Atze J. Bergsma

A central role for TFIID in the pluripotent transcription circuitry

Embryonic stem (ES) cells are pluripotent and characterized by open chromatin and high transcription levels, achieved through auto-regulatory and feed-forward transcription factor loops. ES-cell identity is maintained by a core of factors including Oct4 (also known as Pou5f1), Sox2, Klf4, c-Myc (OSKM) and Nanog, and forced expression of the OSKM factors can reprogram somatic cells into induced pluripotent stem cells (iPSCs) resembling ES cells. These gene-specific factors for RNA-polymerase-II-mediated transcription recruit transcriptional cofactors and chromatin regulators that control access to and activity of the basal transcription machinery on gene promoters. How the basal transcription machinery is involved in setting and maintaining the pluripotent state is unclear. Here we show that knockdown of the transcription factor IID (TFIID) complex affects the pluripotent circuitry in mouse ES cells and inhibits reprogramming of fibroblasts. TFIID subunits and the OSKM factors form a feed-forward loop to induce and maintain a stable transcription state. Notably, transient expression of TFIID subunits greatly enhanced reprogramming. These results show that TFIID is critical for transcription-factor-mediated reprogramming. We anticipate that, by creating plasticity in gene expression programs, transcription complexes such as TFIID assist reprogramming into different cellular states.

Identification and Characterization of Aberrant GAA Pre‐mRNA Splicing in Pompe Disease Using a Generic Approach

Identification of pathogenic variants in monogenic diseases is an important aspect of diagnosis, genetic counseling, and prediction of disease severity. Pathogenic mechanisms involved include changes in gene expression, RNA processing, and protein translation. Variants affecting pre‐mRNA splicing are difficult to predict due to the complex mechanism of splicing regulation. A generic approach to systematically detect and characterize effects of sequence variants on splicing would improve current diagnostic practice. Here, it is shown that such approach is feasible by combining flanking exon RT‐PCR, sequence analysis of PCR products, and exon‐internal quantitative RT‐PCR for all coding exons. Application of this approach to one novel and six previously published variants in the acid‐alpha glucosidase (GAA) gene causing Pompe disease enabled detection of a total of 11 novel splicing events. Aberrant splicing included cryptic splice‐site usage, intron retention, and exon skipping. Importantly, the extent of leaky wild‐type splicing correlated with disease onset and severity. These results indicate that this approach enables sensitive detection and in‐depth characterization of variants affecting splicing, many of which are still unrecognized or poorly understood. The approach is generic and should be adaptable for application to other monogenic diseases to aid in improved diagnostics.

From Cryptic Toward Canonical Pre-mRNA Splicing in Pompe Disease: a Pipeline for the Development of Antisense Oligonucleotides

While 9% of human pathogenic variants have an established effect on pre-mRNA splicing, it is suspected that an additional 20% of otherwise classified variants also affect splicing. Aberrant splicing includes disruption of splice sites or regulatory elements, or creation or strengthening of cryptic splice sites. For the majority of variants, it is poorly understood to what extent and how these may affect splicing. We have identified cryptic splicing in an unbiased manner. Three types of cryptic splicing were analyzed in the context of pathogenic variants in the acid α-glucosidase gene causing Pompe disease. These involved newly formed deep intronic or exonic cryptic splice sites, and a natural cryptic splice that was utilized due to weakening of a canonical splice site. Antisense oligonucleotides that targeted the identified cryptic splice sites repressed cryptic splicing at the expense of canonical splicing in all three cases, as shown by reverse-transcriptase-quantitative polymerase chain reaction analysis and by enhancement of acid α-glucosidase enzymatic activity. This argues for a competition model for available splice sites, including intact or weakened canonical sites and natural or newly formed cryptic sites. The pipeline described here can detect cryptic splicing and correct canonical splicing using antisense oligonucleotides to restore the gene defect.While 9% of human pathogenic variants have an established effect on pre-mRNA splicing, it is suspected that an additional 20% of otherwise classified variants also affect splicing. Aberrant splicing includes disruption of splice sites or regulatory elements, or creation or strengthening of cryptic splice sites. For the majority of variants, it is poorly understood to what extent and how these may affect splicing. We have identified cryptic splicing in an unbiased manner. Three types of cryptic splicing were analyzed in the context of pathogenic variants in the acid α-glucosidase gene causing Pompe disease. These involved newly formed deep intronic or exonic cryptic splice sites, and a natural cryptic splice that was utilized due to weakening of a canonical splice site. Antisense oligonucleotides that targeted the identified cryptic splice sites repressed cryptic splicing at the expense of canonical splicing in all three cases, as shown by reverse-transcriptase-quantitative polymerase chain reaction analysis and by enhancement of acid α-glucosidase enzymatic activity. This argues for a competition model for available splice sites, including intact or weakened canonical sites and natural or newly formed cryptic sites. The pipeline described here can detect cryptic splicing and correct canonical splicing using antisense oligonucleotides to restore the gene defect.

Antisense Oligonucleotides Promote Exon Inclusion and Correct the Common c.-32-13T>G GAA Splicing Variant in Pompe Disease

The most common variant causing Pompe disease is c.-32-13T>G (IVS1) in the acid α-glucosidase (GAA) gene, which weakens the splice acceptor of GAA exon 2 and induces partial and complete exon 2 skipping. It also allows a low level of leaky wild-type splicing, leading to a childhood/adult phenotype. We hypothesized that cis-acting splicing motifs may exist that could be blocked using antisense oligonucleotides (AONs) to promote exon inclusion. To test this, a screen was performed in patient-derived primary fibroblasts using a tiling array of U7 small nuclear RNA (snRNA)-based AONs. This resulted in the identification of a splicing regulatory element in GAA intron 1. We designed phosphorodiamidate morpholino oligomer-based AONs to this element, and these promoted exon 2 inclusion and enhanced GAA enzyme activity to levels above the disease threshold. These results indicate that the common IVS1 GAA splicing variant in Pompe disease is subject to negative regulation, and inhibition of a splicing regulatory element using AONs is able to restore canonical GAA splicing and endogenous GAA enzyme activity.

GAA Deficiency in Pompe Disease Is Alleviated by Exon Inclusion in iPSC-Derived Skeletal Muscle Cells

Pompe disease is a metabolic myopathy caused by deficiency of the acid α-glucosidase (GAA) enzyme and results in progressive wasting of skeletal muscle cells. The c.-32-13T>G (IVS1) GAA variant promotes exon 2 skipping during pre-mRNA splicing and is the most common variant for the childhood/adult disease form. We previously identified antisense oligonucleotides (AONs) that promoted GAA exon 2 inclusion in patient-derived fibroblasts. It was unknown how these AONs would affect GAA splicing in skeletal muscle cells. To test this, we expanded induced pluripotent stem cell (iPSC)-derived myogenic progenitors and differentiated these to multinucleated myotubes. AONs restored splicing in myotubes to a similar extent as in fibroblasts, suggesting that they act by modulating the action of shared splicing regulators. AONs targeted the putative polypyrimidine tract of a cryptic splice acceptor site that was part of a pseudo exon in GAA intron 1. Blocking of the cryptic splice donor of the pseudo exon with AONs likewise promoted GAA exon 2 inclusion. The simultaneous blocking of the cryptic acceptor and cryptic donor sites restored the majority of canonical splicing and alleviated GAA enzyme deficiency. These results highlight the relevance of cryptic splicing in human disease and its potential as therapeutic target for splicing modulation using AONs.

Alternative Splicing in Genetic Diseases: Improved Diagnosis and Novel Treatment Options.

Alternative splicing is an important mechanism to regulate gene expression and to expand the repertoire of gene products in order to accommodate an increase in complexity of multicellular organisms. It needs to be precisely regulated, which is achieved via RNA structure, splicing factors, transcriptional regulation, and chromatin. Changes in any of these factors can lead to disease. These may include the core spliceosome, splicing enhancer/repressor sequences and their interacting proteins, the speed of transcription by RNA polymerase II, and histone modifications. While the basic principle of splicing is well understood, it is still very difficult to predict splicing outcome, due to the multiple levels of regulation. Current molecular diagnostics mainly uses Sanger sequencing of exons, or next-generation sequencing of gene panels or the whole exome. Functional analysis of potential splicing variants is scarce, and intronic variants are often not considered. This likely results in underestimation of the percentage of splicing variants. Understanding how sequence variants may affect splicing is not only crucial for confirmation of diagnosis and for genetic counseling, but also for the development of novel treatment options. These include small molecules, transsplicing, antisense oligonucleotides, and gene therapy. Here we review the current state of molecular mechanisms of splicing regulation and how deregulation can lead to human disease, diagnostics to detect splicing variants, and novel treatment options based on splicing correction.

Identification and characterization of aberrant splicing in Pompe disease using a genetic approach

Identifi cation of pathogenic variants in monogenic diseases is an important aspect of diagnosis, genetic counseling, and prediction of disease severity. Pathogenic mechanisms involved include changes in gene expression, RNA processing, and protein translation. Variants affecting pre-mRNA splicing are diffi cult to predict due to the complex mechanism of splicing regulation. A generic approach to systematically detect and characterize the effects of sequence variants on splicing would improve current diagnostic practice.

Identification and Characterization of Aberrant Splicing in Pompe Disease Using a Generic Approach.

Identifi cation of pathogenic variants in monogenic diseases is an important aspect of diagnosis, genetic counseling, and prediction of disease severity. Pathogenic mechanisms involved include changes in gene expression, RNA processing, and protein translation. Variants affecting pre-mRNA splicing are diffi cult to predict due to the complex mechanism of splicing regulation. A generic approach to systematically detect and characterize the effects of sequence variants on splicing would improve current diagnostic practice.