mutation detection process, Exciton Primers show high signal strength with low background leading to a superior specificity and sensitivity compared to SYBR Green I. Signal strength can be further enhanced using multiple dyes within one Exciton Primer or use of multiple Exciton Primers in the same amplification reaction. Here we demonstrate the use of Exciton Primers for genotyping a single nucleotide polymorphism (SNP) in the VKORC1 locus (-1639G>A) relevant for Warfarin dosing as an example for Exciton Primers mediated genotyping by SmartAmp2. The genotyping assay can use only one labeled Exciton Primer for endpoint detection, or simultaneously by real-time monitoring detect wild-type and mutant alleles in a one-tube reaction using two Exciton Primers having different dyes. Working directly from blood samples, Exciton Primer mediated genotyping by SmartAmp2 offers superior solutions for rapid point-of-care testing.

FOXP1 is an androgen-responsive transcription factor that negatively regulates androgen receptor signaling in prostate cancer cells.

Androgen and androgen receptor (AR) play important roles in the formation and the progression of prostate cancer. AR activates its target genes by recruiting various coregulators and transcriptional factors. Here we show that the FOXP1 forkhead transcription factor is a novel androgen-regulated gene. By sequencing DNA fragments obtained from chromatin immunoprecipitation (ChIP), a bona-fide AR binding site (ARBS) is identified in an intron region of FOXP1 gene. FOXP1 can be induced by androgen in hormone-sensitive prostate cancer LNCaP cells at both mRNA and protein levels. In particular, a smaller FOXP1 variant, FOXP1D, is upregulated in response to androgen. Notably, we demonstrate that FOXP1 directly interacts with AR and negatively regulates AR signaling ligand-dependently, as exemplified by the transcriptional repression of PSA gene regulated by androgen-dependent FOXP1 recruitment on its enhancer region. We show that several other forkhead transcription factors are also androgen-responsive in LNCaP cells. Our study provides a new insight to the function of forkhead transcription factors that modulates AR signaling as an androgen-regulated transcriptional factor, which would contribute to the tumorigenesis of prostate cancer.

Development of a DNA barcode tagging method for monitoring dynamic changes in gene expression by using an ultra high-throughput sequencer.

CAGE (cap analysis of gene expression) is a method for identifying transcription start sites by sequencing the first 20 or 21 nucleotides from the 5' end of capped transcripts, allowing genome-wide promoter analyses to be performed. The potential of the CAGE as a form of expression profiling was limited previously by sequencing technology and the labor-intensive protocol. Here we describe an improved CAGE method for use with a next generation sequencer. This modified method allows the identification of the RNA source of each CAGE tag within a pooled library by introducing DNA tags (barcodes). The method not only drastically improves the sequencing capacity, but also contributes to savings in both time and budget. Additionally, this pooled CAGE tag method enables the dynamic changes in promoter usage and gene expression to be monitored.

Improved systemic delivery of insulin by condensed drug loading in a dimpled suppository.

The development of peptide therapeutics owing to the advances in biotechnology has overcome some unmet medical needs; however, the route of administration is still limited to injections. Systemic delivery of insulin via an enteral route remains a great challenge due to its instability and low mucosal permeability. In this study, we investigated the effect of drug condensation in a suppository on the efficacy of insulin after rectal administration. Suppositories with dimples are prepared by a mold method using a hard fat (Suppocire® AM). Insulin or fluorescein isothiocyanate-dextran (molecular weight: 3,000-5,000) (FD4) as a model of a hydrophilic macromolecule was loaded in the dimples, and sealed with other lipids with different melting points. The in vitro release test showed that the time to 50% drug release depends on the melting point of the lipid for sealing but not on the number of dimples. The suppositories with one-, or three-dimple containing insulin and caprylocaproyl macrogol-8 glyceride (Labrasol®) were administered to rats at 0.5 U/head. The reduction in plasma glucose level was more significant for the one-dimple-type suppository than for the three-dimple-type although the one-dimple-type suppository contained less amount of Labrasol by one-third compared to the three-dimple-type. These results suggest that condensation of an insulin dose in a limited surface area of a suppository improves systemic availability via the rectal route with a reduced amount of an absorption enhancer.