Elsevier

Gene

Volume 294, Issues 1–2, 10 July 2002, Pages 269-277
Gene

Cloning and characterization of the 5′-flanking region of the rat neuron-specific Class III β-tubulin gene

https://doi.org/10.1016/S0378-1119(02)00801-6Get rights and content

Abstract

The promoter regions of several neuron-specific structural proteins (e.g. neurofilaments, peripherin, Tα1-tubulin) have revealed potential regulatory elements that could contribute to the choice of a neuronal phenotype during development. We initiated study of the 5′-flanking region of the rat Class III neuron-specific β-tubulin gene (βIII-tubulin) because this gene is expressed at the time of terminal mitosis only in neurons and thus its promoter should be an excellent tool for studying neuron-specific gene expression during the transition from proliferative progenitor cell to early neuronal differentiation. We identified the minimal promoter region needed to drive expression of the βIII-tubulin gene. This minimal region contains multiple putative binding sites for the transcription factors SP1 and AP2, as well as a central nervous system enhancer regulatory element and an E-box. A primer extension analysis identifies a single transcription start site. We highlight several putative regulatory elements that may modulate the expression of the βIII-tubulin gene in a stage- and tissue-specific manner. In addition, we show that the first 490 bp of the promoter are sufficient to regulate βIII-tubulin gene expression during neuronal differentiation of PCC7 cells.

Introduction

The large variety of cell types that compose the vertebrate nervous system are produced by the coordinated expression of numerous transcription factors that progressively restrict the fates of neural ectodermal cells. An important goal is to identify those factors necessary for specifying each cell type in order to influence neural stem cells to differentiate as cellular replacements to treat congenital and degenerative disorders. A useful approach for elucidating how different cell types are determined is the identification of putative transcription factor binding sites in the flanking regions of phenotype-specific structural genes. Examples include glial fibrillary acidic protein for astrocytes (Brenner, 1994), a myelin-specific protein (PLP) for oligodendrocytes (Berndt et al., 2001) and Tα1-tubulin (Gloster et al., 1994) for neurons.

The Class III β-tubulin gene is another important candidate for discovering factors necessary for the expression of a neuronal fate and/or initiation of neuronal differentiation. In vertebrates there are five β-tubulin genes that encode highly homologous protein classes (Nogales, 2001). These proteins share high amino acid identity, but there are two domains that diverge significantly among the classes (Sullivan et al., 1986). The ‘isotype-defining’ domain at the extreme carboxyl terminus provides functional diversity, and defines six different classes of β-tubulin proteins (Sullivan, 1988). Class I protein is ubiquitously expressed in all tissues; Class II and Class IVa proteins are prominent in brain but also expressed in several other tissues; Class IVb protein is expressed exclusively in the testes; Class V protein is detectable in all cell types except neurons. Class III β-tubulin is specifically expressed in the nervous system (Lewis et al., 1985a) only by neurons (Moody et al., 1989, Lee et al., 1990a, Lee et al., 1990b), with some expression in testis (Lee et al., 1990a). In both chick and mouse, Class III β-tubulin is expressed just prior to or at the terminal mitosis of nearly all developing neurons (Moody et al., 1989, Easter et al., 1993). This suggests that its expression is regulated by elements necessary for committing a neural progenitor cell to a neuron, and for initiating differentiation. Further, this gene is upregulated during sensory axon regeneration (Moskowitz et al., 1993). Thus, the promoter region of this gene should provide an excellent tool for studying these important processes. In addition, the expression of this gene is misregulated in a number of cancers (Katsetos et al., 2001), indicating that understanding its normal regulation could elucidate underlying causes of these malignancies. To provide the necessary background information for understanding its normal and abnormal regulation, we identified, cloned and sequenced ∼5 kb of the 5′-flanking region of the rat Class III neuron-specific β-tubulin (herein referred to as βIII-tubulin) gene. The rat gene was chosen because this animal is an important model for neurodegenerative disease, neural transplants and behavioral assessments of recovery of function (http://www.nih.gov/science/models/rat).

Section snippets

Construction of recombinant plasmids

The 350 bp-long PCR fragment BT-UTR containing the first 293 bp of the 5′-coding region and 57 bp of the 5′-UTR of βIII-tubulin was cloned into the pAMP1 vector (Life Technologies; pAMP1/BT-UTR plasmid). To analyse the 5′ genomic sequence of βIII-tubulin, a 6 kb-long DNA fragment was obtained from a positive P1 clone digested with BamHI and subsequently cloned into the pZero vector (InVitrogen; pZero/BTIII-6.0 plasmid).

For CAT assay studies, three βIII-tubulin promoter constructs were generated

Isolation of the 5′ βIII-tubulin coding region and 5′-UTR

The 5′-coding region and the 5′-UTR of the βIII-tubulin gene were isolated first to design probes that would not hybridize with other highly homologous classes of β-tubulins (Cleveland and Sullivan, 1985, Sullivan and Cleveland, 1986, Sullivan, 1988). A rat βIII-tubulin cDNA clone (isolated in the laboratory of Dr. Anthony Frankfurter, University of Virginia), which lacked the first 210 nucleotides of the coding region and the 5′-UTR, provided primer sequence for the RACE procedure. We obtained

Acknowledgements

This work was supported by NIH Grants NS23158 (S.A.M.) and NS41391 (A.E.C.).

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