The regulation of the processes of myelination and remyelination are poorly understood. Our long-term goals are to better understand these processes. However, the immediate goals are to understand the regulation of a single protein-the P2 protein. We will study the regulation of expression of the human and mouse P2 genes. To accomplish these studies, we have cloned the mouse and human P2 genes and generated a rat Schwann cell line (MT4H1) and will attempt to generate a human Schwann cell line.
In Specific Aim #1, we will identify the important proximal regulatory regions of the mouse P2 gene by transfection into rat Schwann cells. In fact, in the """"""""Preliminary Studies,"""""""" we have identified a TC-rich region which down-regulates P2 expression and a second region that up-regulates P2 expression. Furthermore, we have identified three transcriptional factors which alter P2 expression: c-jun/c-fos, CAAT/enhance binding protein (C/EBP), belonging to the bZip family of transcriptional factors, and HNF-1, aPOU domain protein. How these three transcriptional factors affect P2 expression will be examined in detail. Furthermore, the regions of the promoter that are necessary and sufficient for tissue-specific expression will be determined by transfecting the 5'-deletion constructs into Schwann cells and other cell types, as well as, producing transgenic animals and examining tissue for beta-galactosidase expression.
In Specific Aim #2, we will use strategies previously used to generate rat Schwann cell lines to produce human Schwann cell lines. The cell lines obtained will be characterized by immunocytochemical studies, morphological appearance, biochemical properties, and ability to form myelin when co- cultured with dorsal root ganglia neurites. Only those cells most resembling untransfected human Schwann cells will be used for further studies. Although we have cloned the human P2 gene and know the restriction map, we must still sequence the promoter. Once the sequence is known, studies similar to those undertaken for the mouse P2 promoter will be conducted. These studies should help in understanding the similarities and differences in the regulation of the human and mouse myelin P2 genes. Information about this regulation may provide insights into the processes of myelination and demyelination, which then may be used to help in designing possible therapies to aid recovery in diseases resulting from demyelination or dysmyelination.
| Rutkowski, J L; Kirk, C J; Lerner, M A et al. (1995) Purification and expansion of human Schwann cells in vitro. Nat Med 1:80-3 |
| Bharucha, V A; Peden, K W; Subach, B R et al. (1993) Characterization of the cis-acting elements of the mouse myelin P2 promoter. J Neurosci Res 36:508-19 |
