CTCF as a regulator of alternative splicing: new tricks for an old player

Three decades of research have established the CCCTC-binding factor (CTCF) as a ubiquitously expressed chromatin organizing factor and master regulator of gene expression. A new role for CTCF as a regulator of alternative splicing (AS) has now emerged. CTCF has been directly and indirectly linked to the modulation of AS at the individual transcript and at the transcriptome-wide level. The emerging role of CTCF-mediated regulation of AS involves diverse mechanisms; including transcriptional elongation, DNA methylation, chromatin architecture, histone modifications, and regulation of splicing factor expression and assembly. CTCF thereby appears to not only co-ordinate gene expression regulation but contributes to the modulation of transcriptomic complexity. In this review, we highlight previous discoveries regarding the role of CTCF in AS. In addition, we summarize detailed mechanisms by which CTCF mediates AS regulation. We propose opportunities for further research designed to examine the possible fate of CTCF-mediated alternatively spliced genes and associated biological consequences. CTCF has been widely acknowledged as the ‘master weaver of the genome’. Given its multiple connections, further characterization of CTCF’s emerging role in splicing regulation might extend its functional repertoire towards a ‘conductor of the splicing orchestra’

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

Recent Advances in Cancer Fusion Transcript Detection

Extensive investigation of gene fusions in cancer has led to the discovery of novel biomarkers and therapeutic targets. To date, most studies have neglected chromosomal rearrangement-independent fusion transcripts and complex fusion structures such as double or triple-hop fusions, and fusion-circRNAs. In this review, we untangle fusion-related terminology and propose a classification system involving both gene and transcript fusions. We highlight the importance of RNA-level fusions and how long-read sequencing approaches can improve detection and characterization. Moreover, we discuss novel bioinformatic tools to identify fusions in long-read sequencing data and strategies to experimentally validate and functionally characterize fusion transcripts

The changing paradigm of intron retention: regulation, ramifications and recipes

Intron retention (IR) is a form of alternative splicing that has long been neglected in mammalian systems although it has been studied for decades in non-mammalian species such as plants, fungi, insects and viruses. It was generally assumed that mis-splicing, leading to the retention of introns, would have no physiological consequence other than reducing gene expression by nonsense-mediated decay. Relatively recent landmark discoveries have highlighted the pivotal role that IR serves in normal and disease-related human biology. Significant technical hurdles have been overcome, thereby enabling the robust detection and quantification of IR. Still, relatively little is known about the cis- and trans-acting modulators controlling this phenomenon. The fate of an intron to be, or not to be, retained in the mature transcript is the direct result of the influence exerted by numerous intrinsic and extrinsic factors at multiple levels of regulation. These factors have altered current biological paradigms and provided unexpected insights into the transcriptional landscape. In this review, we discuss the regulators of IR and methods to identify them. Our focus is primarily on mammals, however, we broaden the scope to non-mammalian organisms in which IR has been shown to be biologically relevant

Sensitive Flow Cytometric Analysis Reveals a Novel Type of Parent-of-Origin Effect in the Mouse Genome

AbstractThe discovery of classic parental imprinting came, at least in part, from the analysis of transgene expression in mice [1]. It was noticed that some transgenes were only expressed following paternal transmission [2–4] and that others sometimes showed differential patterns of methylation depending on the parent of origin [5, 6]. Here, we present evidence of a novel and more subtle form of parental imprinting by taking advantage of the highly sensitive detection of murine transgene expression afforded by flow cytometry. We have produced nine lines of transgenic mice carrying a GFP reporter linked to the human α-globin promoter and enhancer elements, which direct expression to erythroid cells. A high proportion of transgenic lines, four of the nine, display significantly lower levels of expression following maternal transmission. Both the percentage of expressing cells and the mean fluorescence in expressing cells are between 10% and 30% lower following maternal transmission. These effects are reversible upon passage through the opposite germline. This finding raises the possibility that differences in the epigenetic state of the maternal and paternal chromosomes in adult somatic cells are more widespread than was previously thought

ZNF265—a novel spliceosomal protein able to induce alternative splicing

The formation of the active spliceosome, its recruitment to active areas of transcription, and its role in pre-mRNA splicing depends on the association of a number of multifunctional serine/arginine-rich (SR) proteins. ZNF265 is an arginine/serine-rich (RS) domain containing zinc finger protein with conserved pre-mRNA splicing protein motifs. Here we show that ZNF265 immunoprecipitates from splicing extracts in association with mRNA, and that it is able to alter splicing patterns of Tra2-β1 transcripts in a dose-dependent manner in HEK 293 cells. Yeast two-hybrid analysis and immunoprecipitation indicated interaction of ZNF265 with the essential splicing factor proteins U1-70K and U2AF35. Confocal microscopy demonstrated colocalization of ZNF265 with the motor neuron gene product SMN, the snRNP protein U1-70K, the SR protein SC35, and with the transcriptosomal components p300 and YY1. Transfection of HT-1080 cells with ZNF265–EGFP fusion constructs showed that nuclear localization of ZNF265 required the RS domain. Alignment with other RS domain–containing proteins revealed a high degree of SR dipeptide conservation. These data show that ZNF265 functions as a novel component of the mRNA processing machinery

Increased chromatin accessibility facilitates intron retention in specific cell differentiation states

Dynamic intron retention (IR) in vertebrate cells is of widespread biological importance. Aberrant IR is associated with numerous human diseases including several cancers. Despite consistent reports demonstrating that intrinsic sequence features can help introns evade splicing, conflicting findings about cell type or condition-specific IR regulation by trans-regulatory and epigenetic mechanisms demand an unbiased and systematic analysis of IR in a controlled experimental setting. We integrated matched mRNA sequencing (RNA-seq), whole-genome bisulfite sequencing (WGBS), nucleosome occupancy methylome sequencing (NOMe-Seq), and chromatin immunoprecipitation sequencing (ChIP-seq) data from primary human myeloid and lymphoid cells. Using these multi-omics data and machine learning we trained two complementary models to determine the role of epigenetic factors in the regulation of IR in cells of the innate immune system. We show that increased chromatin accessibility, as revealed by nucleosome-free regions, contributes substantially to the retention of introns in a cell-specific manner. We also confirm that intrinsic characteristics of introns are key for them to evade splicing. This study suggests an important role of chromatin architecture in IR regulation. With an increasing appreciation that pathogenic alterations are linked to RNA processing, our findings may provide useful insights for the development of novel therapeutic approaches that target aberrant splicing

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

lentiglobin gene therapy for transfusion dependent β thalassemia outcomes from the phase 1 2 northstar and phase 3 northstar 2 studies

Introduction Transfusion-dependent β-thalassemia (TDT) is a severe genetic disease characterized by anemia, iron overload and serious comorbidities for which gene therapy may be an effective treatment option. LentiGlobin gene therapy contains autologous CD34+ hematopoietic stem cells (HSCs) transduced ex vivo with the BB305 lentiviral vector (LVV) encoding β-globin with a T87Q substitution. Objective Evaluate the efficacy and safety of LentiGlobin in patients with TDT in the phase 1/2 Northstar (HGB-204; NCT01745120) and phase 3 Northstar-2 (HGB-207; NCT02906202) studies. Methods Patients with TDT (≥100 mL/kg/yr of red blood cells [RBCs] or ≥8 RBC transfusions/yr) received G-CSF and plerixafor for mobilization and HSCs were transduced with the BB305 LVV. Patients underwent single agent busulfan myeloablative conditioning, were infused with transduced cells, and were followed for engraftment, safety, and efficacy. Statistics are presented as median (min – max). Results As of March 7, 2018, 18 patients (12 – 35 yrs) were treated in Northstar (follow-up 32.1 [23.1 – 41.9] months) and as of May 15, 2018, 11 patients (12 – 24 yrs) were treated in Northstar-2 (follow-up 8.5 [0.3 – 16.2] months). Patients received a median cell dose of 8.0 (5.0 – 19.4) CD34+ cells × 106/kg in both studies. The median time to neutrophil and platelet engraftment in both studies was 19 (14 – 30) days and 44 (19 – 191) days, respectively; 1 patient in Northstar-2 (0.3 months follow-up) had not engrafted at time of analysis. Of 6 patients with platelet engraftment ≥ Day 60, 4 had non-serious bleeding events prior to engraftment. All 6 had intact spleens and 3/6 received G-CSF between Days 0 – 21. Both factors appeared associated with time to platelet engraftment. In Northstar, 8/10 patients with non-β0/β0 genotypes and 2/8 patients with β0/β0 genotypes achieved transfusion independence (TI; weighted average hemoglobin [Hb] ≥ 9 g/dL without RBC transfusions for ≥ 12 months). Median Hb during TI was 10.0 (9.3 – 13.1) g/dL. In Northstar-2, 7/8 patients with non-β0/β0 genotypes and ≥ 6 months follow-up stopped RBC transfusions with Hb of 11.1 – 13.3 g/dL at last visit; the first patient treated achieved TI. Non-hematologic grade ≥ 3 adverse events post-infusion in ≥ 5/29 (15%) patients were stomatitis, febrile neutropenia, and pharyngeal inflammation. Veno-occlusive liver disease attributed to busulfan occurred in 4/29 patients (Table 1). There was no transplant-related mortality, vector-mediated replication competent lentivirus, or clonal dominance. Conclusion In Northstar, 80% of patients with non-β0/β0 genotypes achieved TI and early Northstar-2 data suggest that patients can achieve near-normal Hb without transfusions. The safety profile of LentiGlobin is consistent with myeloablative busulfan conditioning. Longer time to platelet engraftment was observed in few patients, but no graft failure or deaths were reported

Nuclear microRNAs in normal hemopoiesis and cancer

Abstract Since the discovery of microRNAs (miRNAs) in the early 1990s, these small molecules have been increasingly recognized as key players in the regulation of critical biological processes. They have also been implicated in many diverse human diseases. The canonical function of miRNAs is to target the 3′ untranslated region (3′ UTR) of cytoplasmic messenger RNA to post-transcriptionally regulate mRNA and protein levels. It has now been shown that miRNAs can also bind to the promoter regions of genes or primary miRNA transcripts to regulate gene expression. Such observations have indicated the presence of miRNAs in the nucleus and implied additional non-canonical functions. Nevertheless, the role(s) of nuclear miRNAs in normal hemopoiesis and cancer remains elusive despite a burgeoning literature. Herein, we review current knowledge concerning the abundance and/or functions of nuclear miRNAs during blood cell development and cancer biology. We also discuss ongoing challenges in order to provoke further studies into identifying key roles for nuclear miRNAs in the development of other cell lineages and human cancers