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. 2006 Oct;136(10):2463-7.
doi: 10.1093/jn/136.10.2463.

VSports - Alignment of the transcription start site coincides with increased transcriptional activity from the human asparagine synthetase gene following amino acid deprivation of HepG2 cells

Hong Chen  1 , Michael S Kilberg
Affiliations

Alignment of the transcription start site coincides with increased transcriptional activity from the human asparagine synthetase gene following amino acid deprivation of HepG2 cells

Hong Chen et al. J Nutr. 2006 Oct.

VSports - Abstract

Transcription initiation sites of the asparagine synthetase gene were investigated in human hepatoma cells after amino acid limitation by incubation in amino acid-complete minimal essential medium or medium lacking histidine. Cells incubated in complete minimal essential medium had mRNA transcripts with starting positions spanning across the 69 nucleotides immediately upstream of a previously designated transcription start site (+1), whereas the majority of mRNA transcripts started at nucleotide (+1) in cells incubated in histidine-free medium. Similar results were obtained regardless of whether the analysis was by 5' rapid amplification of cDNA ends or a ribonuclease protection assay VSports手机版. Low ASNS mRNA expression in amino acid-complete medium was associated with the wide range of initiation sites, whereas preferred alignment of the general transcription machinery at nucleotide (+1), observed in the amino acid deprived condition, was associated with a concurrent increase in transcription activity. To our knowledge, these results are the first example in a mammalian cell of transcription start selection by nutrient availability. .

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Figures

Figure 1
Figure 1
Analysis of ASNS steady-state mRNA content (A) and gene transcription activity (B) in HepG2 hepatoma cells following histidine deprivation for 8 h. Values are means ± SEM, n = 3 independent experiments with each sample was measured in duplicate.
Figure 2
Figure 2
The 5′ RNA ligase-mediated rapid amplification of cDNA ends (5′ RACE) assay was used to analyze transcription start sites for the ASNS gene in HepG2 hepatoma cells after histidine deprivation for 12 h. A representative gel image of a RACE analysis is labeled to show the broad upper (MEM) and sharper lower (MEM –His) bands that were analyzed by densitometry to obtain the percentage values given in the text.
Figure 3
Figure 3
Sequences of ASNS 5′ RACE products randomly cloned from total RNA isolated from HepG2 cells lacking histidine for 12 h. Sequences representing 1–3 clones are shown by a black line, 4–10 clones by a gray box, and ≥10 clones by a black box. The number of clones is shown to the left of each sequence. The locations of NSRE-1, NSRE-2, the TATA box, and the major +1 transcription start site identified by Greco et al. (15) are shown in the diagram.
Figure 4
Figure 4
Sequencing of 5′ RACE products prepared from gel-purified bands representing the 2 primary ASNS mRNA populations shown in Figure 2. The locations of NSRE-1, NSRE-2, the TATA box, and the major +1 transcription start site identified by Greco et al. (15) are shown in the diagram. Sequences representing 1–3 clones are shown by a black line, whereas the lone sequence showing >3 clones is shown by a black box. The number of clones is shown to the left of each sequence.
Figure 5
Figure 5
Mapping transcription start sites for the human ASNS gene by a ribonuclease protection assay (RPA). Panel A shows a representative gel and the areas of the lanes that were quantified for panel B are indicated by the bracket (upstream mRNA) and the arrow (+1). The control lane, C, shows the completeness of the ribonuclease digestion with yeast RNA; and the lane labeled P shows the undigested probe. Panel B represents the quantification of band intensity from the gel shown in panel A. The open portion of the bars represents the combined intensity of all the bands in the upstream mRNA (bracket in panel A) and the closed portion of the bars represents quantification of the band (arrow in panel A) that corresponds to mRNA starting at the +1 nucleotide.
See this image and copyright information in PMC

References

    1. Kilberg MS, Pan YX, Chen H, Leung-Pineda V. Nutritional control of gene expression: how mammalian cells respond to amino acid limitation. Annu Rev Nutr. 2005;25:59–85. - PMC - PubMed
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    1. Barbosa-Tessmann IP, Pineda VL, Nick HS, Schuster SM, Kilberg MS. Transcriptional regulation of the human asparagine synthetase gene by carbohydrate availability. Biochem J. 1999;339:151–8. - PMC - PubMed

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