A Role for Nuclear Factor I in the Intrinsic Control of Cerebellar Granule Neuron Gene Expression

Wei Wang,Rachel E Stock,Richard M Gronostajski,Yong Wee Wong,Melitta Schachner,Daniel L Kilpatrick

doi:10.1074/jbc.m410370200

Abstract

Nervous system formation requires the elaboration of a complex series of differentiation events in both a spatially and maturation-regulated manner. A fundamental question is how neuronal subtype specification and developmental gene expression are controlled within maturing neurons. The alpha6 subunit of the gamma-aminobutyric acid type A (GABA(A)) receptor (GABRA6) is preferentially expressed in cerebellar granule neurons and is part of an intrinsic program directing their differentiation. We have employed a lentiviral approach to examine the transcriptional mechanisms controlling neuronal subtype-selective expression of this gene. These studies demonstrated that nuclear factor I (NFI) proteins are required for both transgenic GABRA6 promoter activity as well as endogenous expression of this gene in cerebellar granule neurons. Chromatin immunoprecipitation also showed that NFI proteins are bound to the GABRA6 promoter in these cells in vivo. Furthermore, analyses of gene knockout mice revealed that Nfia is specifically required for normal expression of the GABRA6 gene in cerebellar granule neurons. NFI expression and DNA binding activity are highly enriched in granule neurons, implicating this transcription factor family in the neuronal subtype-selective expression of the GABRA6 gene. These studies define a new role for NFI proteins as neuronal subtype-enriched transcriptional regulators that participate in an intrinsic transcriptional program directing the differentiation of cerebellar granule neurons.

Highlights

Fundamental to nervous system development is the specification and maturation of diverse neuronal populations that assemble into a complex synaptic network
Identification of Cerebellar granule neurons (CGNs)-enriched Nuclear Proteins That Bind to the GABRA6 Promoter—Developmental and neuronal subtype-specific regulation of the mouse GABRA6 gene is largely determined at the level of gene transcription [18]
GABRA6 gene expression is readily detectable in differentiated CGNs cultured for 6 days in vitro (6 DIV) [29], which was confirmed in the cultures used here

Summary

Introduction

Fundamental to nervous system development is the specification and maturation of diverse neuronal populations that assemble into a complex synaptic network. These events require the appropriate spatiotemporal expression of an array of different genes during development, and a critical question is the nature of the transcriptional program that controls their proper expression. During CGN differentiation, GABAARs undergo a maturationdependent change from mainly benzodiazepine-sensitive to benzodiazepine-insensitive forms [3] This is associated with a switch in GABAAR subunit expression wherein the ␣2 and ␣3 subunits conferring benzodiazepine sensitivity are down-regulated while the ␣6 subunit is induced [3]. The GABRA6 gene provides an excellent opportunity to investigate intrinsic transcriptional mechanisms controlling granule neuron subtype-specific gene regulation. The present studies demonstrate that NFI proteins are enriched in CGNs and are required for the expression of the GABRA6 gene

Methods

Results

Conclusion