As shown in Fig. 8.1, dimethyl sulphate in vivo footprinting documented that the human AS promoter region immediately upstream of the major transcription start site contains six separate protein-binding sites (Barbosa-Tessmann et al., 2000). Ofthese, five have been implicated in nutrient control of the human AS gene: three GC boxes (GC-I, GC-II and GC-III) and two nutrient-sensing response elements (NSRE-1 and -2). All three GC boxes are required to maintain basal transcription and to obtain maximal activation of the AS gene by amino acid limitation
(Leung-Pineda and Kilberg, 2002). However, when functionally analysed individually, there is not complete redundancy among the three GC sequences and there is a difference in the degree of importance with regard to transcription (GC-III > GC-II > GC-I). In vitro, two of the GC sequences formed protein-DNA complexes (GC-II and GC-III) with either Sp1 or Sp3, but the absolute amount of these complexes and the total pool of either Sp1 or Sp3 protein did not increase following amino acid limitation. In vivo expression of Sp1 and Sp3 in Drosophila SL2 cells, which lack Sp proteins, increased AS promoter activity, but functional differences between the factors were observed (Leung-Pineda and Kilberg, 2002). Sp1 expression increased basal transcription from the AS promoter, but did not cause a further increase when SL2 cells were amino acid deprived. In contrast, Sp3 expression enhanced both the basal and the starvation-induced AS-driven transcription.
Two of the protein-binding sites identified by in vivo footprinting showed changes in protein protection in response to amino acid deprivation (Barbosa-Tessmann et al., 2000). These two sites,
originally labelled sites V and VI, have been renamed NSRE-1 and NSRE-2 (see Fig. 8.1). Single nucleotide mutagenesis throughout the entire region of these two sites has defined the boundaries of these two elements more clearly (Fig. 8.2). The NSRE-1 sequence (5'-TGATGAAAC-3') located from nucleotide -68 to -60 within the AS proximal promoter overlaps the sequence first identified by Guerrini et al. (1993) as having AARE activity. Given that the NSRE-1 sequence is necessary for induction of the AS gene following activation of not only the AAR pathway, but also the ERSR pathway (Barbosa-Tessmann et al., 2000), it is clear that this element is functionally more important than simply as an AARE. To reflect this broader nutrient-detecting capability, the term NSRE-1 was coined. When nuclear extracts from amino acid-starved HepG2 cells were tested by EMSA, increased amounts of protein-NSRE-1 complexes were detected (Barbosa-Tessmann et al., 2000). Furthermore, a second element 5'-GTTACA-3' (nucleotides -48 to -43), positioned 11 nucleotides downstream of the NSRE-1, was shown also to be absolutely required for induction ofthe AS gene by both amino acid and glucose starvation (Barbosa-Tessmann et al., 2000). Once again, single nucleo-tide mutagenesis has defined the boundaries ofthis site (Fig. 8.2), but the sequence does not correspond identically to any known transcription factor consensus sequence. Collectively, the promoter analysis documents that at least three separate ay-regulatory elements are minimally required for an optimal transcriptional response of the AS gene either to amino acid or glucose deprivation, one or more of the GC boxes, NSRE-1 and NSRE-2. The term nutrient-sensing response unit (NSRU) has been coined to describe the collective action of these elements.
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