Amino acid deprivation and endoplasmic reticulum stress induce expression of multiple activating transcription factor-3 mRNA species that, when overexpressed in HepG2 cells, modulate transcription by the human asparagine synthetase promoter.
Transcription from the ASNS (asparagine synthetase) gene is increased in response to either amino acid (amino acid response) or glucose (endoplasmic reticulum stress response) deprivation. These two independent pathways converge on the same set of genomic cis-elements within the ASNS promoter, referred to as nutrient-sensing response element-1 and -2. Chromatin immunoprecipitation analysis provides the first in vivo evidence for activating transcription factor (ATF)-3 binding to the proximal ASNS promoter containing the nutrient-sensing response element-1 sequence. Overexpression of the full-length ATF3 protein caused a concentration-dependent biphasic response in ASNS promoter-driven transcription. Both amino acid limitation and activation of the endoplasmic reticulum stress response by glucose deprivation caused an increase in ATF3 mRNA content. However, reverse transcriptase-PCR analysis revealed that the increase in the ATF3 mRNA species detected by Northern analysis actually encoded both full-length ATF3 and two predicted truncated ATF3 isoforms (ATF3deltaZip2c and ATF3deltaZip3). Based on sequence analysis, one of the predicted truncated proteins (ATF3deltaZip3) is likely incapable of binding DNA; and yet, exogenous expression of the cDNA enhanced starvation-induced or ATF4-activated ASNS transcription, possibly by sequestering corepressor proteins. Collectively, the results provide evidence for a potential role of multiple predicted ATF3 isoforms in the transcriptional regulation of the ASNS gene in response to nutrient deprivation.
Pan
YuanXiang
Y
Department of Biochemistry and Molecular Biology, Centers for Mammalian Genetics and Nutritional Sciences, University of Florida College of Medicine, Gainesville, Florida 32610-0245, USA.
Chen
Hong
H
Siu
Fai
F
Kilberg
Michael S
MS
D051700:Activating Transcription Factor 3; D017398:Alternative Splicing; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001217:Aspartate-Ammonia Ligase / Q000096:biosynthesis* / Q000235:genetics*; D015152:Blotting, Northern; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005786:Gene Expression Regulation; D005947:Glucose / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D051700:Activating Transcription Factor 3; D000596:Amino Acids; C497108:Atf3 protein, rat; D002843:Chromatin; D018076:DNA, Complementary; D020033:Protein Isoforms; D012333:RNA, Messenger; D014157:Transcription Factors; D001217:Aspartate-Ammonia Ligase; D005947:Glucose