Activation of Dynamin I Gene Expression by Sp1 and Sp3 Is Required for Neuronal Differentiation of N1E-115 Cells

Jiyun Yoo,Moon-Jin Jeong,Byoung-Mog Kwon,Man-Wook Hur,Young-Mee Park,Mi Young Han

doi:10.1074/jbc.m111788200

Abstract

Dynamin I is a key molecule required for the recycling of synaptic vesicles in neurons, and it has been known that dynamin I gene expression is induced during neuronal differentiation. Our previous studies established that neuronal restriction of dynamin I gene expression is controlled by Sp1 and nuclear factor-kappaB-like element-1. Here, using a series of deletion constructs and site-directed mutation, we found that transcription of dynamin I gene during neuronal differentiation of N1E-115 cells is controlled primarily by the Sp1 element located between -13 to -4 bp of the dynamin I promoter. Gel shift analysis demonstrated that in addition to Sp1, Sp3 could interact with this Sp1 element. The requirement for Sp family transcription factors in dynamin I gene expression was confirmed by using mithramycin, an inhibitor of Sp1/Sp3 binding. Mithramycin repressed dynamin I gene expression and resulted in blocking of neuronal differentiation of N1E-115 cells. The localization of the dynamin I protein was also restricted in the peripheral region of the nucleus by the mithramycin treatment. Thus, all of our results suggest that induction of dynamin I gene expression during N1E-115 cell differentiation is modulated by Sp1/Sp3 interactions with the dynamin I promoter, and its expression is important for neuronal differentiation of the N1E-115 cells.

Highlights

Dynamin is a GTPase protein that is believed to play a key role in detaching clathrin-coated vesicles from the plasma membrane [1, 2]
To examine whether the activity of the mouse dynamin I promoter we previously cloned is activated during neurite formation, pCATIP931 containing a 931-bp 5Ј-flanking sequence of the mouse dynamin I gene (Ϫ931-ϩ105) fused to the promoterless CAT gene in pCAT-Basic was transfected in N1E-115 cells, and the cells were either grown under control conditions or treated with 1.5% Me2SO for 48 h
We observed that the localization of dynamin is restricted in peripheral region of nuclear in mithramycin-treated cells (Fig. 7, c– e), whereas dynamin I proteins are dispersed in cytoplasm and cell processes including neurites in differentiated cells (Fig. 7b). All of these results definitely suggest that induction of dynamin I gene expression and exact localization are critical for neurite formation

Summary

Introduction

Dynamin is a GTPase protein that is believed to play a key role in detaching clathrin-coated vesicles from the plasma membrane [1, 2]. Mithramycin repressed dynamin I gene expression and resulted in blocking of neuronal differentiation of N1E-115 cells.

Results

Conclusion