LOFAR, the LOw-Frequency ARray, is a new-generation radio interferometer constructed in the north of the Netherlands and across europe. Utilizing a novel phased-array design, LOFAR covers the largely unexplored low-frequency range from 10-240 MHz and provides a number of unique observing capabilities. Spreading out from a core located near the village of Exloo in the northeast of the Netherlands, a total of 40 LOFAR stations are nearing completion. A further five stations have been deployed throughout Germany, and one station has been built in each of France, Sweden, and the UK. Digital beam-forming techniques make the LOFAR system agile and allow for rapid repointing of the telescope as well as the potential for multiple simultaneous observations. With its dense core array and long interferometric baselines, LOFAR achieves unparalleled sensitivity and angular resolution in the low-frequency radio regime. The LOFAR facilities are jointly operated by the International LOFAR Telescope (ILT) foundation, as an observatory open to the global astronomical community. LOFAR is one of the first radio observatories to feature automated processing pipelines to deliver fully calibrated science products to its user community. LOFAR's new capabilities, techniques and modus operandi make it an important pathfinder for the Square Kilometre Array (SKA). We give an overview of the LOFAR instrument, its major hardware and software components, and the core science objectives that have driven its design. In addition, we present a selection of new results from the commissioning phase of this new radio observatory.

SummaryAlthough skeletal muscle cells can be generated from human induced pluripotent stem cells (iPSCs), transgene-free protocols include only limited options for their purification and expansion. In this study, we found that fluorescence-activated cell sorting-purified myogenic progenitors generated from healthy controls and Pompe disease iPSCs can be robustly expanded as much as 5 × 1011-fold. At all steps during expansion, cells could be cryopreserved or differentiated into myotubes with a high fusion index. In vitro, cells were amenable to maturation into striated and contractile myofibers. Insertion of acid α-glucosidase cDNA into the AAVS1 locus in iPSCs using CRISPR/Cas9 prevented glycogen accumulation in myotubes generated from a patient with classic infantile Pompe disease. In vivo, the expression of human-specific nuclear and sarcolemmar antigens indicated that myogenic progenitors engraft into murine muscle to form human myofibers. This protocol is useful for modeling of skeletal muscle disorders and for using patient-derived, gene-corrected cells to develop cell-based strategies.

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.

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.

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease caused by an alanine tract expansion mutation in poly(A) binding protein nuclear 1 (expPABPN1). To model OPMD in a myogenic and physiological context, we generated mouse myoblast cell clones stably expressing either human wild type (WT) or expPABPN1 at low levels. Transgene expression is induced on myotube differentiation and results in formation of insoluble nuclear PABPN1 aggregates that are similar to those observed in patients with OPMD. Quantitative analysis of PABPN1 in myotube cultures revealed that expPABPN1 accumulation and aggregation is greater than that of the WT protein. We found that aggregation of expPABPN1 is more affected than WT PABPN1 by inhibition of proteasome activity. Consistent with this, in myotube cultures expressing expPABPN1, deregulation of the proteasome was identified as the most significantly perturbed pathway. Differences in the accumulation of soluble WT and expPABPN1 were consistent with differences in ubiquitination and rate of protein turnover. This study demonstrates, for the first time to our knowledge, that, in myotubes, the ratio of soluble/insoluble expPABPN1 is significantly lower compared with that of the WT protein. We suggest that this difference can contribute to muscle weakness in OPMD.

Background: Nur77 is an orphan nuclear receptor involved in vascular disease, of which the regulation of activity is poorly understood. Results: FHL2 binds Nur77 and represses its transcriptional activity by inhibition of Nur77 association with DNA. Conclusion: FHL2 acts as a co-repressor of Nur77. Significance: Our data suggest that interaction of FHL2 with Nur77 plays a pivotal role in vascular 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.

SummarySilencing of the FMR1 gene leads to fragile X syndrome, the most common cause of inherited intellectual disability. To study the epigenetic modifications of the FMR1 gene during silencing in time, we used fibroblasts and induced pluripotent stem cells (iPSCs) of an unmethylated full mutation (uFM) individual with normal intelligence. The uFM fibroblast line carried an unmethylated FMR1 promoter region and expressed normal to slightly increased FMR1 mRNA levels. The FMR1 expression in the uFM line corresponds with the increased H3 acetylation and H3K4 methylation in combination with a reduced H3K9 methylation. After reprogramming, the FMR1 promoter region was methylated in all uFM iPSC clones. Two clones were analyzed further and showed a lack of FMR1 expression, whereas the presence of specific histone modifications also indicated a repressed FMR1 promoter. In conclusion, these findings demonstrate that the standard reprogramming procedure leads to epigenetic silencing of the fully mutated FMR1 gene.