HIF2α Upregulates the Migration Factor ODZ1 under Hypoxia in Glioblastoma Stem Cells

Background: Glioblastoma (GBM) remains a major clinical challenge due to its invasive capacity, resistance to treatment, and recurrence. We have previously shown that ODZ1 contributes to glioblastoma invasion and that ODZ1 mRNA levels can be upregulated by epigenetic mechanisms in response to hypoxia. Herein, we have further studied the transcriptional regulation of ODZ1 in GBM stem cells (GSCs) under hypoxic conditions and analyzed whether HIF2α has any role in this regulation. Methods: We performed the experiments in three primary GSC cell lines established from tumor specimens. GSCs were cultured under hypoxia, treated with HIF regulators (DMOG, chetomin), or transfected with specific siRNAs, and the expression levels of ODZ1 and HIF2α were analyzed. In addition, the response of the ODZ1 promoter cloned into a luciferase reporter plasmid to the activation of HIF was also studied. Results: The upregulation of both mRNA and protein levels of HIF2α under hypoxia conditions correlated with the expression of ODZ1 mRNA. Moreover, the knockdown of HIF2α by siRNAs downregulated the expression of ODZ1. We found, in the ODZ1 promoter, a HIF consensus binding site (GCGTG) 1358 bp from the transcription start site (TSS) and a HIF-like site (CCGTG) 826 bp from the TSS. Luciferase assays revealed that the stabilization of HIF by DMOG resulted in the increased activity of the ODZ1 promoter. Conclusions: Our data indicate that the HIF2α-mediated upregulation of ODZ1 helps strengthen the transcriptional control of this migration factor under hypoxia in glioblastoma stem cells. The discovery of this novel transcriptional pathway identifies new targets to develop strategies that may avoid GBM tumor invasion and recurrence.

Alternative splicing of medaka bcl6aa and its repression by Prdm1a and Prdm1b

Bcl6 and Prdm1 (Blimp1) are a pair of transcriptional factors that repressing each other in the mammals. Prdm1 represses the expression of bcl6 by binding a cis-element of bcl6 gene in mammals. The homologs of Bcl6 and Prdm1 have been identified in teleost fish. However, whether these two factors regulate each other by the same way in fish as that in the mammals is not clear. In this study, the regulation of bcl6aa by Prdm1 was investigated in medaka. The mRNA of bcl6aa has three variants (bcl6aaX1-X3) at the 5′-end by alternative splicing with different promoters detected by RT-PCR. The three variants can be detected in adult tissues and developing embryos of medaka. The predicted proteins of Bcl6aaX1-X3 may have modification such as acetylation, C-mannosylation, phosphorylation, and sumoylation in the N-terminuses with different half-lives and relative translation efficiencies. Prdm1a and prdm1b are expressed in the tissues and embryos where and when bcl6aa is expressed. The expression of prdm1a was high while the expression of bcl6aa was low, and vice versa, detected in the spleen after stimulation with LPS or polyI:C. In vitro reporter assay indicated that bcl6aa could be directly repressed by both Prdm1a and Prdm1b in a dosage-dependent manner. After mutation of the key base, G, of all predicted binding sites in the core promoter region of bcl6aa, the repression by Prdm1a and/or Prdm1b disappeared. The consensus binding site of Prdm1 in bcl6aa gene is GAAAA(T/G). These results indicate that both Prdm1a and Prdm1b directly repress the expression of bcl6aa by binding the consensus binding site where the 5′-G is critical in medaka fish.

β-catenin/LEF1/IGF-IIR Signaling Axis Galvanizes the Angiotensin-II- induced Cardiac Hypertrophy

Cardiovascular diseases have a high prevalence worldwide and constitute the leading causes of mortality. Recently, malfunctioning of β-catenin signaling has been addressed in hypertensive heart condition. Ang-II is an important mediator of cardiovascular remodeling processes which not only regulates blood pressure but also leads to pathological cardiac changes. However, the contribution of Ang-II/β-catenin axis in hypertrophied hearts is ill-defined. Employing in vitro H9c2 cells and in vivo spontaneously hypertensive rats (SHR) cardiac tissue samples, western blot analysis, luciferase assays, nuclear-cytosolic protein extracts, and immunoprecipitation assays, we found that under hypertensive condition β-catenin gets abnormally induced that co-activated LEF1 and lead to cardiac hypertrophy changes by up-regulating the IGF-IIR signaling pathway. We identified putative LEF1 consensus binding site on IGF-IIR promoter that could be regulated by β-catenin/LEF1 which in turn modulate the expression of cardiac hypertrophy agents. This study suggested that suppression of β-catenin expression under hypertensive condition could be exploited as a clinical strategy for cardiac pathological remodeling processes.

The Flavonoid Quercetin Induces AP-1 Activation in FRTL-5 Thyroid Cells

Previous studies have shown that quercetin inhibits thyroid function both in vitro and in vivo. An attempt to evaluate the effect of quercetin at the promoter level of the thyroid-specific genes led to the observation that this compound induces the basal activity of the reporter vector. Therefore, the action of quercetin has been evaluated on the basal activity of several reporter vectors: The PGL3 basic, promoter and control vectors from Promega, and a pSV-based chloramphenicol acetyltransferase (CAT) reporter vector. In the Fisher Rat Thyroid cell Line FRTL-5 thyroid cells transiently transfected, quercetin 10 μM increased the basal activity of all the reporter vectors evaluated, although the degree of the effect was significantly different among them. The analysis of the difference among the regulatory regions of these vectors identified the activator protein 1 (AP-1) binding site as one of the potential sites involved in the quercetin effect. Electromobility shift assay experiments showed that the treatment with quercetin induced the binding of a protein complex to an oligonucleotide containing the AP-1 consensus binding site. This is the first study showing an effect of quercetin on AP-1 activity in thyroid cells. Further studies are in progress to understand the role of AP-1 activation in the effects of quercetin on thyroid function.

Modulating Fis and IHF binding specificity, crosstalk and regulatory logic through the engineering of complex promoters

Bacterial promoters are usually formed by multiplecis-regulatory elements recognized by a plethora of transcriptional factors (TFs). From those, global regulators are key elements since these TFs are responsible for the regulation of hundreds of genes in the bacterial genome. For instance, Fis and IHF are two global regulators which play a major role in gene expression control inEscherichia coliand usually multiplecis-regulatory elements for these proteins co-occur at target promoters. Here, we investigated the relationship between the architecture of thecis-regulatory elements for Fis and IHF inE. coli. For this, we constructed 42 synthetic promoter variants harboring consensuscis-elements for Fis and IHF at different distances from a core −35/−10 region and in different numbers and combinations. We first demonstrated that although Fis preferentially recognizes its consensuscis-element, it can also recognize, to some extent, the consensus binding site for IHF, and the same was true for IHF, which was also able of recognizing Fis binding sites. However, changing the arrangement of thecis-elements (i.e., the position or the number of sites) can completely abolish unspecific binding of both TFs. More remarkably, we demonstrate that combiningcis-elements for both TFs could result in Fis and IHF repressed or activated promoters depending on the final architecture of the promoters in an unpredictable way. Taken together, the data presented here demonstrate how small changes in the architecture of bacterial promoters could result in drastic changes in the final regulatory logic of the system, with important implications for the understanding of natural complex promoters in bacteria and their engineering for novel applications.ImportanceThe understanding of the regulatory complex in bacteria is a key issue in modern microbiology. Here, we constructed synthetic bacterial promoters in order to investigate how binding of transcriptional factors to multiple target sites at the promoters can influence gene expression. Our results demonstrate in a systematic way that the arrangement and number of thesecis-regulatory elements are crucial for the final expression dynamics of the target promoters. In particular, we show that TF binding specificity or promiscuity can be modulated using different promoter architectures based on consensuscis-regulatory elements, and that transcriptional repression and activation can also be affected by promoter architecture. These results are relevant both for the understanding of natural systems and for the construction of synthetic circuits for biotechnological applications.

MrpJ Directly RegulatesProteus mirabilisVirulence Factors, Including Fimbriae and Type VI Secretion, during Urinary Tract Infection

ABSTRACTProteus mirabilisis a leading cause of catheter-associated urinary tract infections (CAUTIs) and urolithiasis. The transcriptional regulator MrpJ inversely modulates two critical aspects ofP. mirabilisUTI progression: fimbria-mediated attachment and flagellum-mediated motility. Transcriptome data indicated a network of virulence-associated genes under MrpJ's control. Here, we identify the direct gene regulon of MrpJ and its contribution toP. mirabilispathogenesis, leading to the discovery of novel virulence targets.Chromatinimmunoprecipitation followed by high-throughputsequencing (ChIP-seq) was used for the first time in a CAUTI pathogen to probe forin vivodirect targets of MrpJ. Selected MrpJ-regulated genes were mutated and assessed for their contribution to UTI using a mouse model. ChIP-seq revealed a palindromic MrpJ binding sequence and 78 MrpJ-bound regions, including binding sites upstream of genes involved in motility, fimbriae, and a type VI secretion system (T6SS). A combinatorial mutation approach established the contribution of three fimbriae (fim8A,fim14A, andpmpA) to UTI and a new pathogenic role for the T6SS in UTI progression. In conclusion, this study (i) establishes the direct gene regulon and an MrpJ consensus binding site and (ii) led to the discovery of new virulence genes inP. mirabilisUTI, which could be targeted for therapeutic intervention of CAUTI.

Rab1b overexpression modifies Golgi size and gene expression in HeLa cells and modulates the thyrotrophin response in thyroid cells in culture

Rab1b belongs to the Rab-GTPase family that regulates membrane trafficking and signal transduction systems able to control diverse cellular activities, including gene expression. Rab1b is essential for endoplasmic reticulum–Golgi transport. Although it is ubiquitously expressed, its mRNA levels vary among different tissues. This work aims to characterize the role of the high Rab1b levels detected in some secretory tissues. We report that, in HeLa cells, an increase in Rab1b levels induces changes in Golgi size and gene expression. Significantly, analyses applied to selected genes, KDELR3, GM130 (involved in membrane transport), and the proto-oncogene JUN, indicate that the Rab1b increase acts as a molecular switch to control the expression of these genes at the transcriptional level, resulting in changes at the protein level. These Rab1b-dependent changes require the activity of p38 mitogen-activated protein kinase and the cAMP-responsive element-binding protein consensus binding site in those target promoter regions. Moreover, our results reveal that, in a secretory thyroid cell line (FRTL5), Rab1b expression increases in response to thyroid-stimulating hormone (TSH). Additionally, changes in Rab1b expression in FRTL5 cells modify the specific TSH response. Our results show, for the first time, that changes in Rab1b levels modulate gene transcription and strongly suggest that a Rab1b increase is required to elicit a secretory response.