Postsynaptic chromatin is under neural control at the neuromuscular junction

In adult skeletal muscle, the nicotinic acetylcholine receptor (AChR) specifically accumulates at the neuromuscular junction, to allow neurotransmission. This clustering is paralleled by a compartmentalization of AChR genes expression to subsynaptic nuclei, which acquire a unique gene expression program and a specific morphology in response to neural cues. Our results demonstrate that neural agrin-dependent reprogramming of myonuclei involves chromatin remodelling, histone hyperacetylation and histone hyperphosphorylation. Activation of AChR genes in subsynaptic nuclei is mediated by the transcription factor GABP. Here we demonstrate that upon activation, GABP recruits the histone acetyl transferase (HAT) p300 on the AChR ɛ subunit promoter, whereas it rather recruits the histone deacetylase HDAC1 when the promoter is not activated. Moreover, the HAT activity of p300 is required in vivo for AChR expression. GABP therefore couples chromatin hyperacetylation and AChR activation by neural factors in subsynaptic nuclei

Expression of Pannexin 1 and Pannexin 3 during skeletal muscle development, regeneration, and Duchenne muscular dystrophy

Pannexin 1 (Panx1) and Pannexin 3 (Panx3) are single membrane channels recently implicated in myogenic commitment, as well as myoblast proliferation and differentiation in vitro. However, their expression patterns during skeletal muscle development and regeneration had yet to be investigated. Here, we show that Panx1 levels increase during skeletal muscle development becoming highly expressed together with Panx3 in adult skeletal muscle. In adult mice, Panx1 and Panx3 were differentially expressed in fast- and slow-twitch muscles. We also report that Panx1/PANX1 and Panx3/PANX3 are co-expressed in mouse and human satellite cells, which play crucial roles in skeletal muscle regeneration. Interestingly, Panx1 and Panx3 levels were modulated in muscle degeneration/regeneration, similar to the pattern seen during skeletal muscle development. As Duchenne muscular dystrophy is characterized by skeletal muscle degeneration and impaired regeneration, we next used mild and severe mouse models of this disease and found a significant dysregulation of Panx1 and Panx3 levels in dystrophic skeletal muscles. Together, our results are the first demonstration that Panx1 and Panx3 are differentially expressed amongst skeletal muscle types with their levels being highly modulated during skeletal muscle development, regeneration, and dystrophy. These findings suggest that Panx1 and Panx3 channels may play important and distinct roles in healthy and diseased skeletal muscles

Staufen1 Regulates Multiple Alternative Splicing Events either Positively or Negatively in DM1 Indicating Its Role as a Disease Modifier

Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by an expansion of CUG repeats in the 3' UTR of the DMPK gene. The CUG repeats form aggregates of mutant mRNA, which cause misregulation and/or sequestration of RNA-binding proteins, causing aberrant alternative splicing in cells. Previously, we showed that the multi-functional RNA-binding protein Staufen1 (Stau1) was increased in skeletal muscle of DM1 mouse models and patients. We also showed that Stau1 rescues the alternative splicing profile of pre-mRNAs, e.g. the INSR and CLC1, known to be aberrantly spliced in DM1. In order to explore further the potential of Stau1 as a therapeutic target for DM1, we first investigated the mechanism by which Stau1 regulates pre-mRNA alternative splicing. We report here that Stau1 regulates the alternative splicing of exon 11 of the human INSR via binding to Alu elements located in intron 10. Additionally, using a high-throughput RT-PCR screen, we have identified numerous Stau1-regulated alternative splicing events in both WT and DM1 myoblasts. A number of these aberrant ASEs in DM1, including INSR exon 11, are rescued by overexpression of Stau1. However, we find other ASEs in DM1 cells, where overexpression of Stau1 shifts the splicing patterns away from WT conditions. Moreover, we uncovered that Stau1-regulated ASEs harbour Alu elements in intronic regions flanking the alternative exon more than non-Stau1 targets. Taken together, these data highlight the broad impact of Stau1 as a splicing regulator and suggest that Stau1 may act as a disease modifier in DM1

Expression of mutant Ets protein at the neuromuscular synapse causes alterations in morphology and gene expression.

The localized transcription of several muscle genes at the motor endplate is controlled by the Ets transcription factor GABP. To evaluate directly its contribution to the formation of the neuromuscular junction, we generated transgenic mice expressing a general Ets dominant-negative mutant specifically in skeletal muscle. Quantitative RT-PCR analysis demonstrated that the expression of genes containing an Ets-binding site was severely affected in the mutant mice. Conversely, the expression of other synaptic genes, including MuSK and Rapsyn, was unchanged. In these animals, muscles expressing the mutant transcription factor developed normally, but examination of the post-synaptic morphology revealed marked alterations of both the primary gutters and secondary folds of the neuromuscular junction. Our results demonstrate that Ets transcription factors are crucial for the normal formation of the neuromuscular junction. They further show that Ets-independent mechanisms control the synaptic expression of a distinct set of synaptic genes.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jFLWINinfo:eu-repo/semantics/publishe

Alu elements in Stau1 and non-Stau1 regulated ASEs.

<p>(A) All ASEs were considered for this analysis with the exception of any that had a blank PSI, as described above. Stau1 (≥15% PSI) and non-Stau1 regulated ASEs targets were searched using RepeatMasker software v 4.0.5, to identify the presence of Alu elements in introns flanking the ASE. (B) The subfamilies of Alu elements in Stau1-regulated targets are presented. The locations of Alu elements in Stau1-regulated cassette-type ASE containing targets are represented in a bar graph as the number of Alu elements found in the upstream, downstream or in both introns surrounding the ASE. The locations of these intronic Alu elements were then correlated to the type of splicing event induced by Stau1 overexpression, i.e. exon inclusion or exon skipping.</p

Validation of Stau1-regulated ASEs from RT-PCR splicing screen, in WT and DM1 cell lines.

<p>Total RNA was collected from four GM0 cell lines; WT (GM01653) and DM1 cell lines GM03132 (1700 CTG), GM03987 (500 CTG), and GM03991 (50–80 CTG). RT-PCR was performed to determine splicing ratios of ASE in the (A) <i>INSR</i> (B) <i>hnRNPA2B1</i>, (C) <i>LRRC23</i>, (D) <i>HIF1α</i> mRNA isoforms. ASE is indicated by exon number for each event. Bar graphs show an average of three independent experiments. Error bars represent SEM * = p < 0.5, ** = p < 0.01.</p

Stau1 levels regulate the pre-mRNA splicing of the human <i>INSR</i> in HeLa cells.

<p>(A) pGIPZ (CTRL) or Stau1-HA (Stau1-HA) plasmids were transiently transfected into HeLa cell lines, and total RNA and protein lysate was collected after 48 hours. RT-PCR using primers specific to the human endogenous <i>INSR</i> were used on cDNA synthesized from total RNA to amplify the two isoforms (IR-A and IR-B) of the <i>INSR</i>. Stau1-HA protein levels were assessed by Western blot using HA-specific antibodies, and GAPDH was used as a loading control. (B) shCTRL or shStau1 were transiently transfected into HeLa cell lines and total RNA and protein lysate was collected after 48 hours. RT-PCR was performed to amplify the <i>INSR</i> isoforms. Stau1 protein levels were assessed by Western blot and quantified using GAPDH as a loading control. (C) Representative Western blots showing protein levels of CUGBP1, MBNL1 and hnRNP H in HeLa cells transfected with CTRL, shRNA or Stau1-HA plasmids. GAPDH was used as a loading control. (D) Quantification of Western blot analysis of splicing factors upon Stau1 level modulation. In all cases, bar graphs show an average of ≥3 independent experiments. Error bars represent SEM * = p < 0.05, ** = p < 0.01.</p

Stau1 overexpression regulates the alternative splicing of numerous ASEs which are both beneficial and detrimental in DM1 conditions.

<p>(A) A total of 33 ASEs were considered for this analysis based on the following criteria. First, ASEs that showed a ≥10% ΔPSI from WT to DM1 conditions were selected. From this, ASEs that showed a ≥10% ΔPSI with Stau1 overexpression were selected and identified as either beneficial or detrimental for the DM1 pathology (refer to results). (B) Bar graphs showing the specific ASEs that the overexpression of Stau1-HA under DM1 conditions either returned towards the WT splicing pattern (left bar graph) or continued towards a detrimental effect (right bar graph). (C-E) Comparison of the data obtained from the RT-PCR splicing screen presented here to that of Klinck et al., 2014. The threshold used by Klinck et al., was applied (≥5% PSI) to compare the 163 ASEs used in both screens (Refer to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005827#pgen.1005827.s005" target="_blank">S1 Table</a>). (E) The direction of splicing was determined by identifying whether modification of the splicing proteins (i.e. Stau1 and/or MBNL1/RBFOX1) shifted the splicing pattern of an ASE in similar directions, for example, modification of both splicing factors resulted in an increased splicing of the long isoform.</p

Stau1 regulates splicing of <i>INSR</i> exon 11 through an interaction with Alu elements in intron 10.

<p>(A) The genomic DNA sequence of the human INSR (NG_008852.1) was used to assess the Alu elements located in intron 10. Introns are not to scale, and this is indicated in intron 11 (//). The IR-minigene constructs used in this study are shown here. Previously deleted segments of genomic DNA determined not to influence exon 11 splicing are indicated in intron 11 (Δ symbol), and black dotted lines represent the deleted segment containing the three Alu elements. (B) HeLa cells were transiently transfected with Stau1-HA plasmid and either the WT or ΔAlus IR-minigene. Immunoprecipitation (IP) of Stau1-HA protein was carried out using HA-specific antibodies under RNase-free conditions. Western blot using HA-antibodies show equal amounts of Stau1-HA protein was immunoprecipitated in each condition. RNA was collected and DNase-treated prior to cDNA synthesis. RT-PCR was performed using <i>GAPDH</i> specific primers to demonstrate a lack of non-specific binding of RNA to the beads used for immunoprecipitation. Equal amounts of transfected minigenes were confirmed by performing RT-PCR on the cDNA synthesized from the 10% inputs lysates with primers specific to amplify a portion of the plasmid vector (pSG5) and the IR-minigene (Exon 10), corresponds to the pSG5-E10 labelled band. RT-qPCR was carried out using primers specific to an 115 bp region of intron 10 of the IR-minigene to determine the amount of IR-minigene RNA bound to immunoprecipitated Stau1-HA. Bar graphs show an average of four independent RIP experiments. (C-D) HeLa cells were transiently transfected with a CTRL, Stau1-HA plasmid or shStau1 and either the WT or ΔAlus IR-minigene. IR-minigene splicing was determined by RT-PCR. The average of ≥3 independent experiments was used. Error bars represent SEM * = p < 0.05, ** = p < 0.01, *** = p < 0.001.</p