Project description:PPARγ regulates glucose and lipid homeostasis, insulin signaling and adipocyte differentiation. Here we report the skipping of exon 5 as legitimate splicing event generating PPARγΔ5, a new truncated isoform lacking the ligand binding domain. PPARγΔ5 is endogenously expressed in human adipose tissue and during adipocyte differentiation, lacks the ligand-dependent transactivation ability and acts as dominant negative reducing PPARγ activity. Ligand-mediated PPARγ activation induces exon 5 skipping in a negative feedback loop, suggesting alternative splicing as a new mechanism regulating PPARγ activity. PPARγΔ5 over-expression modifies PPARγ-induced transcriptional network, significantly impairing the differentiation ability of adipocyte precursor cells. Additionally, PPARγΔ5 expression in subcutaneous adipose tissue positively correlates with BMI in two independent cohorts of obese and diabetic patients. From a functional perspective, PPARγΔ5 mimics PPARG dominant negative mutated receptors, possibly contributing to adipose tissue dysfunctions. These findings open unexplored scenario in PPARG regulation and PPARγ-related diseases.
Project description:Peroxisome proliferator-activated receptor g (PPARg) is a nuclear receptor that is a vital regulator of adipogenesis, insulin sensitivity, and lipid metabolism. Activation of PPARg by antidiabetic thiazolidinediones (TZD) reverses insulin resistance but also leads to weight gain that limits the use of these drugs. There are two main PPARg isoforms, but the specific functions of each are not established. Here we generated mouse lines in which endogenous PPARg1 and PPARg2 were epitope-tagged to interrogate isoform-specific genomic binding, and mice deficient in either PPARg1 or PPARg2 to assess isoform-specific gene regulation. Strikingly, although PPARg1 and PPARg2 contain identical DNA binding domains, we uncovered isoform-specific genomic binding sites in addition to shared sites. Moreover, PPARg1 and PPARg2 regulated different set of genes in adipose tissue depots, suggesting distinct roles in adipocyte biology. Indeed, mice with selective deficiency of PPARg1 maintained body temperature better than wild type or PPARg2-deficient mice. Most remarkably, although TZD treatment improved glucose toleranceinsulin resistance in mice lacking either PPARg1 or PPARg2, the PPARg1-deficient mice were protected from TZD-induced body weight gain compared to PPARg2-deficient mice. Thus, PPARg isoforms have specific and separable metabolic functions that may be targeted to improve therapy for insulin resistance and diabetes.
Project description:Ligand-mediated activation of the nuclear hormone receptor PPAR gamma lowers blood pressure and improves glucose tolerance in humans. Two naturally occurring mutations (P467L, V290M) in the ligand binding domain of PPAR gamma have been described in humans that lead to severe insulin resistance and hypertension. Experimental evidence suggests that these mutant versions of PPAR gamma act in a dominant negative fashion. To better understand the molecular mechanisms underlying PPAR gamma action in the vasculature, we determined the global gene expression profile in primary aortic endothelial cells in response to endothelial cell specific expression of a dominant negative isoform of PPAR gamma (V290M).
Project description:The generation of distiThe generation of distinct messenger RNA isoforms through alternative RNA processing influences the expression and function of genes, often in a cell-type specific manner. Here, we assess the regulatory relationships between transcription initiation, alternative splicing, and 3ʹ end site selection. Applying multiple long-read-sequencing approaches to obtain an assembly accurately representing even the longest transcripts from end to end, we quantify mRNA isoform choice in Drosophila and human tissues, including the transcriptionally complex nervous system. We find that in Drosophila brains as well as in human cerebral organoids, 3ʹ end site choice is globally influenced by the site of transcription initiation. “Dominant promoters”, characterized by specific epigenetic signatures including p300/CBP binding, impose a transcriptional constraint to define splice and polyadenylation variants. In vivo deletion or overexpression of dominant promoters as well as CBP/p300 loss disrupted the 3ʹ end expression landscape. Our study demonstrates the crucial impact of TSS choice on the regulation of transcript diversity and tissue identity.ct messenger RNA isoforms through alternative splicing and alternative 3' end formation influences the expression and function of genes, often in a cell-type specific manner. Here, we quantitatively assess the regulatory relationships between transcription initiation and co-transcriptional processing steps, particularly 3' end formation. Applying multiple long-read-sequencing approaches to obtain an assembly accurately representing even the longest mRNA isoforms from end-to-end, we quantify mRNA isoform choice in Drosophila and human tissues, including the transcriptionally complex nervous system. We find that in Drosophila brains as well as in human cerebral organoids, 3' end site choice is globally influenced by the site of transcription start. We define a subset of TSSs, “dominant promoters” that impose a transcriptional constraint to predetermine splice and polyadenylation variants, which are characterized by specific epigenetic signatures. In vivo deletion or overexpression of dominant promoters disrupted the 3' end expression landscape. Our study demonstrates the crucial impact of transcription initiation site choice on the regulation of transcript diversity and tissue identity.
Project description:The generation of distinct messenger RNA isoforms through alternative RNA processing influences the expression and function of genes, often in a cell-type specific manner. Here, we assess the regulatory relationships between transcription initiation, alternative splicing, and 3ʹ end site selection. Applying multiple long-read-sequencing approaches to obtain an assembly accurately representing even the longest transcripts from end to end, we quantify mRNA isoform choice in Drosophila and human tissues, including the transcriptionally complex nervous system. We find that in Drosophila brains as well as in human cerebral organoids, 3ʹ end site choice is globally influenced by the site of transcription initiation. “Dominant promoters”, characterized by specific epigenetic signatures including p300/CBP binding, impose a transcriptional constraint to define splice and polyadenylation variants. In vivo deletion or overexpression of dominant promoters as well as CBP/p300 loss disrupted the 3ʹ end expression landscape. Our study demonstrates the crucial impact of TSS choice on the regulation of transcript diversity and tissue identity.ct messenger RNA isoforms through alternative splicing and alternative 3' end formation influences the expression and function of genes, often in a cell-type specific manner. Here, we quantitatively assess the regulatory relationships between transcription initiation and co-transcriptional processing steps, particularly 3' end formation. Applying multiple long-read-sequencing approaches to obtain an assembly accurately representing even the longest mRNA isoforms from end-to-end, we quantify mRNA isoform choice in Drosophila and human tissues, including the transcriptionally complex nervous system. We find that in Drosophila brains as well as in human cerebral organoids, 3' end site choice is globally influenced by the site of transcription start. We define a subset of TSSs, “dominant promoters” that impose a transcriptional constraint to predetermine splice and polyadenylation variants, which are characterized by specific epigenetic signatures. In vivo deletion or overexpression of dominant promoters disrupted the 3' end expression landscape. Our study demonstrates the crucial impact of transcription initiation site choice on the regulation of transcript diversity and tissue identity.
Project description:Peroxisome proliferator–activated receptor γ (PPARγ) is the central regulator of adipogenesis, and its dysregulation is linked to obesity and metabolic diseases. Identification of the factors that regulate PPARγ expression and activity is therefore crucial for combating obesity. Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor with a known role in xenobiotic detoxification. Recent studies have suggested that AhR also plays essential roles in energy metabolism. However, the detailed mechanisms remain unclear. We previously reported that experiments with adipocyte-specific Cullin 4b (Cul4b)-knockout mice showed that CUL4B suppresses adipogenesis by targeting PPARγ. Here, using immunoprecipitation, ubiquitination, real-time PCR and Gst-pulldown assays, we report that AhR functions as the substrate receptor in CUL4B–RING E3 ubiquitin ligase (CRL4B) complex and is required for recruiting PPARγ. AhR overexpression reduced PPARγ stability and suppressed adipocyte differentiation, and AhR knockdown stimulated adipocyte differentiation in 3T3-L1 cells. Furthermore, we found that two lysine sites on residues 268 and 293 in PPARγ are targeted for CRL4B-mediated ubiquitination, indicating cross-talk between acetylation and ubiquitination. Our findings establish a critical role of AhR in regulating PPARγ stability and suggest that the AhR–PPARγ interaction may represent a potential therapeutic target for managing metabolic diseases arising from PPARγ dysfunction.
Project description:Alternative splicing is a mechanism in eukaryotes by which different forms of messenger RNAs (mRNAs) are generated from the same gene. Identification of alternative splice variants requires the identification of peptides specific for alternative splice forms. For this purpose, we generated a human database which contains only proteotypic tryptic peptides specific for alternative splice forms from Swiss-Prot entries. Using this database allows an easy access to the peptide sequences that matches the unique amino acid sequence of splice variants to MS data. Furthermore, we combined this database without isoform 1-specific peptides with human Swiss-Prot. This combined database can be used as a general database for searching of LC-MS data. LC-MS data derived from in-solution digests of two different cell lines (LNCaP, HeLa), and phosphoproteomics studies were analyzed using these two databases. Several non-isoform 1-specific peptides were found in both cell lines, some of them seemed to be cell line specific. Control and apoptotic phosphoproteomes from Jurkat T cells revealed several non-isoform 1-specific peptides and some of them showed clear quantitative differences between the two states.
Project description:Myotonic dystrophy type 1 (DM1) is a multisystemic genetic disorder caused by a CTG trinucleotide repeat expansion in the 3′ untranslated region of DMPK gene. Heart dysfunctions occur in nearly 80% of DM1 patients and are the second leading cause of disease-related deaths, yet, the underlying mechanisms remain unclear. Herein, we report that upregulation of a non-muscle splice isoform of RNA binding protein RBFOX2 in DM1 heart tissue—due to altered splicing factor and microRNA activities—induces the characteristic cardiac conduction defects detected in DM1 individuals. Mice engineered to express the non-muscle RBFOX2 isoform in heart via tetracycline- inducible transgenesis, or CRISPR/Cas9 targeted genome editing, reproduced DM1- related cardiac-conduction delay and spontaneous episodes of arrhythmia. Furthermore, by integrating RNA binding with cardiac transcriptome datasets of DM1 patients, and mice expressing the non-muscle RBFOX2 isoform, we identified a core network of RBFOX2-driven splicing defects in sodium, potassium, and calcium channels that can alter their rate of ion diffusion and electrophysiological properties. Thus, our results uncover a trans-dominant role for an aberrantly expressed RBFOX2 isoform in DM1 cardiac pathogenesis.
Project description:The generation of distinct messenger RNA isoforms through alternative splicing and alternative 3' end formation influences the expression and function of genes, often in a cell-type specific manner. Here, we quantitatively assess the regulatory relationships between transcription initiation and co-transcriptional processing steps, particularly 3' end formation. Applying multiple long-read-sequencing approaches to obtain an assembly accurately representing even the longest mRNA isoforms from end-to-end, we quantify mRNA isoform choice in Drosophila and human tissues, including the transcriptionally complex nervous system. We find that in Drosophila brains as well as in human cerebral organoids, 3' end site choice is globally influenced by the site of transcription start. We define a subset of TSSs, “dominant promoters” that impose a transcriptional constraint to predetermine splice and polyadenylation variants, which are characterized by specific epigenetic signatures. In vivo deletion or overexpression of dominant promoters disrupted the 3' end expression landscape. Our study demonstrates the crucial impact of transcription initiation site choice on the regulation of transcript diversity and tissue identity.