Regulation of the neuropathy-associated Pmp22 gene by a distal super-enhancer

Abstract

Peripheral nerve myelination is adversely affected in the most common form of the hereditary peripheral neuropathy called Charcot-Marie-Tooth Disease. This form, classified as CMT1A, is caused by a 1.4 Mb duplication on chromosome 17, which includes the abundantly expressed Schwann cell myelin gene, Peripheral Myelin Protein 22 (PMP22). This is one of the most common copy number variants causing neurological disease. Overexpression of Pmp22 in rodent models recapitulates several aspects of neuropathy, and reduction of Pmp22 in such models results in amelioration of the neuropathy phenotype. Recently we identified a potential super-enhancer approximately 90-130 kb upstream of the Pmp22 transcription start sites. This super-enhancer encompasses a cluster of individual enhancers that have the acetylated histone H3K27 active enhancer mark, and coincides with smaller duplications identified in patients with milder CMT1A-like symptoms, where the PMP22 coding region itself was not part of the duplication. In this study, we have utilized genome editing to create a deletion of this super-enhancer to determine its role in Pmp22 regulation. Our data show a significant decrease in Pmp22 transcript expression using allele-specific internal controls. Moreover, the P2 promoter of the Pmp22 gene, which is used in other cell types, is affected, but we find that the Schwann cell-specific P1 promoter is disproportionately more sensitive to loss of the super-enhancer. These data show for the first time the requirement of these upstream enhancers for full Pmp22 expression.

Direct relationship between increased expression and mistrafficking of the Charcot–Marie–Tooth–associated protein PMP22

“…Transcription factor binding and open chromatin markers were found to be much more abundant at the PMP22 superenhancer in myelinating Schwann cells compared with oligodendrocytes (32). Furthermore, deletion of this super-enhancer in a Schwann cell line dramatically reduced PMP22 transcripts (33). Under conditions of CMT1A trisomy, in which the superenhancer and promotors are also duplicated, the level of expressed PMP22 may be even higher.…”

Emerging Therapies for Charcot-Marie-Tooth Inherited Neuropathies

“…Those are small (usually ~19–22 nucleotides long) RNA structures that specifically bind on the transcript messenger RNA (mRNA) of interest in order to either degrade it or block its translation [ 35 , 36 ]. Antisense oligonucleotides (ASOs) are single-stranded synthetic nucleic acids that specifically bind on mRNA sequences and promote their RNAseH-depended degradation [ 37 ]. An alternative to ASOs, the antiparallel triplex-forming oligonucleotides, bind to the major groove of duplex DNA of interest [ 38 ].…”

“…CRISPR/Cas9 was used in a rat SC line in order to delete an upstream region of PMP22 gene, which is predicted to host an enhancer or a promoter of the gene. Deletion of this region resulted in a reduction of Pmp22 mRNA levels [ 37 ]. Likewise, CRISPR/Cas9-mediated deletion of the TATA-box promoter of PMP22 gene in C22 mice using non-viral intraneural injections also downregulated Pmp22 mRNA and improved nerve pathology [ 41 ].…”

Gene Therapy Options as New Treatment for Inherited Peripheral Neuropathy

“…Reducing the protein expression of PMP22 can also be achieved by modulating the transcriptional activity of a gene. Recently, two independent research groups demonstrated the feasibility of PMP22 reduction by disrupting either the promoter or enhancer of PMP22 with gene editing technology [81,82]. The clustered regularly interspaced short palindromic repeats (CRISPR) and related Cas genes are now emerging as essential tools for gene editing [83,84].…”

“…Intriguingly, gene editing technology was applied to disrupt normal genes rather than to correct the mutant gene for CMT1A treatment. Pantera et al [81] investigated the feasibility of gene editing for reducing the transcription of PMP22 in vitro. They deleted the potential super-enhancer or promoter located approximately 90~130 kb upstream of the Pmp22 transcription sites using CRISPR/Cas9 in S16, a rat Schwann cell line, which effectively reduced the mRNA level of PMP22.…”