Identification of Pou5f1, Sox2, and Nanog downstream target genes with statistical confidence by applying a novel algorithm to time course microarray and genome-wide chromatin immunoprecipitation data

<p>Abstract</p> <p>Background</p> <p>Target genes of a transcription factor (TF) <it>Pou5f1 </it>(<it>Oct3/4 </it>or <it>Oct4</it>), which is essential for pluripotency maintenance and self-renewal of embryonic stem (ES) cells, have previously been identified based on their response to <it>Pou5f1 </it>manipulation and occurrence of Chromatin-immunoprecipitation (ChIP)-binding sites in promoters. However, many responding genes with binding sites may not be direct targets because response may be mediated by other genes and ChIP-binding site may not be functional in terms of transcription regulation.</p> <p>Results</p> <p>To reduce the number of false positives, we propose to separate responding genes into groups according to direction, magnitude, and time of response, and to apply the false discovery rate (FDR) criterion to each group individually. Using this novel algorithm with stringent statistical criteria (FDR < 0.2) to a compendium of published and new microarray data (3, 6, 12, and 24 hr after <it>Pou5f1 </it>suppression) and published ChIP data, we identified 420 tentative target genes (TTGs) for <it>Pou5f1</it>. The majority of TTGs (372) were down-regulated after <it>Pou5f1 </it>suppression, indicating that the <it>Pou5f1 </it>functions as an activator of gene expression when it binds to promoters. Interestingly, many activated genes are potent suppressors of transcription, which include polycomb genes, zinc finger TFs, chromatin remodeling factors, and suppressors of signaling. Similar analysis showed that <it>Sox2 </it>and <it>Nanog </it>also function mostly as transcription activators in cooperation with <it>Pou5f1</it>.</p> <p>Conclusion</p> <p>We have identified the most reliable sets of direct target genes for key pluripotency genes – <it>Pou5f1</it>, <it>Sox2</it>, and <it>Nanog</it>, and found that they predominantly function as activators of downstream gene expression. Thus, most genes related to cell differentiation are suppressed indirectly.</p

Breast Cancer Metastasis in the Skin with Hyperkeratotic Pigmentation Caused by Melanocyte Colonization

Pigmented breast cancer in the skin caused by nonneoplastic melanocytes of epidermal origin is a rare condition of metastasis from breast cancer, but the pathogenesis of this phenomenon is almost unknown. In this report, we describe a case of breast cancer metastasis in the skin with prominent hyperkeratotic pigmentation caused by nonneoplastic melanocyte colonization. Immunohistochemical staining revealed that the metastatic tumor cells produced IL-23, which is reported not only to induce IL-17 but also to inhibit cell apoptosis in breast cancer cells, which affects tumor progression. In addition to IL-23, substantial numbers of IL-17-producing cells were detected at the peritumoral area, suggesting that IL-17 might induce not only melanogenesis but also keratinocyte proliferation and tumorigenesis. Our report suggests possible mechanisms of hyperkeratotic pigmentation of breast cancer metastasis in the skin

Differential effects of beta-blockade on dispersion of repolarization in the absence and presence of sympathetic stimulation between the lqt1 and lqt2 forms of congenital long qt syndrome

AbstractObjectivesThis study compared the effects of beta-blockade on transmural and spatial dispersion of repolarization (TDR and SDR, respectively) between the LQT1 and LQT2 forms of congenital long QT syndrome (LQTS).BackgroundThe LQT1 form is more sensitive to sympathetic stimulation and more responsive to beta-blockers than either the LQT2 or LQT3 forms.MethodsEighty-seven-lead, body-surface electrocardiograms (ECGs) were recorded before and after epinephrine infusion (0.1 μg/kg body weight per min) in the absence and presence of oral propranolol (0.5–2.0 mg/kg per day) in 11 LQT1 patients and 11 LQT2 patients. The Q-Tendinterval, the Q-Tpeakinterval and the interval between Tpeakand Tend(Tp-e), representing TDR, were measured and averaged from 87-lead ECGs and corrected by Bazett’s method (corrected Q-Tendinterval [cQTe], corrected Q-Tpeakinterval [cQTp] and corrected interval between Tpeakand Tend[cTp-e]). The dispersion of cQTe(cQTe-D) was obtained among 87 leads and was defined as the interval between the maximum and minimum values of cQTe.ResultsPropranolol in the absence of epinephrine significantly prolonged the mean cQTpvalue but not the mean cQTevalue, thus decreasing the mean cTp-evalue in both LQT1 and LQT2 patients; the differences with propranolol were significantly larger in LQT1 than in LQT2 (p < 0.05). The maximum cQTe, minimum cQTeand cQTe-D were not changed with propranolol. Propranolol completely suppressed the influence of epinephrine in prolonging the mean cQTe, maximum cQTeand minimum cQTevalues, as well as increasing the mean cTp-eand cQTe-D values in both groups.ConclusionsBeta-blockade under normal sympathetic tone produces a greater decrease in TDR in the LQT1 form than in the LQT2 form, explaining the superior effectiveness of beta-blockers in LQT1 versus LQT2. Beta-blockers also suppress the influence of sympathetic stimulation in increasing TDR and SDR equally in LQT1 and LQT2 syndrome

The modeling of Alzheimer's disease by the overexpression of mutant Presenilin 1 in human embryonic stem cells.

Cellular disease models are useful tools for Alzheimer's disease (AD) research. Pluripotent stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), are promising materials for creating cellular models of such diseases. In the present study, we established cellular models of AD in hESCs that overexpressed the mutant Presenilin 1 (PS1) gene with the use of a site-specific gene integration system. The overexpression of PS1 did not affect the undifferentiated status or the neural differentiation ability of the hESCs. We found increases in the ratios of amyloid-β 42 (Aβ42)/Aβ40 and Aβ43/Aβ40. Furthermore, synaptic dysfunction was observed in a cellular model of AD that overexpressed mutant PS1. These results suggest that the AD phenotypes, in particular, the electrophysiological abnormality of the synapses in our AD models might be useful for AD research and drug discovery

Changes in global gene expression during in vitro decidualization of rat endometrial stromal cells

During the preimplantation phase of pregnancy the endometrial stroma differentiates into decidua, a process that implies numerous morphological changes and is an example of physiological transdifferentiation. Here we show that UIII rat endometrial stromal cells cultured in the presence of calf serum acquired morphological features of decidual cells and expressed decidual markers. To identify genes involved in decidualization we compared gene expression patterns of control and decidualized UIII cells using cDNA microarray. We found 322 annotated genes exhibiting significant differences in expression (>3-fold, fold discovery rate (FDR) >0.005), of which 312 have not been previously related to decidualization. Analysis of overrepresented functions revealed that protein synthesis, gene expression, and chromatin architecture and remodeling are the most relevant modified functions during decidualization. Relevant genes are also found in the functional terms differentiation, cell proliferation, signal transduction, and matrix/structural proteins. Several of these new genes involved in decidualization (Csdc2, Trim27, Eef1a1, Bmp1, Wt1, Aes, Gna12, and Men1) are shown to be also regulated in uterine decidua during normal pregnancy. Thus, the UIII cell culture model will allow future mechanistic studies to define the transcriptional network regulating reprogramming of stromal cells into decidual cells.Facultad de Ciencias Exacta

Characterization of the novel mutant A78T-HERG from a long QT syndrome type 2 patient: Instability of the mutant protein and stabilization by heat shock factor 1

Background:The human ether-a-go-go-related gene (HERG) encodes the α-subunit of rapidly activating delayed-rectifier potassium channels. Mutations in this gene cause long QT syndrome type 2 (LQT2). In most cases, mutations reduce the stability of the channel protein, which can be restored by heat shock (HS). Methods: We identified the novel mutant A78T-HERG in a patient with LQT2. The purpose of the current study was to characterize this mutant protein and test whether HS and heat shock factors (HSFs) could stabilize the mutant protein. A78T-HERG and wild-type HERG (WT-HERG) were expressed in HEK293 cells and analyzed by immunoblotting, immunoprecipitation, immunofluorescence, and whole-cell patch clamping. Results: When expressed in HEK293 cells, WT-HERG gave rise to immature and mature forms of the protein at 135 and 155 kDa, respectively. A78T-HERG gave rise only to the immature form, which was heavily ubiquitinated. The proteasome inhibitor MG132 increased the expression of immature A78T-HERG and increased both the immature and mature forms of WT-HERG. WT-HERG, but not A78T-HERG, was expressed on the plasma membrane. In whole-cell patch clamping experiments, depolarizing pulses evoked E4031-sensitive HERG channel currents in cells transfected with WT-HERG, but not in cells transfected with A78T-HERG. The A78V mutant, but not A78G mutant, remained in the immature form similarly to A78T. Maturation of the A78T-HERG protein was facilitated by HS, expression of HSF-1, or exposure to geranyl geranyl acetone. Conclusions: A78T-HERG was characterized by protein instability and reduced expression on the plasma membrane. The stability of the mutant was partially restored by HSF-1, indicating that HSF-1 is a target for the treatment for LQT2 caused by the A78T mutation in HERG

Efficient integration of transgenes into a defined locus in human embryonic stem cells

Random integration is one of the more straightforward methods to introduce a transgene into human embryonic stem (ES) cells. However, random integration may result in transgene silencing and altered cell phenotype due to insertional mutagenesis in undefined gene regions. Moreover, reliability of data may be compromised by differences in transgene integration sites when comparing multiple transgenic cell lines. To address these issues, we developed a genetic manipulation strategy based on homologous recombination and Cre recombinase-mediated site-specific integration. First, we performed gene targeting of the hypoxanthine phosphoribosyltransferase 1 (HPRT) locus of the human ES cell line KhES-1. Next, a gene-replacement system was created so that a circular vector specifically integrates into the targeted HPRT locus via Cre recombinase activity. We demonstrate the application of this strategy through the creation of a tetracycline-inducible reporter system at the HPRT locus. We show that reporter gene expression was responsive to doxycycline and that the resulting transgenic human ES cells retain their self-renewal capacity and pluripotency

A small molecule that promotes cardiac differentiation of human pluripotent stem cells under defined, cytokine- and xeno-free conditions.

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, are potentially useful in regenerative therapies for heart disease. For medical applications, clinical-grade cardiac cells must be produced from hPSCs in a defined, cost-effective manner. Cell-based screening led to the discovery of KY02111, a small molecule that promotes differentiation of hPSCs to cardiomyocytes. Although the direct target of KY02111 remains unknown, results of the present study suggest that KY02111 promotes differentiation by inhibiting WNT signaling in hPSCs but in a manner that is distinct from that of previously studied WNT inhibitors. Combined use of KY02111 and WNT signaling modulators produced robust cardiac differentiation of hPSCs in a xeno-free, defined medium, devoid of serum and any kind of recombinant cytokines and hormones, such as BMP4, Activin A, or insulin. The methodology has potential as a means for the practical production of human cardiomyocytes for regeneration therapies