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A Genome Editing Approach to Studying Pmp22 Enhancer Functionality
Author(s) -
Pantera Harrison,
Moran John,
LopezAnido Camila,
Svaren John
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.584.2
Subject(s) - gene duplication , peripheral myelin protein 22 , enhancer , biology , myelin , gene , crispr , genetics , gene dosage , cas9 , microbiology and biotechnology , transcription factor , gene expression , neuroscience , central nervous system
The function of the peripheral nervous system is dependent on myelination of nerves by Schwann cells. This process is tightly governed by a network of transcription factors during development. Myelin stability 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. This duplication includes the abundantly expressed myelin gene Peripheral Myelin Protein 22 ( Pmp22 ), which is associated with multiple disease states in a gene dosage‐dependent manner. Previous studies have shown that reducing the expression of Pmp22 in rodent models of CMT1A results in amelioration of the neuropathy phenotype found in myelinating Schwann cells in the PNS. In recent work characterizing the mechanisms regulating Pmp22 transcription, we identified a cluster of enhancers approximately 100 kb upstream of the Pmp22 TSS. This upstream cluster was found within a smaller duplication identified in patients with CMT1A‐like symptoms, where the Pmp22 coding region itself was not part of the duplication. This cluster possesses multiple binding sites for factors that are required for Schwann cell development and myelination: Egr2 and Sox10. Previous studies in our lab implicate these sites as Pmp22 enhancers, but until now there has been an absence of evidence of functional importance in achieving the high endogenous levels of Pmp22 expression. Using genome editing tools including CRISPR/Cas9 in a modified rat Schwann cell line that expresses Pmp22 at near‐physiological levels, we have established clonal lines possessing deletions that include these upstream transcription factor‐binding sites. Preliminary data show an observed decrease in Pmp22 transcript expression ostensibly due to the loss of these sites. These data show for the first time the requirement of these upstream enhancers for full Pmp22 expression and alternate promoter usage. Support or Funding Information This work is supported by NIH grant R01 NS083841.