Post-transcriptional microRNA repression of PMP22 dose in severe Charcot-Marie-Tooth disease type 1.

Copy number variation (CNV) may lead to pathological traits, and Charcot-Marie-Tooth disease type 1A (CMT1A), the commonest inherited peripheral neuropathy, is due to a genomic duplication encompassing the dosage-sensitive PMP22 gene. MicroRNAs act as repressors on post-transcriptional regulation of gene expression and in rodent models of CMT1A, overexpression of one such microRNA (miR-29a) has been shown to reduce the PMP22 transcript and protein level. Here we present genomic and functional evidence, for the first time in a human CNV-associated phenotype, of the 3' untranslated region (3'-UTR)-mediated role of microRNA repression on gene expression. The proband of the family presented with an early-onset, severe sensorimotor demyelinating neuropathy and harboured a novel de novo deletion in the PMP22 3'-UTR. The deletion is predicted to include the miR-29a seed binding site and transcript analysis of dermal myelinated nerve fibres using a novel platform, revealed a marked increase in PMP22 transcript levels. Functional evidence from Schwann cell lines harbouring the wild-type and mutant 3'-UTR showed significantly increased reporter assay activity in the latter, which was not ameliorated by overexpression of a miR-29a mimic. This shows the importance of miR-29a in regulating PMP22 expression and opens an avenue for therapeutic drug development.

Modulation of Schwann cell homeostasis by the BAP1 deubiquitinase.

Schwann cell programming during myelination involves transcriptional networks that activate gene expression but also repress genes that are active in neural crest/embryonic differentiation of Schwann cells. We previously found that a Schwann cell-specific deletion of the EED subunit of the Polycomb Repressive Complex (PRC2) led to inappropriate activation of many such genes. Moreover, some of these genes become re-activated in the pro-regenerative response of Schwann cells to nerve injury, and we found premature activation of the nerve injury program in a Schwann cell-specific knockout of Eed. Polycomb-associated histone modifications include H3K27 trimethylation formed by PRC2 and H2AK119 monoubiquitination (H2AK119ub1), deposited by Polycomb repressive complex 1 (PRC1). We recently found dynamic regulation of H2AK119ub1 in Schwann cell genes after injury. Therefore, we hypothesized that H2AK119 deubiquitination modulates the dynamic polycomb repression of genes involved in Schwann cell maturation. To determine the role of H2AK119 deubiquitination, we generated a Schwann cell-specific knockout of the H2AK119 deubiquitinase Bap1 (BRCA1-associated protein). We found that loss of Bap1 causes tomacula formation, decreased axon diameters and eventual loss of myelinated axons. The gene expression changes are accompanied by redistribution of H2AK119ub1 and H3K27me3 modifications to extragenic sites throughout the genome. BAP1 interacts with OGT in the PR-DUB complex, and our data suggest that the PR-DUB complex plays a multifunctional role in repression of the injury program. Overall, our results indicate Bap1 is required to restrict the spread of polycomb-associated histone modifications in Schwann cells and to promote myelin homeostasis in peripheral nerve.

JUN Regulation of Injury-Induced Enhancers in Schwann Cells.

Schwann cells play a critical role after peripheral nerve injury by clearing myelin debris, forming axon-guiding bands of Büngner, and remyelinating regenerating axons. Schwann cells undergo epigenomic remodeling to differentiate into a repair state that expresses unique genes, some of which are not expressed at other stages of Schwann cell development. We previously identified a set of enhancers that are activated in Schwann cells after nerve injury, and we determined whether these enhancers are preprogrammed into the Schwann cell epigenome as poised enhancers before injury. Poised enhancers share many attributes of active enhancers, such as open chromatin, but are marked by repressive histone H3 lysine 27 (H3K27) trimethylation rather than H3K27 acetylation. We find that most injury-induced enhancers are not marked as poised enhancers before injury indicating that injury-induced enhancers are not preprogrammed in the Schwann cell epigenome. Injury-induced enhancers are enriched with AP-1 binding motifs, and the c-JUN subunit of AP-1 had been shown to be critical to drive the transcriptional response of Schwann cells after injury. Using in vivo chromatin immunoprecipitation sequencing analysis in rat, we find that c-JUN binds to a subset of injury-induced enhancers. To test the role of specific injury-induced enhancers, we focused on c-JUN-binding enhancers upstream of the Sonic hedgehog (Shh) gene, which is only upregulated in repair Schwann cells compared with other stages of Schwann cell development. We used targeted deletions in male/female mice to show that the enhancers are required for robust induction of the Shh gene after injury.SIGNIFICANCE STATEMENT The proregenerative actions of Schwann cells after nerve injury depends on profound reprogramming of the epigenome. The repair state is directed by injury-induced transcription factors, like JUN, which is uniquely required after nerve injury. In this study, we test whether the injury program is preprogrammed into the epigenome as poised enhancers and define which enhancers bind JUN. Finally, we test the roles of these enhancers by performing clustered regularly interspaced short palindromic repeat (CRISPR)-mediated deletion of JUN-bound injury enhancers in the Sonic hedgehog gene. Although many long-range enhancers drive expression of Sonic hedgehog at different developmental stages of specific tissues, these studies identify an entirely new set of enhancers that are required for Sonic hedgehog induction in Schwann cells after injury.

H3K27 demethylases are dispensable for activation of Polycomb-regulated injury response genes in peripheral nerve.

The induction of nerve injury response genes in Schwann cells depends on both transcriptional and epigenomic reprogramming. The nerve injury response program is regulated by the repressive histone mark H3K27 trimethylation (H3K27me3), deposited by Polycomb repressive complex 2 (PRC2). Loss of PRC2 function leads to early and augmented induction of the injury response gene network in peripheral nerves, suggesting H3K27 demethylases are required for derepression of Polycomb-regulated nerve injury genes. To determine the function of H3K27 demethylases in nerve injury, we generated Schwann cell-specific knockouts of H3K27 demethylase Kdm6b and double knockouts of Kdm6b/Kdm6a (encoding JMJD3 and UTX). We found that H3K27 demethylases are largely dispensable for Schwann cell development and myelination. In testing the function of H3K27 demethylases after injury, we found early induction of some nerve injury genes was diminished compared with control, but most injury genes were largely unaffected at 1 and 7 days post injury. Although it was proposed that H3K27 demethylases are required to activate expression of the cyclin-dependent kinase inhibitor Cdkn2a in response to injury, Schwann cell-specific deletion of H3K27 demethylases affected neither the expression of this gene nor Schwann cell proliferation after nerve injury. To further characterize the regulation of nerve injury response genes, we found that injury genes are associated with repressive histone H2AK119 ubiquitination catalyzed by PRC1, which declines after injury. Overall, our results indicate H3K27 demethylation is not required for induction of injury response genes and that other mechanisms likely are involved in activating Polycomb-repressed injury genes in peripheral nerve.

Pmp22 super-enhancer deletion causes tomacula formation and conduction block in peripheral nerves.

Copy number variation of the peripheral nerve myelin gene Peripheral Myelin Protein 22 (PMP22) causes multiple forms of inherited peripheral neuropathy. The duplication of a 1.4 Mb segment surrounding this gene in chromosome 17p12 (c17p12) causes the most common form of Charcot-Marie-Tooth disease type 1A, whereas the reciprocal deletion of this gene causes a separate neuropathy termed hereditary neuropathy with liability to pressure palsies (HNPP). PMP22 is robustly induced in Schwann cells in early postnatal development, and several transcription factors and their cognate regulatory elements have been implicated in coordinating the gene's proper expression. We previously found that a distal super-enhancer domain was important for Pmp22 expression in vitro, with particular impact on a Schwann cell-specific alternative promoter. Here, we investigate the consequences of deleting this super-enhancer in vivo. We find that loss of the super-enhancer in mice reduces Pmp22 expression throughout development and into adulthood, with greater impact on the Schwann cell-specific promoter. Additionally, these mice display tomacula formed by excessive myelin folding, a pathological hallmark of HNPP, as have been previously observed in heterozygous Pmp22 mice as well as sural biopsies from patients with HNPP. Our findings demonstrate a mechanism by which smaller copy number variations, not including the Pmp22 gene, are sufficient to reduce gene expression and phenocopy a peripheral neuropathy caused by the HNPP-associated deletion encompassing PMP22.

Interaction between MAOA and FOXP2 in association with autism and verbal communication in a Korean population.

Expression levels of monoamine oxidase A (MAOA), the enzyme that related to monoamine neurotransmitters metabolism such as serotonin, are related to schizophrenia and autism spectrum disorder. Forkhead box protein P2 (FOXP2), a transcription factor, is associated with abnormal language development and is expressed in several areas of the central nervous system in response to serotonin. For this reason, we undertook interaction analysis between MAOA and FOXP2 in autism spectrum disorder, including testing the verbal communication score of the childhood autism rating scale. In interaction analysis, the FOXP2-TCGC (rs12531289-rs1350135-rs10230087-rs2061183) diplotype and MAOA-TCG (rs6323-rs1801291-rs3027407) haplotype were significantly associated with autism spectrum disorder in males. However, when the interaction term was omitted, neither MAOA nor FOXP2 was associated with autism spectrum disorder or verbal communication. These results indicate that language and speech ability is affected by an interaction between FOXP2 and MAOA, but not by either gene separately.

Association of the FGA and SLC6A4 genes with autistic spectrum disorder in a Korean population.

BACKGROUND: Autism spectrum disorder (ASD) is a neurobiological disorder characterized by distinctive impairments in cognitive function, language, and behavior. Linkage and population studies suggest a genetic association between solute carrier family 6 member 4 (SLC6A4) variants and ASD. METHOD: Logistic regression was used to identify associations between single-nucleotide polymorphisms (SNPs) and ASD with 3 alternative models (additive, dominant, and recessive). Linear regression analysis was performed to determine the influence of SNPs on Childhood Autism Rating Scale (CARS) scores as a quantitative phenotype. RESULTS: In the present study, we examined the associations of SNPs in the SLC6A4 gene and the fibrinogen alpha chain (FGA) gene. Logistic regression analysis showed a significant association between the risk of ASD and rs2070025 and rs2070011 in the FGA gene. The gene-gene interaction between SLC6A4 and FGA was not significantly associated with ASD susceptibility. However, polymorphisms in both SLC6A4 and the FGA gene significantly affected the symptoms of ASD. CONCLUSION: Our findings indicate that FGA and SLC6A4 gene interactions may contribute to the phenotypes of ASD rather than the incidence of ASD.