Didier Auboeuf

Tissue distribution and quantification of the expression of mRNAs of peroxisome proliferator-activated receptors and liver X receptor-alpha in humans: no alteration in adipose tissue of obese and NIDDM patients

Members of the peroxisome proliferator–activated receptor (PPAR) family might be involved in pathologies with altered lipid metabolism. They participate in the control of the expression of genes involved in lipid metabolism and adipocyte differentiation. In addition, thiazolidinediones improve insulin resistance in vivo by activating PPARγ. However, little is known regarding their tissue distribution and relative expression in humans. Using a quantitative and sensitive reverse transcription (RT)-competitive polymerase chain reaction (PCR) assay, we determined the distribution and relative mRNA expression of the four PPARs (α, β, γ1, and γ2) and liver X receptor-α (LXRα) in the main tissues implicated in lipid metabolism. PPARα and LXRα were mainly expressed in liver, while PPARγ1 predominated in adipose tissue and large intestine. We found that PPARγ2 mRNA was a minor isoform, even in adipose tissue, thus causing question of its role in humans. PPARβ mRNA was present in all the tissues tested at low levels. In addition, PPARγ mRNA was barely detectable in skeletal muscle, suggesting that improvement of insulin resistance with thiazolidinediones may not result from a direct effect of these agents on PPARγ in muscle. Obesity and NIDDM were not associated with change in PPARs and LXRα expression in adipose tissue. The mRNA levels of PPARγ1, the predominant form in adipocytes, did not correlate with BMI, leptin mRNA levels, or fasting insulinemia in 29 subjects with various degrees of obesity. These results indicated that obesity is not associated with alteration in PPAR gene expression in abdominal subcutaneous adipose tissue in humans.

Widespread Estrogen-Dependent Repression of microRNAs Involved in Breast Tumor Cell Growth

Altered expression of microRNAs (miRNA), an abundant class of small nonprotein-coding RNAs that mostly function as negative regulators of protein-coding gene expression, is common in cancer. Here, we analyze the regulation of miRNA expression in response to estrogen, a steroid hormone that is involved in the development and progression of breast carcinomas and that is acting via the estrogen receptors (ER) transcription factors. We set out to thoroughly describe miRNA expression, by using miRNA microarrays and real-time reverse transcription-PCR (RT-PCR) experiments, in various breast tumor cell lines in which estrogen signaling has been induced by 17beta-estradiol (E(2)). We show that the expression of a broad set of miRNAs decreases following E(2) treatment in an ER-dependent manner. We further show that enforced expression of several of the repressed miRNAs reduces E(2)-dependent cell growth, thus linking expression of specific miRNAs with estrogen-dependent cellular response. In addition, a transcriptome analysis revealed that the E(2)-repressed miR-26a and miR-181a regulate many genes associated with cell growth and proliferation, including the progesterone receptor gene, a key actor in estrogen signaling. Strikingly, miRNA expression is also regulated in breast cancers of women who had received antiestrogen neoadjuvant therapy. Overall, our data indicate that the extensive alterations in miRNA regulation upon estrogen signaling pathway play a key role in estrogen-dependent functions and highlight the utility of considering miRNA expression in the understanding of antiestrogen resistance of breast cancer.

The expression of ob gene is not acutely regulated by insulin and fasting in human abdominal subcutaneous adipose tissue.

The regulation of ob gene expression in abdominal subcutaneous adipose tissue was investigated using a reverse transcription-competitive PCR method to quantify the mRNA level of leptin. Leptin mRNA level was highly correlated with the body mass index of 26 subjects (12 lean, 7 non-insulin-dependent diabetic, and 7 obese patients). The effect of fasting on ob gene expression was investigated in 10 subjects maintained on a hypocaloric diet (1045 KJ/d) for 5 d. While their metabolic parameters significantly changed (decrease in insulinemia, glycemia, and resting metabolic rate and increase in plasma ketone bodies), the caloric restriction did not modify the leptin mRNA level in the adipose tissue. To verify whether insulin regulates ob gene expression, six lean subjects underwent a 3-h euglycemic hyperinsulinemic (846 +/- 138 pmol/liter) clamp. Leptin and Glut 4 mRNA levels were quantified in adipose tissue biopsies taken before and at the end of the clamp. Insulin infusion produced a significant threefold increase in Glut 4 mRNA while leptin mRNA was not affected. It is concluded that ob gene expression is not acutely regulated by insulin or by metabolic factors related to fasting in human abdominal subcutaneous adipose tissue.

Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy

Myotonic dystrophy is the most common muscular dystrophy in adults and the first recognized example of an RNA-mediated disease. Congenital myotonic dystrophy (CDM1) and myotonic dystrophy of type 1 (DM1) or of type 2 (DM2) are caused by the expression of mutant RNAs containing expanded CUG or CCUG repeats, respectively. These mutant RNAs sequester the splicing regulator Muscleblind-like-1 (MBNL1), resulting in specific misregulation of the alternative splicing of other pre-mRNAs. We found that alternative splicing of the bridging integrator-1 (BIN1) pre-mRNA is altered in skeletal muscle samples of people with CDM1, DM1 and DM2. BIN1 is involved in tubular invaginations of membranes and is required for the biogenesis of muscle T tubules, which are specialized skeletal muscle membrane structures essential for excitation-contraction coupling. Mutations in the BIN1 gene cause centronuclear myopathy, which shares some histopathological features with myotonic dystrophy. We found that MBNL1 binds the BIN1 pre-mRNA and regulates its alternative splicing. BIN1 missplicing results in expression of an inactive form of BIN1 lacking phosphatidylinositol 5-phosphate–binding and membrane-tubulating activities. Consistent with a defect of BIN1, muscle T tubules are altered in people with myotonic dystrophy, and membrane structures are restored upon expression of the normal splicing form of BIN1 in muscle cells of such individuals. Finally, reproducing BIN1 splicing alteration in mice is sufficient to promote T tubule alterations and muscle weakness, a predominant feature of myotonic dystrophy.

Coactivator/corepressor ratios modulate PR-mediated transcription by the selective receptor modulator RU486

Selective receptor modulators, such as the antiprogestin RU486, are known to exhibit partial agonist activities in a cell-type-dependent manner. Employing an in vitro chromatin transcription system that recapitulates progesterone receptor (PR)-mediated transcription in vivo, we have investigated the molecular basis by which the antiprogestin RU486 regulates transcription in a cell-type-specific manner. We have compared the effects of RU486 on PR-dependent transcription in vitro using T47D and HeLa cell nuclear extracts. RU486 exhibits a differential ability to activate transcription within these two cell types. The differential effect on transcription correlates with different ratios of endogenous coactivators/corepressors in these cells. Unlike agonist-bound PR that interacts only with coactivators such as steroid receptor coactivator-1 (SRC-1), RU486-bound PR binds to both coactivator SRC-1 and corepressor silencing mediator for retinoid and thyroid hormone receptor (SMRT) in vitro. Both SRC-1 and SMRT have the capacity to modulate RU486-dependent activity. Moreover, a change in the relative levels of SRC-1 and SMRT contained in our chromatin transcription system modulates agonist/antagonist effects of RU486 on transcription by PR. Our data indicate that the ability of RU486 to activate transcription is modulated by the ratio of coactivators to corepressors and substantiate the important roles of coregulators in the regulation of steroid receptor mediated transactivation in response to selective receptor modulators.

CoAA, a nuclear receptor coactivator protein at the interface of transcriptional coactivation and RNA splicing.

ABSTRACT We have shown that steroid hormones coordinately control gene transcriptional activity and splicing decisions in a promoter-dependent manner. Our hypothesis is that a subset of hormonally recruited coregulators involved in regulation of promoter transcriptional activity also directly participate in alternative RNA splicing decisions. To gain insight into the molecular mechanisms by which transcriptional coregulators could control splicing decisions, we focused our attention on a recently identified coactivator, CoAA. This heterogeneous nuclear ribonucleoprotein (hnRNP)-like protein interacts with the transcriptional coregulator TRBP, a protein recruited to target promoters through interactions with activated nuclear receptors. Using transcriptional and splicing reporter genes driven by different promoters, we observed that CoAA mediates transcriptional and splicing effects in a promoter-preferential manner. We compared the activity of CoAA to the activity of other hnRNP-related proteins that, like CoAA, contain two N-terminal RNA recognition motifs (RRMs) followed by a C-terminal auxiliary domain and either have or have not been implicated in transcriptional control. By swapping either CoAA RRMs or the CoAA auxiliary domain with the corresponding domains of the proteins selected, we showed that depending on the promoter, the RRMs and the auxiliary domain of CoAA are differentially engaged in transcription. This contributes to the promoter-preferential effects mediated by CoAA on RNA splicing during the course of steroid hormone action.

Acute regulation by insulin of phosphatidylinositol-3-kinase, Rad, Glut 4, and lipoprotein lipase mRNA levels in human muscle.

We have investigated the acute regulation by insulin of the mRNA levels of nine genes involved in insulin action, in muscle biopsies obtained before and at the end of a 3-h euglycemic hyperinsulinemic clamp. Using reverse transcription-competitive PCR, we have measured the mRNAs encoding the two insulin receptor variants, the insulin receptor substrate-1, the p85alpha subunit of phosphatidylinositol-3-kinase, Ras associated to diabetes (Rad), the glucose transporter Glut 4, glycogen synthase, 6-phosphofructo-l-kinase, lipoprotein lipase, and the hormone-sensitive lipase. Insulin infusion induced a significant increase in the mRNA level of Glut 4 (+56 +/- 13%), Rad (+96 +/- 25%), the p85alpha subunit of phosphatidylinositol-3-kinase (+92 +/- 18%) and a decrease in the lipoprotein lipase mRNA level (-49 +/- 5%), while the abundance of the other mRNAs was unaffected. The relative expression of the two insulin receptor variants was not modified. These results demonstrate an acute coordinated regulation by insulin of the expression of genes coding key proteins involved in its action in human skeletal muscle and suggest that Rad and the p85alpha regulatory subunit of phosphatidylinositol-3-kinase can be added to the list of the genes controlled by insulin.

Cotranscriptional exon skipping in the genotoxic stress response

Pre-mRNA splicing is functionally coupled to transcription, and genotoxic stresses can enhance alternative exon inclusion by affecting elongating RNA polymerase II. We report here that various genotoxic stress inducers, including camptothecin (CPT), inhibit the interaction between Ewings sarcoma proto-oncoprotein (EWS), an RNA polymerase II–associated factor, and YB-1, a spliceosome-associated factor. This results in the cotranscriptional skipping of several exons of the MDM2 gene, which encodes the main p53 ubiquitin ligase. This reversible exon skipping participates in the regulation of MDM2 expression that may contribute to the accumulation of p53 during stress exposure and its rapid shut-off when stress is removed. Finally, a splicing-sensitive microarray identified numerous exons that are skipped in response to CPT and EWS–YB-1 depletion. These data demonstrate genotoxic stress-induced alteration of the communication between the transcriptional and splicing machineries, which results in widespread exon skipping and plays a central role in the genotoxic stress response.

Splicing factor and exon profiling across human tissues

It has been shown that alternative splicing is especially prevalent in brain and testis when compared to other tissues. To test whether there is a specific propensity of these tissues to generate splicing variants, we used a single source of high-density microarray data to perform both splicing factor and exon expression profiling across 11 normal human tissues. Paired comparisons between tissues and an original exon-based statistical group analysis demonstrated after extensive RT-PCR validation that the cerebellum, testis, and spleen had the largest proportion of differentially expressed alternative exons. Variations at the exon level correlated with a larger number of splicing factors being expressed at a high level in the cerebellum, testis and spleen than in other tissues. However, this splicing factor expression profile was similar to a more global gene expression pattern as a larger number of genes had a high expression level in the cerebellum, testis and spleen. In addition to providing a unique resource on expression profiling of alternative splicing variants and splicing factors across human tissues, this study demonstrates that the higher prevalence of alternative splicing in a subset of tissues originates from the larger number of genes, including splicing factors, being expressed than in other tissues.

Regulation of alternative splicing by the ATP-dependent DEAD-Box RNA helicase p72

ABSTRACT Although a number of ATP-dependent RNA helicases are important for constitutive RNA splicing, no helicases have been implicated in alternative RNA splicing. Here, we show that the abundant DEAD-box RNA helicase p72, but not its close relative p68, affects the splicing of alternative exons containing AC-rich exon enhancer elements. The effect of p72 was tested by using mini-genes that undergo different types of alternative splicing. When the concentration of p72 was increased in transient transfections, the inclusion of enhancer-containing CD44 alternative exons v4 and v5 increased using a mini-gene that contained these exons and their flanking introns inserted into a β-globin gene. Other types of alternative splicing were not impacted by altering p72 concentrations. Mutation of the p72 helicase ATP-binding site or deletion of the carboxy-terminal region of the protein reduced the ability of the transfected protein to affect CD44 variable exon splicing. Use of in vitro extracts overexpressing p72 indicated that p72 becomes associated with complexes containing precursor RNA. Helicases have been implicated both in altering RNA-RNA interactions and in remodeling RNA-protein complexes. CD44 exon v4 contains a potential internal secondary structure element that base pairs the 5′ splice site with a region inside the exon located between enhancer elements. Mutations that destroyed this complementarity modestly increased inclusion in the absence of p72 but still responded to increasing p72 concentration like the wild-type exon, suggesting that p72 might have effects on protein-RNA interactions. In agreement with this hypothesis, p72 was not able to restore the inclusion of an exon mutated for its major enhancer element. Our results suggest that RNA helicases may be important alternative splicing regulatory factors.