A genome-wide search for epigenetically regulated genes in zebra finch using MethylCap-seq and RNA-seq

Learning and memory formation are known to require dynamic CpG (de)methylation and gene expression changes. Here, we aimed at establishing a genome-wide DNA methylation map of the zebra finch genome, a model organism in neuroscience, as well as identifying putatively epigenetically regulated genes. RNA-and MethylCap-seq experiments were performed on two zebra finch cell lines in presence or absence of 5-aza-2'-deoxycytidine induced demethylation. First, the MethylCap-seq methodology was validated in zebra finch by comparison with RRBS-generated data. To assess the influence of (variable) methylation on gene expression, RNA-seq experiments were performed as well. Comparison of RNA-seq and MethylCap-seq results showed that at least 357 of the 3,457 AZA-upregulated genes are putatively regulated by methylation in the promoter region, for which a pathway analysis showed remarkable enrichment for neurological networks. A subset of genes was validated using Exon Arrays, quantitative RT-PCR and CpG pyrosequencing on bisulfite-treated samples. To our knowledge, this study provides the first genome-wide DNA methylation map of the zebra finch genome as well as a comprehensive set of genes of which transcription is under putative methylation control

Molecular Dissection of FUS Points at Synergistic Effect of Low-Complexity Domains in Toxicity

Summary: RNA-binding protein aggregation is a pathological hallmark of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). To gain better insight into the molecular interactions underlying this process, we investigated FUS, which is mutated and aggregated in both ALS and FTLD. We generated a Drosophila model of FUS toxicity and identified a previously unrecognized synergistic effect between the N-terminal prion-like domain and the C-terminal arginine-rich domain to mediate toxicity. Although the prion-like domain is generally considered to mediate aggregation of FUS, we find that arginine residues in the C-terminal low-complexity domain are also required for maturation of FUS in cellular stress granules. These data highlight an important role for arginine-rich domains in the pathology of RNA-binding proteins. : Protein aggregation is a hallmark of ALS. Bogaert et al. describe the molecular interactions between disordered regions of the FUS protein driving its liquid phase behavior, maturation, and neurotoxicity. These findings highlight the physicochemical interactions driving FUS phase separation and give us insights into its misregulation in disease. Keywords: amyotrophic lateral sclerosis, frontotemporal lobar degeneration, phase transition, LLPS, protein aggregation, FUS, prion-like domain, low-complexity domain, intrinsically disordered protei

Molecular Dissection of FUS Points at Synergistic Effect of Low-Complexity Domains in Toxicity

RNA-binding protein aggregation is a pathological hallmark of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). To gain better insight into the molecular interactions underlying this process, we investigated FUS, which is mutated and aggregated in both ALS and FTLD. We generated a Drosophila model of FUS toxicity and identified a previously unrecognized synergistic effect between the N-terminal prion-like domain and the C-terminal arginine-rich domain to mediate toxicity. Although the prion-like domain is generally considered to mediate aggregation of FUS, we find that arginine residues in the C-terminal low-complexity domain are also required for maturation of FUS in cellular stress granules. These data highlight an important role for arginine-rich domains in the pathology of RNA-binding proteins.status: publishe

DNA Methylation Regulates Transcription Factor-Specific Neurodevelopmental but Not Sexually Dimorphic Gene Expression Dynamics in Zebra Finch Telencephalon

Song learning in zebra finches (Taeniopygia guttata) is a prototypical example of a complex learned behavior, yet knowledge of the underlying molecular processes is limited. Therefore, we characterized transcriptomic (RNA-sequencing) and epigenomic (RRBS, reduced representation bisulfite sequencing; immunofluorescence) dynamics in matched zebra finch telencephalon samples of both sexes from 1 day post hatching (1 dph) to adulthood, spanning the critical period for song learning (20 and 65 dph). We identified extensive transcriptional neurodevelopmental changes during postnatal telencephalon development. DNA methylation was very low, yet increased over time, particularly in song control nuclei. Only a small fraction of the massive differential expression in the developing zebra finch telencephalon could be explained by differential CpG and CpH DNA methylation. However, a strong association between DNA methylation and age-dependent gene expression was found for various transcription factors (i.e., OTX2, AR, and FOS) involved in neurodevelopment. Incomplete dosage compensation, independent of DNA methylation, was found to be largely responsible for sexually dimorphic gene expression, with dosage compensation increasing throughout life. In conclusion, our results indicate that DNA methylation regulates neurodevelopmental gene expression dynamics through steering transcription factor activity, but does not explain sexually dimorphic gene expression patterns in zebra finch telencephalon

Drosophila screen connects nuclear transport genes to DPR pathology in c9ALS/FTD

Hexanucleotide repeat expansions in C9orf72 are the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD) (c9ALS/FTD). Unconventional translation of these repeats produces dipeptide repeat proteins (DPRs) that may cause neurodegeneration. We performed a modifier screen in Drosophila and discovered a critical role for importins and exportins, Ran-GTP cycle regulators, nuclear pore components, and arginine methylases in mediating DPR toxicity. These findings provide evidence for an important role for nucleocytoplasmic transport in the pathogenic mechanism of c9ALS/FTD.status: publishe

HDAC6 inhibition reverses axonal transport defects in motor neurons derived from FUS-ALS patients

International audienceAmyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disorder due to selective loss of motor neurons (MNs). Mutations in the fused in sarcoma (FUS) gene can cause both juvenile and late onset ALS. We generated and characterized induced pluripotent stem cells (iPSCs) from ALS patients with different FUS mutations, as well as from healthy controls. Patient-derived MNs show typical cytoplasmic FUS pathology, hypoexcitability, as well as progressive axonal transport defects. Axonal transport defects are rescued by CRISPR/Cas9-mediated genetic correction of the FUS mutation in patient-derived iPSCs. Moreover, these defects are reproduced by expressing mutant FUS in human embryonic stem cells (hESCs), whereas knockdown of endogenous FUS has no effect, confirming that these pathological changes are mutant FUS dependent. Pharmacological inhibition as well as genetic silencing of histone deacetylase 6 (HDAC6) increase α-tubulin acetylation, endo-plasmic reticulum (ER)–mitochondrial overlay, and restore the axonal transport defects in patient-derived MNs

HDAC6 inhibition reverses axonal transport defects in motor neurons derived from FUS-ALS patients

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disorder due to selective loss of motor neurons (MNs). Mutations in the fused in sarcoma (FUS) gene can cause both juvenile and late onset ALS. We generated and characterized induced pluripotent stem cells (iPSCs) from ALS patients with different FUS mutations, as well as from healthy controls. Patient-derived MNs show typical cytoplasmic FUS pathology, hypoexcitability, as well as progressive axonal transport defects. Axonal transport defects are rescued by CRISPR/Cas9-mediated genetic correction of the FUS mutation in patient-derived iPSCs. Moreover, these defects are reproduced by expressing mutant FUS in human embryonic stem cells (hESCs), whereas knockdown of endogenous FUS has no effect, confirming that these pathological changes are mutant FUS dependent. Pharmacological inhibition as well as genetic silencing of histone deacetylase 6 (HDAC6) increase α-tubulin acetylation, endoplasmic reticulum (ER)-mitochondrial overlay, and restore the axonal transport defects in patient-derived MNs.Amyotrophic lateral sclerosis (ALS) leads to selective loss of motor neurons. Using motor neurons derived from induced pluripotent stem cells from patients with ALS and FUS mutations, the authors demonstrate that axonal transport deficits that are observed in these cells can be rescued by HDAC6 inhibition.status: publishe