Study of FoxA Pioneer Factor at Silent Genes Reveals Rfx-Repressed Enhancer at Cdx2 and a Potential Indicator of Esophageal Adenocarcinoma Development

Understanding how silent genes can be competent for activation provides insight into development as well as cellular reprogramming and pathogenesis. We performed genomic location analysis of the pioneer transcription factor FoxA in the adult mouse liver and found that about one-third of the FoxA bound sites are near silent genes, including genes without detectable RNA polymerase II. Virtually all of the FoxA-bound silent sites are within conserved sequences, suggesting possible function. Such sites are enriched in motifs for transcriptional repressors, including for Rfx1 and type II nuclear hormone receptors. We found one such target site at a cryptic “shadow” enhancer 7 kilobases (kb) downstream of the Cdx2 gene, where Rfx1 restricts transcriptional activation by FoxA. The Cdx2 shadow enhancer exhibits a subset of regulatory properties of the upstream Cdx2 promoter region. While Cdx2 is ectopically induced in the early metaplastic condition of Barrett's esophagus, its expression is not necessarily present in progressive Barrett's with dysplasia or adenocarcinoma. By contrast, we find that Rfx1 expression in the esophageal epithelium becomes gradually extinguished during progression to cancer, i.e, expression of Rfx1 decreased markedly in dysplasia and adenocarcinoma. We propose that this decreased expression of Rfx1 could be an indicator of progression from Barrett's esophagus to adenocarcinoma and that similar analyses of other transcription factors bound to silent genes can reveal unanticipated regulatory insights into oncogenic progression and cellular reprogramming

Sarco/Endoplasmic Reticulum Ca2+-ATPases (SERCA) Contribute to GPCR-Mediated Taste Perception

The sense of taste is important for providing animals with valuable information about the qualities of food, such as nutritional or harmful nature. Mammals, including humans, can recognize at least five primary taste qualities: sweet, umami (savory), bitter, sour, and salty. Recent studies have identified molecules and mechanisms underlying the initial steps of tastant-triggered molecular events in taste bud cells, particularly the requirement of increased cytosolic free Ca2+ concentration ([Ca2+]c) for normal taste signal transduction and transmission. Little, however, is known about the mechanisms controlling the removal of elevated [Ca2+]c from the cytosol of taste receptor cells (TRCs) and how the disruption of these mechanisms affects taste perception. To investigate the molecular mechanism of Ca2+ clearance in TRCs, we sought the molecules involved in [Ca2+]c regulation using a single-taste-cell transcriptome approach. We found that Serca3, a member of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) family that sequesters cytosolic Ca2+ into endoplasmic reticulum, is exclusively expressed in sweet/umami/bitter TRCs, which rely on intracellular Ca2+ release for signaling. Serca3-knockout (KO) mice displayed significantly increased aversive behavioral responses and greater gustatory nerve responses to bitter taste substances but not to sweet or umami taste substances. Further studies showed that Serca2 was mainly expressed in the T1R3-expressing sweet and umami TRCs, suggesting that the loss of function of Serca3 was possibly compensated by Serca2 in these TRCs in the mutant mice. Our data demonstrate that the SERCA family members play an important role in the Ca2+ clearance in TRCs and that mutation of these proteins may alter bitter and perhaps sweet and umami taste perception

Necdin, a p53-Target Gene, Is an Inhibitor of p53-Mediated Growth Arrest

In vitro, cellular immortalization and transformation define a model for multistep carcinogenesis and current ongoing challenges include the identification of specific molecular events associated with steps along this oncogenic pathway. Here, using NIH3T3 cells, we identified transcriptionally related events associated with the expression of Polyomavirus Large-T antigen (PyLT), a potent viral oncogene. We propose that a subset of these alterations in gene expression may be related to the early events that contribute to carcinogenesis. The proposed tumor suppressor Necdin, known to be regulated by p53, was within a group of genes that was consistently upregulated in the presence of PyLT. While Necdin is induced following p53 activation with different genotoxic stresses, Necdin induction by PyLT did not involve p53 activation or the Rb-binding site of PyLT. Necdin depletion by shRNA conferred a proliferative advantage to NIH3T3 and PyLT-expressing NIH3T3 (NIHLT) cells. In contrast, our results demonstrate that although overexpression of Necdin induced a growth arrest in NIH3T3 and NIHLT cells, a growing population rapidly emerged from these arrested cells. This population no longer showed significant proliferation defects despite high Necdin expression. Moreover, we established that Necdin is a negative regulator of p53-mediated growth arrest induced by nutlin-3, suggesting that Necdin upregulation could contribute to the bypass of a p53-response in p53 wild type tumors. To support this, we characterized Necdin expression in low malignant potential ovarian cancer (LMP) where p53 mutations rarely occur. Elevated levels of Necdin expression were observed in LMP when compared to aggressive serous ovarian cancers. We propose that in some contexts, the constitutive expression of Necdin could contribute to cancer promotion by delaying appropriate p53 responses and potentially promote genomic instability

Differential modulation of the lactisole 'Sweet Water Taste' by sweeteners.

Pre-exposure to taste stimuli and certain chemicals can cause water to have a taste. Here we studied further the 'sweet water taste' (SWT) perceived after exposure to the sweet taste inhibitor lactisole. Experiment 1 investigated an incidental observation that presenting lactisole in mixture with sucrose reduced the intensity of the SWT. The results confirmed this observation and also showed that rinsing with sucrose after lactisole could completely eliminate the SWT. The generalizability of these findings was investigated in experiment 2 by presenting 5 additional sweeteners before, during, or after exposure to lactisole. The results found with sucrose were replicated with fructose and cyclamate, but the 3 other sweeteners were less effective suppressors of the SWT, and the 2 sweeteners having the highest potency initially enhanced it. A third experiment investigated these interactions on the tongue tip and found that the lactisole SWT was perceived only when water was actively flowed across the tongue. The same experiment yielded evidence against the possibility that suppression of the SWT following exposure to sweeteners is an aftereffect of receptor activation while providing additional support for a role of sweetener potency. Collectively these results provide new evidence that complex inhibitory and excitatory interactions occur between lactisole and agonists of the sweet taste receptor TAS1R2-TAS1R3. Receptor mechanisms that may be responsible for these interactions are discussed in the context of the current model of the SWT and the possible contribution of allosteric modulation

Comprehensive Analysis of Mouse Bitter Taste Receptors Reveals Different Molecular Receptive Ranges for Orthologous Receptors in Mice and Humans

One key to animal survival is the detection and avoidance of potentially harmful compounds by their bitter taste. Variable numbers of taste 2 receptor genes expressed in the gustatory end organs enable bony vertebrates (Euteleostomi) to recognize numerous bitter chemicals. It is believed that the receptive ranges of bitter taste receptor repertoires match the profiles of bitter chemicals that the species encounter in their diets. Human and mouse genomes contain pairs of orthologous bitter receptor genes that have been conserved throughout evolution. Moreover, expansions in both lineages generated species-specific sets of bitter taste receptor genes. It is assumed that the orthologous bitter taste receptor genes mediate the recognition of bitter toxins relevant for both species, whereas the lineage-specific receptors enable the detection of substances differently encountered by mice and humans. By challenging 34 mouse bitter taste receptors with 128 prototypical bitter substances in a heterologous expression system, we identified cognate compounds for 21 receptors, 19 of which were previously orphan receptors. We have demonstrated that mouse taste 2 receptors, like their human counterparts, vary greatly in their breadth of tuning, ranging from very broadly to extremely narrowly tuned receptors. However, when compared with humans, mice possess fewer broadly tuned receptors and an elevated number of narrowly tuned receptors, supporting the idea that a large receptor repertoire is the basis for the evolution of specialized receptors. Moreover, we have demonstrated that sequence-orthologous bitter taste receptors have distinct agonist profiles. Species-specific gene expansions have enabled further diversification of bitter substance recognition spectra

Lactisole sweet water taste requires flowing water rinses.

<p>Shown are log₁₀ ratings of the lactisole SWT experienced on the tongue tip in experiment 3 under conditions of <i>Still</i> vs. <i>Flowing</i> water rinses. The dotted line indicates the level of “barely detectable” sweetness. Vertical bars are the SEs of the log₁₀ means.</p

Lactisole sweet water taste is unaffected by prior sweet receptor activation.

<p>Log₁₀ sweetness ratings are shown for solutions of <b>(a)</b> sucrose and <b>(b)</b> neotame sampled with the tongue tip in 4 conditions: Suc or Neot + Lac: a <i>still</i> lactisole + sweetener mixture (white bars); Lac→Suc or Neot: a <i>still</i> solution of lactisole before a <i>still</i> solution of the sweetener (gray bars); Suc or Neot + Lac → H₂O: a <i>still</i> sweetener + lactisole mixture followed by a <i>flowing</i> water rinse (hatched white bars); and Lac→Suc or Neot→H₂O: a <i>still</i> solution of lactisole before a <i>still</i> solution of the sweetener followed by a <i>flowing</i> water rinse (hatched gray bars). Shown for comparison is the SWT produced by a <i>flowing</i> water rinse after lactisole alone (dashed line). Vertical bars are the standard errors of the log₁₀ means (SEMs).</p

Effects of different sweeteners on the lactisole sweet water taste.

<p>Five different sweeteners (CYCL = cyclamate, FRUC = fructose, SACC = saccharin, SUCRA = sucralose, NHDC = neohesperidin dihydrochalcon) were presented in the 2 treatment conditions of experiment 2: in mixture with lactisole <b>(a-e)</b> or after lactisole <b>(f-j)</b>. The data for the lactisole SWT is shown in each graph (open circles) for comparison with the SWT produced in the treatment conditions (filled circles). In graphs <b>(a-e)</b> L/M on the x-axis indicates that in the treatment condition the mixture was presented on the first trial. Asterisks in graphs (d) and (e) indicate significantly higher SWT on the first water rinse after the mixture compared to the SWT after lactisole alone (Tukey HSD, p<0.05). In graphs <b>(f-j)</b> W/S indicates that in the treatment condition the sweetener was presented on the second trial. Thus in <b>(f-j)</b> the data on the second trial reflects the sweetness evoked by the sweetener, and the remaining 4 trials indicate the SWT. Arrows below the x-axis highlight the large differences in suppression of the SWT during the first water rinse after each sweetener compared to the second water rinse after lactisole alone. The horizontal dotted line in each graph indicates a “barely detectable” level of sweetness. Vertical bars are SEs of the log₁₀ means.</p

Decay in sweetness produced by sweeteners alone across water rinses.

<p>Main graph: Peak sweetness ratings for the 5 sweeteners of experiment 2 when presented alone and during 5 water rinses. S and W’s on the x-axis indicate exposures to the sweeteners and water rinses, respectively. The horizontal dotted line indicates a “barely detectable” level of sweetness; vertical bars are SEs of the log₁₀ means. Inset: residual sweetness during the first water rinse expressed as a percentage of the initial sweetness reported for each of the sweeteners.</p