Stimulation of myelin basic protein gene transcription by Fyn tyrosine kinase for myelination

Abstract

Myelin is synthesized about the time of birth. The Src-family tyrosine kinase Fyn is involved in the initial events of myelination. Fyn is present in myelin-forming cells and is activated through stimulation of cell surface receptors such as large myelin-associated glycoprotein (L-MAG). Here we show that Fyn stimulates transcription of the myelin basic protein (MBP) gene for myelination. MBP is a major component of the myelin membrane. In 4-week-old Fyn-deficient mice, MBP is significantly reduced, and electron microscopic analysis showed that myelination is delayed, compared with wild-type mice. The Fyn-deficient mice had thinner, more irregular myelin than the wild-type. We found that Fyn stimulates the promoter activity of the MBP gene by approximately sevenfold. The region responsible for the transactivation by Fyn is located between nucleotides -675 and -647 with respect to the transcription start site. Proteins binding to this region were found by gel shift study, and the binding activity correlates with Fyn activity during myelination. These results suggest that transactivation of the MBP gene by Fyn is important for myelination.

Fig. 1.

Electron microscopic analysis of myelin in 4-week-old Fyn-deficient and wild-type mice. Typical electron micrographs of spinal cord sections of 4-week-old Fyn-deficient (Fyn^−/−) and wild-type mice are shown. Scale bars, 1 μm. 5000× magnification.

Fig. 2.

Amount of MBP in wild-type and Fyn-deficient mice. Equal amounts of brain or spinal cord lysates obtained from 4-week-old wild-type and Fyn-deficient mice were subjected to immunoblotting with anti-MBP. The amounts of MBP were determined by densitometric scanning of the corresponding bands. Results are presented as densitometric units normalized to the value for the wild type. Error bars represent SEM (n = 6).

Fig. 3.

Activation of the MBP gene transcription by Fyn. Deletion constructs of the MBP promoter–CAT and the transactivation by Fyn (fold activation) are shown. The indicated base pair is from the transcription start site. CV1 cells were transfected with each MBP promoter–CAT plasmid and pME18S vector or Fyn expression plasmid. Enzyme activities in the presence of Fyn were normalized against the activities with pME18S vector. The results are expressed as means ± SD of five separate experiments.

Fig. 4.

Identification of the sequence required for Fyn-dependent transactivation of the MBP promoter. The deletion constructs of the MBP promoter contain positions indicated on thetop line. Enzyme activities in the presence of Fyn were normalized against the activities with pME18S vector (fold activation). The results are expressed as means ± SD of three separate experiments. The sequence used for the experiments depicted in Figures6 and 7 is shown at the top.

Fig. 5.

Deletion of the MBP promoter between −687 and –656 eliminates transactivation by Fyn. The deletion constructs of the MBP promoter contain positions indicated on the top line. Enzyme activities in the presence of Fyn were normalized against the activities with pME18S vector (fold activation). The results are expressed as means ± SD of three separate experiments.

Fig. 6.

The sequence between bp –675 and –647 of the MBP promoter is responsible for Fyn-dependent transactivation. The sequence between bp –675 and –647 of the MBP promoter was fused to the CAT reporter gene with TK promoter (A,TK-FRS-CAT) and transfected with pME18S vector (−) or Fyn expression plasmid (+) into CV1 cells. Results of the CAT assay are shown as fold activation by Fyn (B). Means ± SD.

Fig. 7.

Developmental regulation of protein binding to the Fyn response element. A, Nuclear extracts were prepared from CV1 cells and incubated with end-labeled Fyn response element (−) in the presence of unlabeled specific (S) or nonspecific (N) competitors. Thearrow indicates a band specific for the Fyn response element. B, Nuclear extracts were prepared from brains of 4-d-old (P4) and 30-d-old (P30) mice and incubated with Fyn response element. The arrowindicates a developmentally regulated band specific for the Fyn response element.

Fig. 8.

Schematic illustration for the role of the signaling through Fyn PTK in myelination. The signal from neuron–oligodendrocyte interaction (such as from L-MAG) activates Fyn PTK. Fyn phosphorylates its substrates and stimulates transcription factors. Thus, MBP gene transcription is stimulated and the amount of MBP increases, which is necessary for forming myelin. The Fyn response element and the positions of the interferon-γ response element core sequence (γ-IRE CS) and NF-IL6 core sequence (NF-IL6 CS) are indicated with their consensus sequences. Single letter code: W = Aor T, K = G orT, Y = C orT, N = A orG or C or T.