Mistry, Mohini Shashikant; (2001) Transcriptional regulation of the neuron-restrictive silencer factor. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Neuron-restrictive silencer factor (NRSF), also known as RE1-silencing transcription factor (REST), is a DNA binding protein that silences the expression of multiple neurone-specific, terminal differentiation genes. During embryogenesis NRSF/REST is expressed at high levels in most non-neural tissues, but is downregulated during differentiation of neural progenitor cells into neurones. The proposed role of this transcription factor is to restrict the expression of neurone-specific genes to the nervous system. However NRSF/REST is also important in directing the proper spatial and temporal expression of NRSE/RE1-bearing genes within the developing peripheral and central nervous systems and this has implications for neuronal development. Furthermore in mature neurones the expression of low levels of NRSF/REST mRNA and the alteration of mRNA levels, in response to kainic acid, implies this transcription factor may also be involved in neuronal plasticity. To understand the mechanisms underlying transcriptional control of the NRSF/REST gene, the promoter region was isolated and characterised. A 6.5 kb genomic fragment of the mouse gene containing three exons (exon IA, exon IB and exon IC), that correspond to alternatively spliced 5' non-coding exons, was isolated. Reverse transcription-PCR identified splice variants in mouse fibroblast and neuroblastoma cell lines. Each splice variant contained one of the 5' non-coding exons spliced onto the first coding exon (exon II) resulting in transcripts with three different 5' untranslated regions (UTR). Splice variants containing type A, 5' UTR (corresponding to exon IA sequence) were by far the most abundant. Using 5' RACE PCR, RNase protection and in vitro transcription assays, two clusters of transcription initiation sites were identified within exon IA. Transient transfection analysis of the 6.5 kb genomic fragment, fused upstream of the luciferase reporter gene, showed this region was sufficient to direct cell-specific reporter gene expression in 3T3 fibroblast, Neuro2a neuroblastoma, PC12 pheochromocytoma cell lines and primary neuroepithelial cell cultures. Testing a series of 5' and 3' deletions within the 6.5 kb fragment in transient transfections led to the identification of several positive and negative cis-acting regulatory domains, some of which act in a cell-specific manner. An enhancer region, spanning the 3' end of exon IA and part of the first intron, was crucial for exon IA promoter activity. Characterisation of this proximal enhancer region revealed the presence of a sequence with similarity to the NRSE/RE1 motif, however NRSF/REST does not appear to bind. It is proposed that the differential levels of NRSF/REST gene expression in non-neural and neuronal cells are achieved by the combinatorial action of the cell-specific enhancer and repressor elements that are present within the mouse NRSF/REST gene.

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