PTBP1-activated co-transcriptional splicing controls epigenetic status of pluripotent stem cells.

Many spliceosomal introns are excised from nascent transcripts emerging from RNA polymerase II (RNA Pol II). The extent of cell-type-specific regulation and possible functions of such co-transcriptional events remain poorly understood. We examined the role of the RNA-binding protein PTBP1 in this process using an acute depletion approach followed by the analysis of chromatin- and RNA Pol II-associated transcripts. We show that PTBP1 activates the co-transcriptional excision of hundreds of introns, a surprising effect given that this protein is known to promote intron retention. Importantly, some co-transcriptionally activated introns fail to complete their splicing without PTBP1. In a striking example, retention of a PTBP1-dependent intron triggers nonsense-mediated decay of transcripts encoding DNA methyltransferase DNMT3B. We provide evidence that this regulation facilitates the natural decline in DNMT3B levels in developing neurons and protects differentiation-specific genes from ectopic methylation. Thus, PTBP1-activated co-transcriptional splicing is a widespread phenomenon mediating epigenetic control of cellular identity.

An 840 kb distant upstream enhancer is a crucial regulator of catecholamine-dependent expression of the Bdnf gene in astrocytes.

Brain-derived neurotrophic factor (BDNF) plays a fundamental role in the developing and adult nervous system, contributing to neuronal survival, differentiation, and synaptic plasticity. Dysregulation of BDNF synthesis, secretion or signaling has been associated with many neurodevelopmental, neuropsychiatric, and neurodegenerative disorders. Although the transcriptional regulation of the Bdnf gene has been extensively studied in neurons, less is known about the regulation and function of BDNF in non-neuronal cells. The most abundant type of non-neuronal cells in the brain, astrocytes, express BDNF in response to catecholamines. However, genetic elements responsible for this regulation have not been identified. Here, we investigated four potential Bdnf enhancer regions and based on reporter gene assays, CRISPR/Cas9 engineering and CAPTURE-3C-sequencing we conclude that a region 840 kb upstream of the Bdnf gene regulates catecholamine-dependent expression of Bdnf in rodent astrocytes. We also provide evidence that this regulation is mediated by CREB and AP1 family transcription factors. This is the first report of an enhancer coordinating the transcription of Bdnf gene in non-neuronal cells.

Regulation potential of transcribed simple repeated sequences in developing neurons.

Simple repeated sequences (SRSs), defined as tandem iterations of microsatellite- to satellite-sized DNA units, occupy a substantial part of the human genome. Some of these elements are known to be transcribed in the context of repeat expansion disorders. Mounting evidence suggests that the transcription of SRSs may also contribute to normal cellular functions. Here, we used genome-wide bioinformatics approaches to systematically examine SRS transcriptional activity in cells undergoing neuronal differentiation. We identified thousands of long noncoding RNAs containing >200-nucleotide-long SRSs (SRS-lncRNAs), with hundreds of these transcripts significantly upregulated in the neural lineage. We show that SRS-lncRNAs often originate from telomere-proximal regions and that they have a strong potential to form multivalent contacts with a wide range of RNA-binding proteins. Our analyses also uncovered a cluster of neurally upregulated SRS-lncRNAs encoded in a centromere-proximal part of chromosome 9, which underwent an evolutionarily recent segmental duplication. Using a newly established in vitro system for rapid neuronal differentiation of induced pluripotent stem cells, we demonstrate that at least some of the bioinformatically predicted SRS-lncRNAs, including those encoded in the segmentally duplicated part of chromosome 9, indeed increase their expression in developing neurons to readily detectable levels. These and other lines of evidence suggest that many SRSs may be expressed in a cell type and developmental stage-specific manner, providing a valuable resource for further studies focused on the functional consequences of SRS-lncRNAs in the normal development of the human brain, as well as in the context of neurodevelopmental disorders.

Alternative splicing coupled to nonsense-mediated decay coordinates downregulation of non-neuronal genes in developing mouse neurons.

BACKGROUND: The functional coupling between alternative pre-mRNA splicing (AS) and the mRNA quality control mechanism called nonsense-mediated decay (NMD) can modulate transcript abundance. Previous studies have identified several examples of such a regulation in developing neurons. However, the systems-level effects of AS-NMD in this context are poorly understood. RESULTS: We developed an R package, factR2, which offers a comprehensive suite of AS-NMD analysis functions. Using this tool, we conducted a longitudinal analysis of gene expression in pluripotent stem cells undergoing induced neuronal differentiation. Our analysis uncovers hundreds of AS-NMD events with significant potential to regulate gene expression. Notably, this regulation is significantly overrepresented in specific functional groups of developmentally downregulated genes. Particularly strong association with gene downregulation is detected for alternative cassette exons stimulating NMD upon their inclusion into mature mRNA. By combining bioinformatic analyses with CRISPR/Cas9 genome editing and other experimental approaches we show that NMD-stimulating cassette exons regulated by the RNA-binding protein PTBP1 dampen the expression of their genes in developing neurons. We also provided evidence that the inclusion of NMD-stimulating cassette exons into mature mRNAs is temporally coordinated with NMD-independent gene repression mechanisms. CONCLUSIONS: Our study provides an accessible workflow for the discovery and prioritization of AS-NMD targets. It further argues that the AS-NMD pathway plays a widespread role in developing neurons by facilitating the downregulation of functionally related non-neuronal genes.

Protocol for auxin-inducible depletion of the RNA-binding protein PTBP1 in mouse embryonic stem cells.

Inducible degradation of proteins of interest provides a powerful approach for functional studies. Here, we present a protocol for tightly controlled depletion of the RNA-binding protein PTBP1 in mouse embryonic stem cells (ESCs). We describe steps for establishing an ESC line expressing doxycycline-inducible auxin receptor protein OsTIR1 and tagging endogenous Ptbp1 alleles using CRISPR-Cas9 and homology-directed repair reagents. We then detail procedures for assaying the efficiency of inducible PTBP1 knockdown by immunoblotting. This protocol is adaptable for other protein targets. For complete details on the use and execution of this protocol, please refer to Iannone et al.1.

Intronic enhancer region governs transcript-specific Bdnf expression in rodent neurons.

Brain-derived neurotrophic factor (BDNF) controls the survival, growth, and function of neurons both during the development and in the adult nervous system. Bdnf is transcribed from several distinct promoters generating transcripts with alternative 5' exons. Bdnf transcripts initiated at the first cluster of exons have been associated with the regulation of body weight and various aspects of social behavior, but the mechanisms driving the expression of these transcripts have remained poorly understood. Here, we identify an evolutionarily conserved intronic enhancer region inside the Bdnf gene that regulates both basal and stimulus-dependent expression of the Bdnf transcripts starting from the first cluster of 5' exons in mouse and rat neurons. We further uncover a functional E-box element in the enhancer region, linking the expression of Bdnf and various pro-neural basic helix-loop-helix transcription factors. Collectively, our results shed new light on the cell-type- and stimulus-specific regulation of the important neurotrophic factor BDNF.

CRABP1 provides high malignancy of transformed mesenchymal cells and contributes to the pathogenesis of mesenchymal and neuroendocrine tumors.

CRABP1 (cellular retinoic acid binding protein 1) belongs to the family of fatty acid binding proteins. Retinoic acid binding is the only known functional activity of this protein. The role of CRABP1 in human carcinogenesis remains poorly understood. Here, for the first time we demonstrated pro-metastatic and pro-tumorigenic activity of CRABP1 in mesenchymal tumors. Further functional analysis revealed that the pro-tumorigenic effect of CRABP1 does not depend on retinoic acid binding activity. These results suggest that CRABP1 could have an alternative intracellular functional activity that contributes to the high malignancy of transformed mesenchymal cells. Microarray analysis detected CRABP1-mediated alterations in the expression of about 100 genes, including those encoding key regulatory proteins. CRABP1 is ubiquitously expressed in monophasic synovial sarcomas, while in biphasic synovial sarcomas it is expressed uniquely by the spindle cells of the aggressive mesenchymal component. High level of CRABP1 expression is associated with lymph node metastasis and poor differentiation/high grade of pancreatic neuroendocrine tumors (pNETs). Presented data suggest CRABP1 as a promising biomarker of pNETs' clinical behavior. Our results give the first evidence of pro-tumorigenic and pro-metastatic activity of CRABP1 in mesenchymal and neuroendocrine tumors.