Structural variation in the human genome has emerged as a major cause of disease as genomic data have accumulated. One of the most common structural variants associated with human disease causes the heritable neuropathy known as Charcot-Marie-Tooth (CMT) disease type 1A. This 1.4 Mb duplication causes nearly half of the CMT cases that are genetically diagnosed. The PMP22 gene is highly induced in Schwann cells during development, although its precise role in myelin formation and homeostasis is still under active investigation. The PMP22 gene can be considered as a nucleoprotein complex with enzymatic activity to produce the PMP22 transcript, and the complex is allosterically regulated by transcription factors that respond to intracellular signals and epigenomic modifications. The control of PMP22 transcript levels has been one of the major therapeutic targets of therapy development, and this review summarizes those approaches as well as efforts to characterize the regulation of the PMP22 gene.
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.
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.
The sodium hydrogen exchanger (NHE1) plays a role in intracellular pH homeostasis and acts as scaffolding anchor for a diverse set of proteins. The extended carboxyl terminus is phosphorylated by seven known protein kinases. Phosphorylation site of NHE1 by RhoA Kinase (Rock) was recently defined and found critical for a number of cellular functions. However, the impact of Rock phosphorylation on NHE1‐related signaling for a range of agonists has not been determined. Here we examine three different signaling pathways and the role Rock phosphorylation of NHE1. Using stably expressing tagged NHE1 we identify proteins co‐immunoprecipitating with NHE1 in control and agonist stimulated cells. Cellular function of adhesion and proliferation is also presented. Finally the influence of Rock phosphorylation of NHE1 on cellular migration was determined for three signaling pathways using an impedance‐based assay. This work was supported with funds from NSF MCB‐0817784
The Na+‐H+ exchanger isoform 1 (NHE1) is a transmembrane protein that regulates a range of cellular functions essential for cancer progression including cell adhesion, proliferation and migration. The calcineurin B homologous proteins (CHP1 and CHP2) appear to be essential cofactors to support NHE1 function. The CHP1 and CHP 2 binding domain on NHE1 is the same, amino acids 515 to 530. CHP2 is expressed primarily in tumor cells where it binds to NHE1 with a 5‐10 fold higher affinity than CHP1. CHP2 expression in tumor cells supports increased invasion and migration. Here we investigate the ability of mutations to a key amino acid in the binding domain (N519) to alter CHP2 binding to NHE1 in vivo. Two distinct site directed mutations to NHE1, N519A and N519D, have been constructed along with the accompanying stable cell lines expressing NHE1 with these mutations. We will present data evaluating CHP2 binding to NHE1 in these cells using a GFP‐CHP construct. We will also evaluate how a change in CHP2 binding alters adhesion, proliferation, and migration in mutant expressing cells as compared to cells expressing wild‐type NHE1.
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