As of this writing, nine genes are known to be stimulated transcriptionally by TonEBP: SMIT1, BGT1, TauT, AR, NTE, HSP70, AQP2, UT-A, and TNFa. Unpublished work from the authors' laboratory and others indicates that there are more genes regulated by TonEBP, that is, TonEBP target genes. Identification of all the TonEBP target genes is essential to understanding the biology of TonEBP and osmotic regulation. This section describes strategies used to identify novel TonEBP target genes.
The first step is to identify genes whose mRNA abundance is increased in response to hypertonicity. Cultured cells can be treated with hypertonic medium and induced mRNA can be searched using functional genomic analyses such as cDNA microarray analysis or targeted gene analysis using Northern analysis, RPA, or quantitative PCR. In the kidney, increased mRNA abundance in response to water deprivation is a good place to start. Likewise, in the brain, mRNA induction in response to systemic hyperna-tremia is an excellent clue. For those candidate genes identified in organs or tissues, it is desirable to find a cell culture model in which the candidate gene is induced by hypertonicity (an increase in effective osmolality) but not by hyperosmolality made by ineffective osmolytes such as urea or ethanol. This is because TonEBP is induced by hypertonicity but not by hyperosmolality per se. One should also be aware that switching to extreme hypertonicity, for example, from isotonic medium to 600 mOsm/kg or higher, results in cell death. However, most cells tolerate a switch to mild hypertonicity of up to 500 mOsm/kg, which can be made by the addition of 100 to 120 mM NaCl to isotonic medium. In addition, one should remember that the kinetics of mRNA induction varies: SMIT1 mRNA induces fast (within 6 h), whereas AR mRNA takes longer (more than 6 h).
Cellular evidence that TonEBP is involved in the induction of a candidate gene in response to hypertonicity can be obtained using cultured cells. In general, loss of function is more convincing. Effects of TonEBP knockdown using specific siRNA (see earlier discussion) on induction can be examined. Alternatively, cell lines derived from TonEBP-/- animals can be used. If cells can be transfected efficiently, effects of transient overexpression of TonEBP can be examined. If necessary, inhibitory effects due to expression of DN-TonEBP can be demonstrated as well. These experiments will provide a functional relationship between TonEBP and induction of a candidate gene in response to hypertonicity.
Direct action of TonEBP on the promoter of the candidate gene can be established in a variety of ways. As discussed previously, we doubt that ChIP will yield satisfactory results for TonEBP. Instead, in vivo footprinting analysis can be performed to detect in situ binding to the suspected TonEBP-binding sites in response to hypertonicity (Miyakawa et al., 1998). Finally, the promoter can be analyzed using a reporter gene. All of these techniques require definitive identification of the start of the gene. Start sites for transcription as annotated by NCBI should be examined carefully. If there are solid studies on definition of the transcription start site published, one can use the information. For the vast majority of genes, this information is lacking. Sometimes the NCBI annotation is misleading. For example, in the current annotation of the human SMIT1 gene (SLC5A3) in the NCBI, the first exon is not recognized: the second exon is annotated as the only transcriptional unit. Although we reported that the first intron is «25 kB (Mallee et al., 1997), this information is not recognized in the NCBI annotation. This illustrates that the transcription start site should be mapped to correctly identify the true promoter of a gene. We next describe primer extension analysis to map the transcription start site and construction of the promoter—reporter.
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