Lllii

I Basal transcription (Fed)

□ Induced transcription (-His)

WTCAGGCATGATGAAACTTCCCGCACGCGTTACAGGAGCC

NSRE-1

NSRE-2

Fig. 8.2. Single nucleotide mutagenesis to define the boundaries of the NSRE-1 and NSRE-2 regulatory sites. Mutagenesis and transient expression were carried out as described by Barbosa-Tessmann et al. (2000). Nucleotides -173 to +51 of the human AS gene were linked to the human growth hormone gene (GH) as a reporter and transcription was determined by analysing the GH mRNA content relative to that for the co-transfection control, which was the lacZ gene driven by the cytomegalovirus promoter. The cells were incubated for 18 h in either complete minimal essential medium (MEM) for the data labelled 'Fed' or in MEM lacking histidine (labelled '-His'). For the bars without a standard deviation symbol, the data represent a single analysis and only one nucleotide substitution was tested, purine for purine, pyrimidine for pyrimidine. For the sites with standard deviation bars, the results are representative of three independent experiments, and the wild-type nucleotide was changed to at least two of the three other nucleotides. The latter sites were tested more extensively to define better the boundaries of the NSRE-1- and NSRE-2-binding sites.

As described above, the human CHOP gene is activated by the AAR and ERSR pathways through two completely independent sets of cis-acting elements within the CHOP promoter region, the ERSE-1 at -93 to -75 (Yoshida et al., 2000) and the AARE at -302 to -310 (Bruhat et al., 2000). As just described, the human AS gene is also transcriptionally activated in response to the AAR and ERSR pathways, but, in contrast to CHOP, this activation occurs through the same set of cis-acting elements that function together to make up the NSRU (Barbosa-Tessmann et al, 2000). Interestingly, the NSRE-1 has a high degree of sequence identity with the AARE within the CHOP promoter, differing by only two nucleotides (Fig. 8.3). Although the AARE sequence in the CHOP promoter occurs on the opposite strand to that in the AS promoter, these sequences function as enhancer elements in that activation ofthe genes is observed regardless of orientation (Barbosa-Tessmann et al., 2000). Beyond the two-nucleotide difference in sequence between the CHOP AARE and the AS NSRE-1, the major difference between the promoters that results in the mechanistic difference in response to the ERSR pathway appears to be the presence of the second cis-acting element, NSRE-2 (Barbosa-Tessmann et al., 2000), which the CHOP promoter lacks. Given the similarity between the AS NSRE-1 sequence and the AARE in the CHOP promoter, one might speculate that elimination of the AS NSRE-2 sequence would block activation of the AS gene by the ERSR pathway, but permit retention of activation by the AAR pathway. However, mutagenesis of only the NSRE-2 sequence results in complete loss of responsiveness to both nutrient-regulated pathways (Barbosa-Tessmann et al., 2000). For reasons unknown, the presence of the NSRE-1-like AARE sequence in the CHOP promoter is sufficient to permit transcriptional induction via the AAR pathway, whereas the closely related NSRE-1 sequence is not. Interestingly, insertion of the AS NSRE-2 sequence into the human CHOP promoter, 11 nucleotides downstream from the CHOP AARE, conveys responsiveness to the ERSR pathway (Bruhat et al, 2002).

The CHOP AARE and the AS NSRE-1 also appear to bind different transcription factors. As described above, the CHOP binds both ATF-2 and C/EBPb in vitro but, in vivo, only ATF-2 appears to be functionally associated with this sequence (Bruhat et al, 2000). In contrast, the AS NSRE-1 sequence does not bind ATF-2 in vitro, but does bind C/EBPb (Siu et al, 2001). Furthermore, overexpression ofC/EBPb up-regulates both basal and induced transcription through the NSRE-1 sequence (Siu et al., 2000). The significance of the differences in transcription factor binding at the CHOP AARE and the AS NSRE-1 is unknown, but it is suggested that there is heterogeneity in the upstream signalling steps in a single AAR pathway or, possibly, multiple AAR pathways. Future studies contrasting these two genes and their transcription control by amino acids will be interesting and informative.

Summary and Future Research Opportunities

The investigation of nutrient control in mammalian cells, especially by amino acids, is still in its infancy. The full complement of target genes that respond to protein/amino acid deprivation is not yet known, but should be delineated in the near future by DNA microchip and array technology. Of course, as is already clear, the list of genes

CHOP

nt -310

5'-ATTGCATCA-3 3'-TAACGTAGT-5'

nt -302

Fig. 8.3. Comparison of the sequences for the AS NSRE-1 and the CHOP AARE regulatory sites. The cis-elements that mediate the AAR in the human AS and CHOP promoters are shown. The double-stranded sequences and the corresponding nucleotide numbers relative to the transcription start sites are illustrated. Note that the two elements are orientated on opposite strands.

that are transcriptionally activated and the list of mRNA species that are translationally controlled will differ, possibly significantly. The mechanisms responsible for these transcriptional and trans-lational events also remain to be elucidated more fully. Some of the steps are beginning to emerge and a few of the proteins required have been identified but, from the presentation above contrasting CHOP and AS, it is clear that many more proteins are involved and must still be identified. Finally, there are many questions unanswered regarding the initial sensors of amino acid deprivation and the subsequent signal transduction pathways that are activated. The investigation and characterization of these processes in normal homeostasis and in disease represents an interesting and important study in molecular nutrition.

Acknowledgements

This work was supported by grants to M.S.K. from the National Institutes ofHealth (DK-52064, DK-59315).

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