Regulation of alternative splicing through coupling with transcription and chromatin structure.

Alternative precursor messenger RNA (pre-mRNA) splicing plays a pivotal role in the flow of genetic information from DNA to proteins by expanding the coding capacity of genomes. Regulation of alternative splicing is as important as regulation of transcription to determine cell- and tissue-specific features, normal cell functioning, and responses of eukaryotic cells to external cues. Its importance is confirmed by the evolutionary conservation and diversification of alternative splicing and the fact that its deregulation causes hereditary disease and cancer. This review discusses the multiple layers of cotranscriptional regulation of alternative splicing in which chromatin structure, DNA methylation, histone marks, and nucleosome positioning play a fundamental role in providing a dynamic scaffold for interactions between the splicing and transcription machineries. We focus on evidence for how the kinetics of RNA polymerase II (RNAPII) elongation and the recruitment of splicing factors and adaptor proteins to chromatin components act in coordination to regulate alternative splicing.

Epigenetics in alternative pre-mRNA splicing.

Alternative splicing plays critical roles in differentiation, development, and disease and is a major source for protein diversity in higher eukaryotes. Analysis of alternative splicing regulation has traditionally focused on RNA sequence elements and their associated splicing factors, but recent provocative studies point to a key function of chromatin structure and histone modifications in alternative splicing regulation. These insights suggest that epigenetic regulation determines not only what parts of the genome are expressed but also how they are spliced.

The degree of enhancer or promoter activity is reflected by the levels and directionality of eRNA transcription.

Gene expression is regulated by promoters, which initiate transcription, and enhancers, which control their temporal and spatial activity. However, the discovery that mammalian enhancers also initiate transcription questions the inherent differences between enhancers and promoters. Here, we investigate the transcriptional properties of enhancers during Drosophila embryogenesis using characterized developmental enhancers. We show that while the timing of enhancer transcription is generally correlated with enhancer activity, the levels and directionality of transcription are highly varied among active enhancers. To assess how this impacts function, we developed a dual transgenic assay to simultaneously measure enhancer and promoter activities from a single element in the same embryo. Extensive transgenic analysis revealed a relationship between the direction of endogenous transcription and the ability to function as an enhancer or promoter in vivo, although enhancer RNA (eRNA) production and activity are not always strictly coupled. Some enhancers (mainly bidirectional) can act as weak promoters, producing overlapping spatio-temporal expression. Conversely, bidirectional promoters often act as strong enhancers, while unidirectional promoters generally cannot. The balance between enhancer and promoter activity is generally reflected in the levels and directionality of eRNA transcription and is likely an inherent sequence property of the elements themselves.

Alternative splicing: a pivotal step between eukaryotic transcription and translation.

Alternative splicing was discovered simultaneously with splicing over three decades ago. Since then, an enormous body of evidence has demonstrated the prevalence of alternative splicing in multicellular eukaryotes, its key roles in determining tissue- and species-specific differentiation patterns, the multiple post- and co-transcriptional regulatory mechanisms that control it, and its causal role in hereditary disease and cancer. The emerging evidence places alternative splicing in a central position in the flow of eukaryotic genetic information, between transcription and translation, in that it can respond not only to various signalling pathways that target the splicing machinery but also to transcription factors and chromatin structure.

Accurate genome-wide predictions of spatio-temporal gene expression during embryonic development.

Comprehensive information on the timing and location of gene expression is fundamental to our understanding of embryonic development and tissue formation. While high-throughput in situ hybridization projects provide invaluable information about developmental gene expression patterns for model organisms like Drosophila, the output of these experiments is primarily qualitative, and a high proportion of protein coding genes and most non-coding genes lack any annotation. Accurate data-centric predictions of spatio-temporal gene expression will therefore complement current in situ hybridization efforts. Here, we applied a machine learning approach by training models on all public gene expression and chromatin data, even from whole-organism experiments, to provide genome-wide, quantitative spatio-temporal predictions for all genes. We developed structured in silico nano-dissection, a computational approach that predicts gene expression in >200 tissue-developmental stages. The algorithm integrates expression signals from a compendium of 6,378 genome-wide expression and chromatin profiling experiments in a cell lineage-aware fashion. We systematically evaluated our performance via cross-validation and experimentally confirmed 22 new predictions for four different embryonic tissues. The model also predicts complex, multi-tissue expression and developmental regulation with high accuracy. We further show the potential of applying these genome-wide predictions to extract tissue specificity signals from non-tissue-dissected experiments, and to prioritize tissues and stages for disease modeling. This resource, together with the exploratory tools are freely available at our webserver http://find.princeton.edu, which provides a valuable tool for a range of applications, from predicting spatio-temporal expression patterns to recognizing tissue signatures from differential gene expression profiles.

A DNA intercalating dye-based RT-qPCR alternative to diagnose SARS-CoV-2.

Early detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been proven crucial during the efforts to mitigate the effects of the COVID-19 pandemic. Several diagnostic methods have emerged in the past few months, each with different shortcomings and limitations. The current gold standard, RT-qPCR using fluorescent probes, relies on demanding equipment requirements plus the high costs of the probes and specific reaction mixes. To broaden the possibilities of reagents and thermocyclers that could be allocated towards this task, we have optimized an alternative strategy for RT-qPCR diagnosis. This is based on a widely used DNA-intercalating dye and can be implemented with several different qPCR reagents and instruments. Remarkably, the proposed qPCR method performs similarly to the broadly used TaqMan-based detection, in terms of specificity and sensitivity, thus representing a reliable tool. We think that, through enabling the use of vast range of thermocycler models and laboratory facilities for SARS-CoV-2 diagnosis, the alternative proposed here can increase dramatically the testing capability, especially in countries with limited access to costly technology and reagents.

Ret Receptor Has Distinct Alterations and Functions in Breast Cancer.

Ret receptor tyrosine kinase is a proto-oncogene that participates in development of various cancers. Several independent studies have recently identified Ret as a key player in breast cancer. Although Ret overexpression and function have been under investigation, mainly in estrogen receptor positive breast cancer, a more comprehensive analysis of the impact of recurring Ret alterations in breast cancer is needed. This review consolidates the current knowledge of Ret alterations and their potential effects in breast cancer. We discuss and integrate data on Ret changes in different breast cancer subtypes and potential function in progression, as well as the participation of distinct Ret network signaling partners in these processes. We propose that it will be essential to define a shared molecular feature of tumors with alteration in Ret receptor, be this at the genetic level or via overexpression in order to design effective therapies to target the Ret pathway. Here we review experimental evidence from basic research and pre-clinical studies concentrating on Ret alterations as potential biomarkers for recurrence, and we discuss the possibility that targeting the Ret pathway might in the future become a treatment for breast cancer.

Intragenic epigenetic changes modulate NCAM alternative splicing in neuronal differentiation.

Alternative splicing contributes to cell type-specific transcriptomes. Here, we show that changes in intragenic chromatin marks affect NCAM (neural cell adhesion molecule) exon 18 (E18) alternative splicing during neuronal differentiation. An increase in the repressive marks H3K9me2 and H3K27me3 along the gene body correlated with inhibition of polymerase II elongation in the E18 region, but without significantly affecting total mRNA levels. Treatment with the general DNA methylation inhibitor 5-azacytidine and BIX 01294, a specific inhibitor of H3K9 dimethylation, inhibited the differentiation-induced E18 inclusion, pointing to a role for repressive marks in sustaining NCAM splicing patterns typical of mature neurons. We demonstrate that intragenic deployment of repressive chromatin marks, induced by intronic small interfering RNAs targeting NCAM intron 18, promotes E18 inclusion in undifferentiated N2a cells, confirming the chromatin changes observed upon differentiation to be sufficient to induce alternative splicing. Combined with previous evidence that neuronal depolarization causes H3K9 acetylation and subsequent E18 skipping, our results show how two alternative epigenetic marks regulate NCAM alternative splicing and E18 levels in different cellular contexts.

Transcriptional elongation and alternative splicing.

Alternative splicing has emerged as a key contributor to proteome diversity, highlighting the importance of understanding its regulation. In recent years it became apparent that splicing is predominantly cotranscriptional, allowing for crosstalk between these two nuclear processes. We discuss some of the links between transcription and splicing, with special emphasis on the role played by transcription elongation in the regulation of alternative splicing events and in particular the kinetic model of alternative splicing regulation. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.

Coupling between transcription and alternative splicing.

The scenario of alternative splicing regulation is far more complex than the classical picture of a pre-mRNA being processed post-transcriptionally in more than one way. Introns are efficiently removed while transcripts are still being synthesized, supporting the idea of a co-transcriptional regulation of alternative splicing. Evidence of a functional coupling between splicing and transcription has recently emerged as it was observed that properties of one process may affect the outcome of the other. Co-transcriptionality is thought to improve splicing efficiency and kinetics by directing the nascent pre-mRNA into proper spliceosome assembly and favoring splicing factor recruitment. Two models have been proposed to explain the coupling of transcription and alternative splicing: in the recruitment model, promoters and pol II status affect the recruitment to the transcribing gene of splicing factors or bifunctional factors acting on both transcription and splicing; in the kinetic model, differences in the elongation rate of pol II would determine the timing in which splicing sites are presented, and thus the outcome of alternative splicing decisions. In the later model, chromatin structure has emerged as a key regulator. Although definitive evidence for transcriptionally coupled alternative splicing alterations in tumor development or cancer pathogenesis is still missing, many alternative splicing events altered in cancer might be subject to transcription-splicing coupling regulation.

The serine/arginine-rich protein SF2/ASF regulates protein sumoylation.

Protein modification by conjugation of small ubiquitin-related modifier (SUMO) is involved in diverse biological functions, such as transcription regulation, subcellular partitioning, stress response, DNA damage repair, and chromatin remodeling. Here, we show that the serine/arginine-rich protein SF2/ASF, a factor involved in splicing regulation and other RNA metabolism-related processes, is a regulator of the sumoylation pathway. The overexpression of this protein stimulates, but its knockdown inhibits SUMO conjugation. SF2/ASF interacts with Ubc9 and enhances sumoylation of specific substrates, sharing characteristics with already described SUMO E3 ligases. In addition, SF2/ASF interacts with the SUMO E3 ligase PIAS1 (protein inhibitor of activated STAT-1), regulating PIAS1-induced overall protein sumoylation. The RNA recognition motif 2 of SF2/ASF is necessary and sufficient for sumoylation enhancement. Moreover, SF2/ASF has a role in heat shock-induced sumoylation and promotes SUMO conjugation to RNA processing factors. These results add a component to the sumoylation pathway and a previously unexplored role for the multifunctional SR protein SF2/ASF.

Mechanisms of robustness in gene regulatory networks involved in neural development.

The functions of living organisms are affected by different kinds of perturbation, both internal and external, which in many cases have functional effects and phenotypic impact. The effects of these perturbations become particularly relevant for multicellular organisms with complex body patterns and cell type heterogeneity, where transcriptional programs controlled by gene regulatory networks determine, for example, the cell fate during embryonic development. Therefore, an essential aspect of development in these organisms is the ability to maintain the functionality of their genetic developmental programs even in the presence of genetic variation, changing environmental conditions and biochemical noise, a property commonly termed robustness. We discuss the implication of different molecular mechanisms of robustness involved in neurodevelopment, which is characterized by the interplay of many developmental programs at a molecular, cellular and systemic level. We specifically focus on processes affecting the function of gene regulatory networks, encompassing transcriptional regulatory elements and post-transcriptional processes such as miRNA-based regulation, but also higher order regulatory organization, such as gene network topology. We also present cases where impairment of robustness mechanisms can be associated with neurodevelopmental disorders, as well as reasons why understanding these mechanisms should represent an important part of the study of gene regulatory networks driving neural development.

Neuronal cell depolarization induces intragenic chromatin modifications affecting NCAM alternative splicing.

In search for physiological pathways affecting alternative splicing through its kinetic coupling with transcription, we found that membrane depolarization of neuronal cells triggers the skipping of exon 18 from the neural cell adhesion molecule (NCAM) mRNA, independently of the calcium/calmodulin protein kinase IV pathway. We show that this exon responds to RNA polymerase II elongation, because its inclusion is increased by a slow polymerase II mutant. Depolarization affects the chromatin template in a specific way, by causing H3K9 hyper-acetylation restricted to an internal region of the NCAM gene surrounding the alternative exon. This intragenic histone hyper-acetylation is not paralleled by acetylation at the promoter, is associated with chromatin relaxation, and is linked to H3K36 tri-methylation. The effects on acetylation and splicing fully revert when the depolarizing conditions are withdrawn and can be both duplicated and potentiated by the histone deacetylase inhibitor trichostatin A. Our results are consistent with a mechanism involving the kinetic coupling of splicing and transcription in response to depolarization through intragenic epigenetic changes on a gene that is relevant for the differentiation and function of neuronal cells.

Non-coding RNA Expression, Function, and Variation during Drosophila Embryogenesis.

Long non-coding RNAs (lncRNAs) can often function in the regulation of gene expression during development; however, their generality as essential regulators in developmental processes and organismal phenotypes remains unclear. Here, we performed a tailored investigation of lncRNA expression and function during Drosophila embryogenesis, interrogating multiple stages, tissue specificity, nuclear localization, and genetic backgrounds. Our results almost double the number of annotated lncRNAs expressed at these embryonic stages. lncRNA levels are generally positively correlated with those of their neighboring genes, with little evidence of transcriptional interference. Using fluorescent in situ hybridization, we report the spatiotemporal expression of 15 new lncRNAs, revealing very dynamic tissue-specific patterns. Despite this, deletion of selected lncRNA genes had no obvious developmental defects or effects on viability under standard and stressed conditions. However, two lncRNA deletions resulted in modest expression changes of a small number of genes, suggesting that they fine-tune expression of non-essential genes. Several lncRNAs have strain-specific expression, indicating that they are not fixed within the population. This intra-species variation across genetic backgrounds may thereby be a useful tool to distinguish rapidly evolving lncRNAs with as yet non-essential roles.

Promoter shape varies across populations and affects promoter evolution and expression noise.

Animal promoters initiate transcription either at precise positions (narrow promoters) or dispersed regions (broad promoters), a distinction referred to as promoter shape. Although highly conserved, the functional properties of promoters with different shapes and the genetic basis of their evolution remain unclear. Here we used natural genetic variation across a panel of 81 Drosophila lines to measure changes in transcriptional start site (TSS) usage, identifying thousands of genetic variants affecting transcript levels (strength) or the distribution of TSSs within a promoter (shape). Our results identify promoter shape as a molecular trait that can evolve independently of promoter strength. Broad promoters typically harbor shape-associated variants, with signatures of adaptive selection. Single-cell measurements demonstrate that variants modulating promoter shape often increase expression noise, whereas heteroallelic interactions with other promoter variants alleviate these effects. These results uncover new functional properties of natural promoters and suggest the minimization of expression noise as an important factor in promoter evolution.

Alternative Splicing of G9a Regulates Neuronal Differentiation.

Chromatin modifications are critical for the establishment and maintenance of differentiation programs. G9a, the enzyme responsible for histone H3 lysine 9 dimethylation in mammalian euchromatin, exists as two isoforms with differential inclusion of exon 10 (E10) through alternative splicing. We find that the G9a methyltransferase is required for differentiation of the mouse neuronal cell line N2a and that E10 inclusion increases during neuronal differentiation of cultured cells, as well as in the developing mouse brain. Although E10 inclusion greatly stimulates overall H3K9me2 levels, it does not affect G9a catalytic activity. Instead, E10 increases G9a nuclear localization. We show that the G9a E10(+) isoform is necessary for neuron differentiation and regulates the alternative splicing pattern of its own pre-mRNA, enhancing E10 inclusion. Overall, our findings indicate that by regulating its own alternative splicing, G9a promotes neuron differentiation and creates a positive feedback loop that reinforces cellular commitment to differentiation.

Connections between chromatin signatures and splicing.

Splicing and alternative splicing are involved in the expression of most human genes, playing key roles in differentiation, cell cycle progression, and development. Misregulation of splicing is frequently associated to disease, which imposes a better understanding of the mechanisms underlying splicing regulation. Accumulated evidence suggests that multiple trans-acting factors and cis-regulatory elements act together to determine tissue-specific splicing patterns. Besides, as splicing is often cotranscriptional, a complex picture emerges in which splicing regulation not only depends on the balance of splicing factor binding to their pre-mRNA target sites but also on transcription-associated features such as protein recruitment to the transcribing machinery and elongation kinetics. Adding more complexity to the splicing regulation network, recent evidence shows that chromatin structure is another layer of regulation that may act through various mechanisms. These span from regulation of RNA polymerase II elongation, which ultimately determines splicing decisions, to splicing factor recruitment by specific histone marks. Chromatin may not only be involved in alternative splicing regulation but in constitutive exon recognition as well. Moreover, splicing was found to be necessary for the proper 'writing' of particular chromatin signatures, giving further mechanistic support to functional interconnections between splicing, transcription and chromatin structure. These links between chromatin configuration and splicing raise the intriguing possibility of the existence of a memory for splicing patterns to be inherited through epigenetic modifications.

Perturbation of chromatin structure globally affects localization and recruitment of splicing factors.

Chromatin structure is an important factor in the functional coupling between transcription and mRNA processing, not only by regulating alternative splicing events, but also by contributing to exon recognition during constitutive splicing. We observed that depolarization of neuroblastoma cell membrane potential, which triggers general histone acetylation and regulates alternative splicing, causes a concentration of SR proteins in nuclear speckles. This prompted us to analyze the effect of chromatin structure on splicing factor distribution and dynamics. Here, we show that induction of histone hyper-acetylation results in the accumulation in speckles of multiple splicing factors in different cell types. In addition, a similar effect is observed after depletion of the heterochromatic protein HP1α, associated with repressive chromatin. We used advanced imaging approaches to analyze in detail both the structural organization of the speckle compartment and nuclear distribution of splicing factors, as well as studying direct interactions between splicing factors and their association with chromatin in vivo. The results support a model where perturbation of normal chromatin structure decreases the recruitment efficiency of splicing factors to nascent RNAs, thus causing their accumulation in speckles, which buffer the amount of free molecules in the nucleoplasm. To test this, we analyzed the recruitment of the general splicing factor U2AF65 to nascent RNAs by iCLIP technique, as a way to monitor early spliceosome assembly. We demonstrate that indeed histone hyper-acetylation decreases recruitment of U2AF65 to bulk 3' splice sites, coincident with the change in its localization. In addition, prior to the maximum accumulation in speckles, ∼20% of genes already show a tendency to decreased binding, while U2AF65 seems to increase its binding to the speckle-located ncRNA MALAT1. All together, the combined imaging and biochemical approaches support a model where chromatin structure is essential for efficient co-transcriptional recruitment of general and regulatory splicing factors to pre-mRNA.

RNA Polymerase II Elongation at the Crossroads of Transcription and Alternative Splicing.

The elongation phase of transcription lies at the core of several simultaneous and coupled events leading to alternative splicing regulation. Although underestimated in the past, it is at this phase of the transcription cycle where complexes affecting the transcription machinery itself, chromatin structure, posttranscriptional gene regulation and pre-mRNA processing converge to regulate each other or simply to consolidate higher-order complexes and functions. This paper focuses on the multiple processes that take place during transcription elongation which ultimately regulate the outcome of alternative splicing decisions.

Intragenic chromatin modifications: a new layer in alternative splicing regulation.

The multiple steps that contribute to gene expression in the metazoan nucleus are highly integrated and regulated. Most of pre-mRNA processing is believed to occur co-transcriptionally and choices regarding alternative processing reactions are influenced by transcription. Several articles published in the last year unveiled a connection between chromatin structure and the splicing process, strengthening a view in which the dynamic of intragenic chromatin modifications has an important role regulating alternative splicing (AS) choices. We have recently shown that both neuronal cell depolarization and the use of double stranded small RNAs targeting intragenic regions can modulate AS through chromatin remodeling, taking advantage of the kinetic coupling between splicing and transcription. Here, we discuss the many ways in which intragenic chromatin can participate in alternative splicing regulation.