Florent Hubé

The steroid receptor RNA activator is the first functional RNA encoding a protein

The steroid receptor RNA activator (SRA) has previously been characterized as belonging to the growing family of functional non‐coding RNAs. However, we recently reported the Western blot detection of a putative endogenous SRA protein (SRAP) in breast cancer cells. Herein, we successfully suppressed the expression of this protein through specific RNA interference assay, unequivocally confirming its existence. Moreover, using database searches and Western blot analysis, we also showed that SRAP is highly conserved among chordata. Overall, our results suggest that SRA is the first example of a new class of functional RNAs also able to encode a protein.

Dnmt3b recruitment through E2F6 transcriptional repressor mediates germ-line gene silencing in murine somatic tissues

Methylation of cytosine residues within the CpG dinucleotide in mammalian cells is an important mediator of gene expression, genome stability, X-chromosome inactivation, genomic imprinting, chromatin structure, and embryonic development. The majority of CpG sites in mammalian cells is methylated in a nonrandom fashion, raising the question of how DNA methylation is distributed along the genome. Here, we focused on the functions of DNA methyltransferase-3b (Dnmt3b), of which deregulated activity is linked to several human pathologies. We generated Dnmt3b hypomorphic mutant mice with reduced catalytic activity, which first revealed a deregulation of Hox genes expression, consistent with the observed homeotic transformations of the posterior axis. In addition, analysis of deregulated expression programs in Dnmt3b mutant embryos, using DNA microarrays, highlighted illegitimate activation of several germ-line genes in somatic tissues that appeared to be linked directly to their hypomethylation in mutant embryos. We provide evidence that these genes are direct targets of Dnmt3b. Moreover, the recruitment of Dnmt3b to their proximal promoter is dependant on the binding of the E2F6 transcriptional repressor, which emerges as a common hallmark in the promoters of genes found to be up-regulated as a consequence of impaired Dnmt3b activity. Therefore, our results unraveled a coordinated regulation of genes involved in meiosis, through E2F6-dependant methylation and transcriptional silencing in somatic tissues.

Increasing the relative expression of endogenous non-coding Steroid Receptor RNA Activator (SRA) in human breast cancer cells using modified oligonucleotides

Products of the Steroid Receptor RNA Activator gene (SRA1) have the unusual property to modulate the activity of steroid receptors and other transcription factors both at the RNA (SRA) and the protein (SRAP) level. Balance between these two genetically linked entities is controlled by alternative splicing of intron-1, whose retention alters SRAP reading frame. We have previously found that both fully-spliced SRAP-coding and intron-1-containing non-coding SRA RNAs co-exist in breast cancer cell lines. Herein, we report a significant (Students t-test, P < 0.003) higher SRA–intron-1 relative expression in breast tumors with higher progesterone receptor contents. Using an antisense oligoribonucleotide, we have successfully reprogrammed endogenous SRA splicing and increased SRA RNA–intron-1 relative level in T5 breast cancer cells. This increase is paralleled by significant changes in the expression of genes such as plasminogen urokinase activator and estrogen receptor beta. Estrogen regulation of other genes, including the anti-metastatic NME1 gene, is also altered. Overall, our results suggest that the balance coding/non-coding SRA transcripts not only characterizes particular tumor phenotypes but might also, through regulating the expression of specific genes, be involved in breast tumorigenesis and tumor progression.

The steroid receptor RNA activator protein is expressed in breast tumor tissues

The steroid receptor RNA activator (SRA) was originally described as the first functional noncoding RNA able to specifically coactivate the activity of steroid receptors. We previously demonstrated the existence in breast cancer cell lines of new SRA isoforms that, as opposed to the first cloned SRA RNA, encode for a 236–amino acid protein, SRAP. To investigate the possible implications of the coding SRA RNA and SRAP expression on breast cancer progression, we examined by Western blot analysis 74 primary breast tumors of patients subsequently treated with tamoxifen. Patients whose primary tumors were positive for SRAP expression (n = 24) had a significantly (Kaplan‐Meier survival curve p = 0.047) lower likelihood of dying from recurrent disease than SRAP‐negative patients (n = 50). We generated 2 cell lines, SRAP‐V5‐High.A and SRAP‐V5‐High.B, by stably overexpressing SRAP in the estrogen receptor‐positive MCF‐7 breast cancer cell line. Transient transfection experiments, performed using a luciferase reporter gene under the control of an estrogen‐responsive element, revealed decreased sensitivity to estradiol but no additional sensitivity to tamoxifen in SRAP‐overexpressing cells. Overall, our data suggest that the presence of both coding SRA RNA and its corresponding SRAP modifies the activity of estrogen receptor in breast cancer cells and that SRAP could be a new clinical marker for breast cancer. Further studies are needed to define the respective mechanisms of action and the roles of SRA RNA and protein in breast tumorigenesis and tumor progression.

Expression of small breast epithelial mucin (SBEM) protein in tissue microarrays (TMAs) of primary invasive breast cancers

Aims:  Small breast epithelial mucin (SBEM) is a recently described gene product that shows promise as a new breast biomarker. The aim was to investigate for the first time SBEM protein expression in a large cohort (n = 300) of invasive breast cancers, its relationship to established clinical variables and its association with clinical outcome.

Demonstration of a Tissue Factor Pathway Inhibitor 2 Messenger RNA Synthesis by Pure Villous Cytotrophoblast Cells Isolated from Term Human Placentas

Abstract Tissue factor pathway inhibitor 2 (TFPI-2), a Kunitz-type proteinase inhibitor, might play an important role during placenta growth by regulating trophoblast invasion and differentiation. Many TFPI-2 transcripts have been detected in syncytiotrophoblast cells, but conflicting results have been reported concerning TFPI-2 synthesis by the cytotrophoblast. To address this issue, we developed a method to isolate pure preparations of human villous cytotrophoblast cells from normal term placentas, and the synthesis of tissue factor, TFPI-1, and TFPI-2 mRNAs was then evaluated. Cells were isolated by trypsin-DNase-EDTA digestion, followed by Percoll gradient separation and immunodepletion of human leukocyte antigen-positive cells. The quality of villous cytotrophoblast cells was verified by electron microscopy. Purity of cell preparations was assessed by labeling cells with GB25, a monoclonal antibody specific to villous trophoblast cells, and by checking the absence of contaminating cells using anti-CD9 antibody. The lack of hCG, CD32 mRNA, and tissue factor mRNA also indicated the absence of contaminating cells. Using competitive reverse transcription polymerase chain reaction, we showed that freshly isolated villous cytotrophoblast cells synthesized significant levels of TFPI-1 mRNA and larger amounts of TFPI-2 mRNA. TFPI-1 and TFPI-2 mRNA synthesis remained unchanged when cytotrophoblast cells were cultured in complete medium and evolved as a multinucleated syncytiotrophoblast. These results indicate that the villous cytotrophoblast and syncytiotrophoblast are both important sites of TFPI-2 synthesis in the human placenta. This study also indicates that tissue factor detection should be used systematically to check the purity of cytotrophoblast cell preparations because it allows detection of contamination by monocytes/macrophages and by syncytial fragments.

Demonstration of inducible TFPI-2 mRNA synthesis in BeWo and JEG-3 trophoblast cells using a competitive RT-PCR.

Tissue factor pathway inhibitor-2 (TFPI-2) displays structural similarities with TFPI-1, the major inhibitor of tissue factor (TF)/, factor VIIa. It is synthesized mostly by syncytiotrophoblast in the placenta, but its physiological functions are not fully understood. We studied the synthesis of TFPI-2 mRNA and that of TFPI-1 and TF in three human trophoblast cell lines, JAR, BeWo, and JEG-3. We first developed specific competitive reverse transcription-polymerase chain reaction (RT-PCR) assays for each gene studied using human umbilical vein endothelial cells (HUVEC). The three trophoblast cell lines strongly synthesized TF mRNA whereas the synthesis of TFPI-1 mRNA was very low. TFPI-2 mRNA was not detected in unstimulated or stimulated JAR cells. In contrast, JEG-3 and, to a lesser extent, BeWo produced significant amounts of TFPI-2 mRNA, which were significantly increased after stimulation with phorbol 12-myristate 13-acetate (PMA). However, tumor necrosis factor-alpha (TNF-alpha) had no effect on this synthesis. JEG-3 and BeWo are thus two cell lines that could be used to study TFPI-2 gene regulation and to investigate the role of TF, TFPI-1, and TFPI-2 during trophoblast differentiation.

Identification of an octamer-binding site controlling the activity of the small breast epithelial mucin gene promoter.

e human small breast epithelial mucin (SBEM) gene has been identified as being preferentially expressed in mammary epithelial cells and over-expressed in breast tumors. In this report, we have characterized the promoter of SBEM gene in order to identify sequences responsible for this strong mammary expression. A series of SBEM promoter/luciferase constructs were transiently transfected into both breast (MCF-7, BT-20) and non-breast (HeLa and HepG2) cell lines. In addition to the minimal promoter and to a repressor region, we have identified an 87-bp sequence (-357/-270) driving a strong breast-specific expression. Site-directed mutagenesis of a putative octamer-binding transcription factor binding site located within this latter region led to a strong decrease of the transcriptional activity of the SBEM promoter. Furthermore, transient over-expression of Oct1 and Oct2 not only increased SBEM promoter reporter activity, but also enhanced endogenous SBEM mRNA level. Overall, the data suggest that octamer-binding transcription factors participate in the strong expression of SBEM gene in breast tissues. Clarifying the SBEM gene regulation will help to dissect mechanisms underlying transcription of normal breast and breast cancer-associated genes.

The promoter competition assay (PCA): a new approach to identify motifs involved in the transcriptional activity of reporter genes.

Identifying particular motifs responsible for promoter activity is a crucial step toward the development of new gene-based preventive and therapeutic strategies. However, to date, experimental methods to study promoter activity remain limited. We present in this report a promoter competition assay designed to identify, within a given promoter region, motifs critical for its activity. This assay consists in co-transfecting the promoter to be analyzed and double-stranded oligonucleotides which will compete for the binding of transcription factors. Using the recently characterized SBEM promoter as model, we first delineated the feasibility of the method and optimized the experimental conditions. We then identified, within an 87-bp region responsible for a strong expression of the reporter gene, an octamer-binding site essential for its transcriptional regulation. The importance of this motif has been confirmed by site-directed mutagenesis. The promoter competition assay appears to be a fast and efficient approach to identify, within a given promoter sequence, sites critical for its activity.

“Pocket-sized RNA-Seq”: A Method to Capture New Mature microRNA Produced from a Genomic Region of Interest

Currently, the discovery of new small ncRNAs requires high throughput methods even in the case of focused research on the regulation of specific genes or set of genes. We propose herein a simple, rapid, efficient, and cost effective method to clone and sequence single, yet unknown, small ncRNA. This technique that we called “Pocket-sized RNA-Seq” or psRNA-seq is based on in vitro transcription, RNA pull down and adapted RACE-PCR methods that allow its implementation using either available commercial kits or in-house reagents.