Ctcf haploinsufficiency mediates intron retention in a tissue-specific manner.

CTCF is a master regulator of gene transcription and chromatin organisation with occupancy at thousands of DNA target sites genome-wide. While CTCF is essential for cell survival, CTCF haploinsufficiency is associated with tumour development and hypermethylation. Increasing evidence demonstrates CTCF as a key player in several mechanisms regulating alternative splicing (AS), however, the genome-wide impact of Ctcf dosage on AS has not been investigated. We examined the effect of Ctcf haploinsufficiency on gene expression and AS in five tissues from Ctcf hemizygous (Ctcf+/-) mice. Reduced Ctcf levels caused distinct tissue-specific differences in gene expression and AS in all tissues. An increase in intron retention (IR) was observed in Ctcf+/- liver and kidney. In liver, this specifically impacted genes associated with cytoskeletal organisation, splicing and metabolism. Strikingly, most differentially retained introns were short, with a high GC content and enriched in Ctcf binding sites in their proximal upstream genomic region. This study provides new insights into the effects of CTCF haploinsufficiency on organ transcriptomes and the role of CTCF in AS regulation.

Challenges in defining the role of intron retention in normal biology and disease.

RNA sequencing has revealed a striking diversity in transcriptomic complexity, to which alternative splicing is a major contributor. Intron retention (IR) is a conserved form of alternative splicing that was originally overlooked in normal mammalian physiology and development, due mostly to difficulties in its detection. IR has recently been revealed as an independent mechanism of controlling and enhancing the complexity of gene expression. IR facilitates rapid responses to biological stimuli, is involved in disease pathogenesis, and can generate novel protein isoforms. Many challenges, however, remain in detecting and quantifying retained introns and in determining their effects on cellular phenotype. In this review, we provide an overview of these challenges, and highlight approaches that can be used to address them.

Identification of miR-29b targets using 3-cyanovinylcarbazole containing mimics.

MicroRNAs (miRNAs) are highly conserved ∼22 nt small noncoding RNAs that bind partially complementary sequences in target transcripts. MicroRNAs regulate both translation and transcript stability, and play important roles in development, cellular homeostasis, and disease. There are limited approaches available to agnostically identify microRNA targets transcriptome-wide, and methods using miRNA mimics, which in principle identify direct miRNA:transcript pairs, have low sensitivity and specificity. Here, we describe a novel method to identify microRNA targets using miR-29b mimics containing 3-cyanovinylcarbazole (CNVK), a photolabile nucleoside analog. We demonstrate that biotin-tagged, CNVK-containing miR-29b (CNVK-miR-29b) mimics are nontoxic in cell culture, associate with endogenous mammalian Argonaute2, are sensitive for known targets and recapitulate endogenous transcript destabilization. Partnering CNVK-miR-29b with ultra-low-input RNA sequencing, we recover ∼40% of known miR-29b targets and find conservation of the focal adhesion and apoptotic target pathways in mouse and human. We also identify hundreds of novel targets, including NRAS, HOXA10, and KLF11, with a validation rate of 71% for a subset of 73 novel target transcripts interrogated using a high-throughput luciferase assay. Consistent with previous reports, we show that both endogenous miR-29b and CNVK-miR-29b are trafficked to the nucleus, but find no evidence of nuclear-specific miR-29b transcript binding. This may indicate that miR-29b nuclear sequestration is a regulatory mechanism in itself. We suggest that CNVK-containing small RNA mimics may find applicability in other experimental models.

Patterns and correlates of consumer protection tool use by Australian online gambling customers.

OBJECTIVE: Consumer Protection Tools (CPTs; e.g., deposit limits, timeouts) are provided by gambling sites to assist customers to gamble without harms. We aimed to understand how CPTs are used, and by which customers, which is essential to determine their effectiveness. METHOD: We examined the account data of 39,853 customers (median age = 33 years; 84% male) across six Australian wagering sites over 1 year (2018/07/01-2019/06/30). RESULTS: Most (83%) customers did not use any CPTs, with low rates of use for deposit limits (15.8%), timeouts (0.55%-1.57%), and self-exclusion tools (0.16%-0.57%) observed. Requiring customers to set a deposit limit or opt-out of setting one led to substantial increases in limit setting. Many customers who used limits later changed them, typically by increasing or removing them. Non-CPT users and deposit limit users were similar in their demographic and gambling characteristics, while comparatively, timeout and/or self-exclusion users were younger and displayed more risky gambling behaviors (e.g., higher net loss and betting frequency). CONCLUSIONS: Our findings suggest that voluntary deposit limits have inherent limitations in addressing harmful behaviors if consumers can easily increase or remove limits. The study suggests that greater efforts are needed to encourage CPT use among a broad customer base, including default limits requiring opt-out, greater restrictions on increasing or remove limits, and more persuasive communication of the benefits of timeouts. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Disease-specific oligodendrocyte lineage cells arise in multiple sclerosis.

Multiple sclerosis (MS) is characterized by an immune system attack targeting myelin, which is produced by oligodendrocytes (OLs). We performed single-cell transcriptomic analysis of OL lineage cells from the spinal cord of mice induced with experimental autoimmune encephalomyelitis (EAE), which mimics several aspects of MS. We found unique OLs and OL precursor cells (OPCs) in EAE and uncovered several genes specifically alternatively spliced in these cells. Surprisingly, EAE-specific OL lineage populations expressed genes involved in antigen processing and presentation via major histocompatibility complex class I and II (MHC-I and -II), and in immunoprotection, suggesting alternative functions of these cells in a disease context. Importantly, we found that disease-specific oligodendroglia are also present in human MS brains and that a substantial number of genes known to be susceptibility genes for MS, so far mainly associated with immune cells, are expressed in the OL lineage cells. Finally, we demonstrate that OPCs can phagocytose and that MHC-II-expressing OPCs can activate memory and effector CD4-positive T cells. Our results suggest that OLs and OPCs are not passive targets but instead active immunomodulators in MS. The disease-specific OL lineage cells, for which we identify several biomarkers, may represent novel direct targets for immunomodulatory therapeutic approaches in MS.

Transcriptional Convergence of Oligodendrocyte Lineage Progenitors during Development.

Pdgfra+ oligodendrocyte precursor cells (OPCs) arise in distinct specification waves during embryogenesis in the central nervous system (CNS). It is unclear whether there is a correlation between these waves and different oligodendrocyte (OL) states at adult stages. Here, we present bulk and single-cell transcriptomics resources providing insights on how transitions between these states occur. We found that post-natal OPCs from brain and spinal cord present similar transcriptional signatures. Moreover, post-natal OPC progeny of E13.5 Pdgfra+ cells present electrophysiological and transcriptional profiles similar to OPCs derived from subsequent specification waves, indicating that Pdgfra+ pre-OPCs rewire their transcriptional network during development. Single-cell RNA-seq and lineage tracing indicates that a subset of E13.5 Pdgfra+ cells originates cells of the pericyte lineage. Thus, our results indicate that embryonic Pdgfra+ cells in the CNS give rise to distinct post-natal cell lineages, including OPCs with convergent transcriptional profiles in different CNS regions.

Human iPSC-Derived Cerebellar Neurons from a Patient with Ataxia-Telangiectasia Reveal Disrupted Gene Regulatory Networks.

Ataxia-telangiectasia (A-T) is a rare genetic disorder caused by loss of function of the ataxia-telangiectasia-mutated kinase and is characterized by a predisposition to cancer, pulmonary disease, immune deficiency and progressive degeneration of the cerebellum. As animal models do not faithfully recapitulate the neurological aspects, it remains unclear whether cerebellar degeneration is a neurodevelopmental or neurodegenerative phenotype. To address the necessity for a human model, we first assessed a previously published protocol for the ability to generate cerebellar neuronal cells, finding it gave rise to a population of precursors highly enriched for markers of the early hindbrain such as EN1 and GBX2, and later more mature cerebellar markers including PTF1α, MATH1, HOXB4, ZIC3, PAX6, and TUJ1. RNA sequencing was used to classify differentiated cerebellar neurons generated from integration-free A-T and control induced pluripotent stem cells. Comparison of RNA sequencing data with datasets from the Allen Brain Atlas reveals in vitro-derived cerebellar neurons are transcriptionally similar to discrete regions of the human cerebellum, and most closely resemble the cerebellum at 22 weeks post-conception. We show that patient-derived cerebellar neurons exhibit disrupted gene regulatory networks associated with synaptic vesicle dynamics and oxidative stress, offering the first molecular insights into early cerebellar pathogenesis of ataxia-telangiectasia.

Amiodarone induces cell wall channel formation in yeast Hansenula polymorpha.

The yeast cell wall is constantly remodeled to enable cell growth and division. In this study, we describe a novel type of cell wall modification. We report that the drug amiodarone induces rapid channel formation within the cell wall of the yeast Hansenula polymorpha. Light microscopy shows that shortly after adding amiodarone, spherical structures, which can be stained with DNA binding dyes, form on the cell surface. Electron microphotographs show that amiodarone induces the formation of channels 50-80 nm in diameter in the cell wall that appear to be filled with intracellular material. Using fluorescent microscopy, we demonstrate MitoTracker-positive DNA-containing structures visibly extruded from the cells through these channels. We speculate that the observed channel formation acts to enable the secretion of mitochondrial material from the cell under stressful conditions, thus enabling adaptive changes to the extracellular environment.

RevSex duplication-induced and sex-related differences in the SOX9 regulatory region chromatin landscape in human fibroblasts.

It was recently shown that duplications of the RevSex element, located 0.5 Mb upstream of SOX9, cause XX-disorder of sex development (DSD), and that deletions cause XY-DSD. To explore how a 148 kb RevSex duplication could have turned on gonadal SOX9 expression in the absence of SRY in an XX-male, we examined the chromatin landscape in primary skin fibroblast cultures from the index, his RevSex duplication-carrier father and six controls. The ENCODE project supports the notion that chromatin state maps show overlap between different cell types, i.e., that our study of fibroblasts could be of biological relevance. We examined the SOX9 regulatory region by high-resolution ChIP-on-chip experiments (a kind of "chromatin-CGH") and DNA methylation investigations. The RevSex duplication was associated with chromatin changes predicting better accessibility of the SRY-responsive TESCO enhancer region 14-15 kb upstream of SOX9. Four kb downstream of the TESCO evolutionary conserved region, a peak of the enhancer/promoter-associated H3K4me3 mark was found together with a major dip of the repressive H3K9me3 chromatin mark. Similar differences were also found when three control males were compared with three control females. A marked male/female difference was a more open chromatin signature in males starting ~400 kb upstream of SOX9 and increasing toward the SOX9 promoter. In the RevSex duplication-carrier father, two positions of DNA hypomethylation were also found, one corresponding to the H3K4me3 peak mentioned above. Our results suggest that the RevSex duplication could operate by inducing long-range epigenetic changes. Furthermore, the differences in chromatin state maps between males and females suggest that the Y chromosome or X chromosome dosage may affect chromatin conformation, i.e., that sex-dependent gene regulation may take place by chromatin modification.