Isoforms of the TAL1 transcription factor have different roles in hematopoiesis and cell growth.

T-cell acute lymphoblastic leukemia (T-ALL) protein 1 (TAL1) is a central transcription factor in hematopoiesis. The timing and level of TAL1 expression orchestrate the differentiation to specialized blood cells and its overexpression is a common cause of T-ALL. Here, we studied the 2 protein isoforms of TAL1, short and long, which are generated by the use of alternative promoters as well as by alternative splicing. We analyzed the expression of each isoform by deleting an enhancer or insulator, or by opening chromatin at the enhancer location. Our results show that each enhancer promotes expression from a specific TAL1 promoter. Expression from a specific promoter gives rise to a unique 5' UTR with differential regulation of translation. Moreover, our study suggests that the enhancers regulate TAL1 exon 3 alternative splicing by inducing changes in the chromatin at the splice site, which we demonstrate is mediated by KMT2B. Furthermore, our results indicate that TAL1-short binds more strongly to TAL1 E-protein partners and functions as a stronger transcription factor than TAL1-long. Specifically TAL1-short has a unique transcription signature promoting apoptosis. Finally, when we expressed both isoforms in mice bone marrow, we found that while overexpression of both isoforms prevents lymphoid differentiation, expression of TAL1-short alone leads to hematopoietic stem cell exhaustion. Furthermore, we found that TAL1-short promoted erythropoiesis and reduced cell survival in the CML cell line K562. While TAL1 and its partners are considered promising therapeutic targets in the treatment of T-ALL, our results show that TAL1-short could act as a tumor suppressor and suggest that altering TAL1 isoform's ratio could be a preferred therapeutic approach.

Splicing to Keep Cycling: The Importance of Pre-mRNA Splicing during the Cell Cycle.

Pre-mRNA splicing is a fundamental process in mammalian gene expression, and alternative splicing plays an extensive role in generating protein diversity. Because the majority of genes undergo pre-mRNA splicing, most cellular processes depend on proper spliceosome function. We focus on the cell cycle and describe its dependence on pre-mRNA splicing and accurate alternative splicing. We outline the key cell-cycle factors and their known alternative splicing isoforms. We discuss different levels of pre-mRNA splicing regulation such as post-translational modifications and changes in the expression of splicing factors. We describe the effect of chromatin dynamics on pre-mRNA splicing during the cell cycle. In addition, we focus on spliceosome component SF3B1, which is mutated in many types of cancer, and describe the link between SF3B1 and its inhibitors and the cell cycle.

A journey in the world of craniofacial development: From 1968 to the future.

This article is based on my talk at the meeting "3rd Advances in Craniosynostosis: Basic Science to Clinical Practice", held at University College, London, on 25 August 2023. It describes my contribution, together with that of my research team and external collaborators, to the field of craniofacial development. This began with my PhD research on the effects of excess vitamin A in rat embryos, which led to a study of normal as well as abnormal formation of the cranial neural tube. Many techniques for analysing morphogenetic change became available to me over the years: whole embryo culture, scanning and transmission electron microscopy, cell division analysis, immunohistochemistry and biochemical analysis of the extracellular matrix. The molecular revolution of the 1980s, and key collaborations with international research teams, enabled functional interpretation of some of the earlier morphological observations and required a change of experimental species to the mouse. Interactions between the molecular and experimental analysis of craniofacial morphogenesis in my laboratory with specialists in molecular genetics and clinicians brought my research journey near to my original aim: to contribute to a better understanding of the causes of human congenital anomalies.

KDM3A regulates alternative splicing of cell-cycle genes following DNA damage.

Changes in the cellular environment result in chromatin structure alteration, which in turn regulates gene expression. To learn about the effect of the cellular environment on the transcriptome, we studied the H3K9 demethylase KDM3A. Using RNA-seq, we found that KDM3A regulates the transcription and alternative splicing of genes associated with cell cycle and DNA damage. We showed that KDM3A undergoes phosphorylation by PKA at serine 265 following DNA damage, and that the phosphorylation is important for proper cell-cycle regulation. We demonstrated that SAT1 alternative splicing, regulated by KDM3A, plays a role in cell-cycle regulation. Furthermore we found that KDM3A's demethylase activity is not needed for SAT1 alternative splicing regulation. In addition, we identified KDM3A's protein partner ARID1A, the SWI/SNF subunit, and SRSF3 as regulators of SAT1 alternative splicing and showed that KDM3A is essential for SRSF3 binding to SAT1 pre-mRNA. These results suggest that KDM3A serves as a sensor of the environment and an adaptor for splicing factor binding. Our work reveals chromatin sensing of the environment in the regulation of alternative splicing.

Regulation of alternative splicing by p300-mediated acetylation of splicing factors.

Splicing of precursor mRNA (pre-mRNA) is an important regulatory step in gene expression. Recent evidence points to a regulatory role of chromatin-related proteins in alternative splicing regulation. Using an unbiased approach, we have identified the acetyltransferase p300 as a key chromatin-related regulator of alternative splicing. p300 promotes genome-wide exon inclusion in both a transcription-dependent and -independent manner. Using CD44 as a paradigm, we found that p300 regulates alternative splicing by modulating the binding of splicing factors to pre-mRNA. Using a tethering strategy, we found that binding of p300 to the CD44 promoter region promotes CD44v exon inclusion independently of RNAPII transcriptional elongation rate. Promoter-bound p300 regulates alternative splicing by acetylating splicing factors, leading to exclusion of hnRNP M from CD44 pre-mRNA and activation of Sam68. p300-mediated CD44 alternative splicing reduces cell motility and promotes epithelial features. Our findings reveal a chromatin-related mechanism of alternative splicing regulation and demonstrate its impact on cellular function.

Pyruvate dehydrogenase has a major role in mast cell function, and its activity is regulated by mitochondrial microphthalmia transcription factor.

BACKGROUND: We have recently observed that oxidative phosphorylation-mediated ATP production is essential for mast cell function. Pyruvate dehydrogenase (PDH) is the main regulator of the Krebs cycle and is located upstream of the electron transport chain. However, the role of PDH in mast cell function has not been described. Microphthalmia transcription factor (MITF) regulates the development, number, and function of mast cells. Localization of MITF to the mitochondria and its interaction with mitochondrial proteins has not been explored. OBJECTIVE: We sought to explore the role played by PDH in mast cell exocytosis and to determine whether MITF is localized in the mitochondria and involved in regulation of PDH activity. METHODS: Experiments were performed in vitro by using human and mouse mast cells, as well as rat basophil leukemia cells, and in vivo in mice. The effect of PDH inhibition on mast cell function was examined. PDH interaction with MITF was measured before and after immunologic activation. Furthermore, mitochondrial localization of MITF and its effect on PDH activity were determined. RESULTS: PDH is essential for immunologically mediated degranulation of mast cells. After activation, PDH is serine dephosphorylated. In addition, for the first time, we show that MITF is partially located in the mitochondria and interacts with PDH. This interaction is dependent on the phosphorylation state of PDH. Furthermore, mitochondrial MITF regulates PDH activity. CONCLUSION: The association of mitochondrial MITF with PDH emerges as an important regulator of mast cell function. Our findings indicate that PDH could arise as a new target for the manipulation of allergic diseases.

LysRS serves as a key signaling molecule in the immune response by regulating gene expression.

Lysyl-tRNA synthetase (LysRS) was found to produce diadenosine tetraphosphate (Ap(4)A) in vitro more than two decades ago. Here, we used LysRS silencing in mast cells in combination with transfected normal and mutated LysRS to demonstrate in vivo the critical role played by LysRS in the production of Ap(4)A in response to immunological challenge. Upon such challenge, LysRS was phosphorylated on serine 207 in a MAPK-dependent manner, released from the multisynthetase complex, and translocated into the nucleus. We previously demonstrated that LysRS forms a complex with MITF and its repressor Hint-1, which is released from the complex by its binding to Ap(4)A, enabling MITF to transcribe its target genes. Here, silencing LysRS led to reduced Ap(4)A production in immunologically activated cells, which resulted in a lower level of MITF inducible genes. Our data demonstrate that specific LysRS serine 207 phosphorylation regulates Ap(4)A production in immunologically stimulated mast cells, thus implying that LysRS is a key mediator in gene regulation.

Erbin is a negative modulator of cardiac hypertrophy.

ErbB2 interacting protein (Erbin) is a widely expressed protein and participates in inhibition of several intracellular signaling pathways. Its mRNA has been found to be present in relatively high levels in the heart. However, its physiological role in the heart has not been explored. In the present work, we elucidated the role of Erbin in cardiac hypertrophy. Cardiac hypertrophy was induced in mice either by isoproterenol administration or by aortic constriction. The level of Erbin was significantly decreased in both models. Erbin(-/-) mice rapidly develop decompensated cardiac hypertrophy, and following severe pressure overload all Erbin(-/-) mice died from heart failure. Down-regulation of Erbin expression was also observed in biopsies derived from human failing hearts. It is known that Erbin inhibits Ras-mediated activation of the extracellular signal-regulated kinase (ERK) by binding to Soc-2 suppressor of clear homolog (Shoc2). Our data clearly show that ERK phosphorylation is enhanced in the heart tissues of Erbin(-/-) mice. Furthermore, we clearly demonstrate here that Erbin associates with Shoc2 in both whole hearts and in cardiomyocytes, and that in the absence of Erbin, Raf is phosphorylated and binds Shoc2, resulting in ERK phosphorylation. In conclusion, Erbin is an inhibitor of pathological cardiac hypertrophy, and this inhibition is mediated, at least in part, by modulating ERK signaling.

The Journal of Anatomy: origin and evolution.

The Journal of Anatomy was launched 150 years ago as the Journal of Anatomy and Physiology, in an age when anatomy and physiology were not regarded as separate disciplines. European science in general was advancing rapidly at the time (it was 7 years after publication of Darwin's Origin of Species), and the recent demise of the Natural History Review meant that there was no English language publication covering these subjects. The founding editors were George Murray Humphry of Cambridge and William Turner of Edinburgh, together with Alfred Newton of Cambridge and Edward Perceval Wright of Dublin (the last two served only for a year). The pivotal event leading to the Journal's foundation was the 1866 meeting of the British Association, at which Humphry delivered the 'Address in Physiology' (printed in the first issue). Turner, who was also present at the 1866 British Association meeting, remained as a member of the editorial team for 50 years and was a major contributor of Journal articles. The title was changed to Journal of Anatomy in October 1916, when it was taken under the wing, in terms of both management and ownership, by the Anatomical Society. This article reviews the early years of the Journal's publication in more detail than later years because of the historical interest of this less familiar material. The subject matter, which has remained surprisingly consistent over the years, is illustrated by examples from some notable contributions. The evolution of illustration techniques is surveyed from 1866 to the present day; the final section provides brief summaries of all of the chief editors.

Mitochondrial STAT3 plays a major role in IgE-antigen-mediated mast cell exocytosis.

BACKGROUND: The involvement of mitochondrial oxidative phosphorylation (OXPHOS) in mast cell exocytosis was recently suggested by the finding that mitochondria translocate to exocytosis sites upon mast cell activation. In parallel, mitochondrial signal transducer and activator of transcription 3 (STAT3) was found to be involved in ATP production. However, the regulation of mitochondrial STAT3 function and its connection to mast cell exocytosis is unknown. OBJECTIVE: We sought to explore the role played by mitochondrial STAT3 in mast cell exocytosis. METHODS: Experiments were performed in vitro with human and mouse mast cells and rat basophilic leukemia (RBL) cells and in vivo in mice. OXPHOS activity was measured after immunologic activation. The expression of STAT3, extracellular signal-regulated kinase 1/2, and protein inhibitor of activated STAT3 in the mitochondria during mast cell activation was determined, as was the effect of STAT3 inhibition on OXPHOS activity and mast cell function. RESULTS: Here we show that mitochondrial STAT3 is essential for immunologically mediated degranulation of human and mouse mast cells and RBL cells. Additionally, in IgE-antigen-activated RBL cells, mitochondrial STAT3 was phosphorylated on serine 727 in an extracellular signal-regulated kinase 1/2-dependent manner, which was followed by induction of OXPHOS activity. Furthermore, the endogenous inhibitor of STAT3, protein inhibitor of activated STAT3, was found to inhibit OXPHOS activity in the mitochondria, resulting in inhibition of mast cell degranulation. Moreover, mice injected with Stattic, a STAT3 inhibitor, had a significant decrease in histamine secretion. CONCLUSION: These results provide the first evidence of a regulatory role for mitochondrial STAT3 in mast cell functions, and therefore mitochondrial STAT3 could serve as a new target for the manipulation of allergic diseases.

The spectrum of pre-mRNA splicing in autism.

Disruptions in spatiotemporal gene expression can result in atypical brain function. Specifically, autism spectrum disorder (ASD) is characterized by abnormalities in pre-mRNA splicing. Abnormal splicing patterns have been identified in the brains of individuals with ASD, and mutations in splicing factors have been found to contribute to neurodevelopmental delays associated with ASD. Here we review studies that shed light on the importance of splicing observed in ASD and that explored the intricate relationship between splicing factors and ASD, revealing how disruptions in pre-mRNA splicing may underlie ASD pathogenesis. We provide an overview of the research regarding all splicing factors associated with ASD and place a special emphasis on five specific splicing factors-HNRNPH2, NOVA2, WBP4, SRRM2, and RBFOX1-known to impact the splicing of ASD-related genes. In the discussion of the molecular mechanisms influenced by these splicing factors, we lay the groundwork for a deeper understanding of ASD's complex etiology. Finally, we discuss the potential benefit of unraveling the connection between splicing and ASD for the development of more precise diagnostic tools and targeted therapeutic interventions. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Evolution and Genomics > Computational Analyses of RNA RNA-Based Catalysis > RNA Catalysis in Splicing and Translation.

DNMT3B splicing dysregulation mediated by SMCHD1 loss contributes to DUX4 overexpression and FSHD pathogenesis.

Structural maintenance of chromosomes flexible hinge domain-containing 1 (SMCHD1) is a noncanonical SMC protein and an epigenetic regulator. Mutations in SMCHD1 cause facioscapulohumeral muscular dystrophy (FSHD), by overexpressing DUX4 in muscle cells. Here, we demonstrate that SMCHD1 is a key regulator of alternative splicing in various cell types. We show how SMCHD1 loss causes splicing alterations of DNMT3B, which can lead to hypomethylation and DUX4 overexpression. Analyzing RNA sequencing data from muscle biopsies of patients with FSHD and Smchd1 knocked out cells, we found mis-splicing of hundreds of genes upon SMCHD1 loss. We conducted a high-throughput screen of splicing factors, revealing the involvement of the splicing factor RBM5 in the mis-splicing of DNMT3B. Subsequent RNA immunoprecipitation experiments confirmed that SMCHD1 is required for RBM5 recruitment. Last, we show that mis-splicing of DNMT3B leads to hypomethylation of the D4Z4 region and to DUX4 overexpression. These results suggest that DNMT3B mis-splicing due to SMCHD1 loss plays a major role in FSHD pathogenesis.

The enigma of the role of protein inhibitor of activated STAT3 (PIAS3) in the immune response.

Protein inhibitor of activated STAT3 (PIAS3), the main cellular inhibitor of signal transducers and activator of transcription 3 (STAT3), has been described as a modulator of DNA binding transcription factors. The exploration of the emerging roles of PIAS3 in immune regulation is a growing and fascinating field. Recent discoveries have shed new light on the key role of PIAS3 in the regulation of transcriptional activity, and on the molecular mechanism involved. These findings suggest that the known functions of this signalling molecule are merely the "tip of the iceberg". This article reviews the challenging questions regarding the link between PIAS3 and the intracellular signalling in immune cells. Some of the known functions of PIAS3 that potentially modulate key proteins in the immune system will also be discussed.

Transcription factor E3, a major regulator of mast cell-mediated allergic response.

BACKGROUND: Microphthalmia transcription factor, an MiT transcription family member closely related to transcription factor E3 (TFE3), is essential for mast cell development and survival. TFE3 was previously reported to play a role in the functions of B and T cells; however, its role in mast cells has not yet been explored. OBJECTIVE: We sought to explore the role played by TFE3 in mast cell function. METHODS: Mast cell numbers were evaluated by using toluidine blue staining. FACS analysis was used to determine percentages of Kit and FcεRI double-positive cells in the peritoneum of wild-type (WT) and TFE3 knockout (TFE3(-/-)) mice. Cytokine and inflammatory mediator secretion were measured in immunologically activated cultured mast cells derived from either knockout or WT mice. In vivo plasma histamine levels were measured after immunologic triggering of these mice. RESULTS: No significant differences in mast cell numbers between WT and TFE3(-/-) mice were observed in the peritoneum, lung, and skin. However, TFE3(-/-) mice showed a marked decrease in the number of Kit(+) and FcεRI(+) peritoneal and cultured mast cells. Surface expression levels of FcεRI in TFE3(-/-) peritoneal mast cells was significantly lower than in control cells. Cultured mast cells derived from TFE3(-/-) mice showed a marked decrease in degranulation and mediator secretion. In vivo experiments showed that the level of plasma histamine in TFE3(-/-) mice after an allergic trigger was substantially less than that seen in WT mice. CONCLUSION: TFE3 is a novel regulator of mast cell functions and as such could emerge as a new target for the manipulation of allergic diseases.

Nprl3 is required for normal development of the cardiovascular system.

C16orf35 is a conserved and widely expressed gene lying adjacent to the human α-globin cluster in all vertebrate species. In-depth sequence analysis shows that C16orf35 (now called NPRL3) is an orthologue of the yeast gene Npr3 (nitrogen permease regulator 3) and, furthermore, is a paralogue of its protein partner Npr2. The yeast Npr2/3 dimeric protein complex senses amino acid starvation and appropriately adjusts cell metabolism via the TOR pathway. Here we have analysed a mouse model in which expression of Nprl3 has been abolished using homologous recombination. The predominant effect on RNA expression appears to involve genes that regulate protein synthesis and cell cycle, consistent with perturbation of the mTOR pathway. Embryos homozygous for this mutation die towards the end of gestation with a range of cardiovascular defects, including outflow tract abnormalities and ventriculoseptal defects consistent with previous observations, showing that perturbation of the mTOR pathway may affect development of the myocardium. NPRL3 is a candidate gene for harbouring mutations in individuals with developmental abnormalities of the cardiovascular system.

A specific epitope of protein inhibitor of activated STAT3 is responsible for the induction of apoptosis in rat transformed mast cells.

Protein inhibitor of activated STAT3 (PIAS3) functions in vivo as a key molecule in suppressing the transcriptional activity of both microphthalmia transcription factor (MITF) and STAT3, two transcription factors that play a major role in the development, phenotypic expression, and survival of mast cells and melanocytes. In the present study we have investigated the role played by PIAS3 in the regulation of cell cycle in mast cells and melanocytes. We have characterized the biological role of a 23-aa domain derived from PIAS3 that induces apoptosis in these cells by inhibiting the transcriptional activity of both MITF and STAT3. This PIAS3 inhibitor peptide could serve as the beginning of an in depth study for the development of peptide inhibitors for MITF and STAT3.

VEGFA's distal enhancer regulates its alternative splicing in CML.

Enhancer demethylation in leukemia has been shown to lead to overexpression of genes which promote cancer characteristics. The vascular endothelial growth factor A (VEGFA) enhancer, located 157 Kb downstream of its promoter, is demethylated in chronic myeloid leukemia (CML). VEGFA has several alternative splicing isoforms with different roles in cancer progression. Since transcription and splicing are coupled, we wondered whether VEGFA enhancer activity can also regulate the gene's alternative splicing to contribute to the pathology of CML. Our results show that mutating the VEGFA +157 enhancer promotes exclusion of exons 6a and 7 and activating the enhancer by tethering a chromatin activator has the opposite effect. In line with these results, CML patients present with high expression of +157 eRNA and inclusion of VEGFA exons 6a and 7. In addition, our results show that the positive regulator of RNAPII transcription elongation, CCNT2, binds VEGFA's promoter and enhancer, and its silencing promotes exclusion of exons 6a and 7 as it slows down RNAPII elongation rate. Thus our results suggest that VEGFA's +157 enhancer regulates its alternative splicing by increasing RNAPII elongation rate via CCNT2. Our work demonstrates for the first time a connection between an endogenous enhancer and alternative splicing regulation of its target gene.

The evolution of human artistic creativity.

Creating visual art is one of the defining characteristics of the human species, but the paucity of archaeological evidence means that we have limited information on the origin and evolution of this aspect of human culture. The components of art include colour, pattern and the reproduction of visual likeness. The 2D and 3D art forms that were created by Upper Palaeolithic Europeans at least 30,000 years ago are conceptually equivalent to those created in recent centuries, indicating that human cognition and symbolling activity, as well as anatomy, were fully modern by that time. The origins of art are therefore much more ancient and lie within Africa, before worldwide human dispersal. The earliest known evidence of 'artistic behaviour' is of human body decoration, including skin colouring with ochre and the use of beads, although both may have had functional origins. Zig-zag and criss-cross patterns, nested curves and parallel lines are the earliest known patterns to have been created separately from the body; their similarity to entopic phenomena (involuntary products of the visual system) suggests a physiological origin. 3D art may have begun with human likeness recognition in natural objects, which were modified to enhance that likeness; some 2D art has also clearly been influenced by suggestive features of an uneven surface. The creation of images from the imagination, or 'the mind's eye', required a seminal evolutionary change in the neural structures underpinning perception; this change would have had a survival advantage in both tool-making and hunting. Analysis of early tool-making techniques suggests that creating 3D objects (sculptures and reliefs) involves their cognitive deconstruction into a series of surfaces, a principle that could have been applied to early sculpture. The cognitive ability to create art separate from the body must have originated in Africa but the practice may have begun at different times in genetically and culturally distinct groups both within Africa and during global dispersal, leading to the regional variety seen in both ancient and recent art. At all stages in the evolution of artistic creativity, stylistic change must have been due to rare, highly gifted individuals.

Microphthalmia transcription factor isoforms in mast cells and the heart.

The microphthalmia transcription factor (Mitf) is critical for the survival and differentiation of a variety of cell types. While on the transcript level it has been noted that melanocytes and cardiomyocytes express specific Mitf isoforms, mast cells express several isoforms, mainly Mitf-H and Mitf-MC, whose function has not been thoroughly investigated. We found that in mast cells the expression of the specific Mitf isoforms is dependent on physiological stimuli that cause a major shifting of promoter usage and internal splicing. For example, activation of the c-kit signaling pathway almost totally abolished one of the main splice isoforms. Since cardiomyocytes express only the Mitf-H isoform, they were an ideal system to determine this isoform's physiological role. We identified that the expression of myosin light-chain 1a (MLC-1a) is regulated by Mitf-H. Interestingly, the transactivation of MLC-1a by Mitf-H in cardiomyocytes is decreased by overexpression of the splice form with exon 6a. In conclusion, we found that there is physiological switching of Mitf isoforms and that the promoter context and the cell context have a combined influence on gene expression programs.