Roles of coactivators in hypoxic induction of the erythropoietin gene.
BACKGROUND
Hypoxia-inducible expression of the erythropoietin (EPO) gene is mediated principally by hypoxia-inducible factor 2alpha (HIF-2alpha) in Hep3B cells under physiologic conditions. How/whether p300/CBP and the members of p160 coactivator family potentiate hypoxic induction of endogenous EPO and other HIF-2alpha and hypoxia-inducible factor 1alpha (HIF-1alpha) target genes remains unclear.


RESULTS
We demonstrate, using chromatin immunoprecipitation (ChIP) analysis, that the histone acetyl transferase (HAT) coactivators p300, SRC-1 and SRC-3 are recruited to the 3' enhancer of the EPO gene upon hypoxic stimulation, and that each associates with the enhancer in a periodic fashion. Hypoxia induced acetylation of the EPO gene 5' promoter at histone 4 and lysine 23 of histone 3. Knocking down SRC-3, but not SRC-1 or SRC-2, using short interfering RNAs (siRNAs), reduced EPO transcriptional activity. Knocking down p300 resulted in dramatic down-regulation of hypoxic stimulation of EPO gene transcription, negated recruitment of RNA polymerase II to the gene's promoter, and eliminated hypoxia-stimulated acetylation at the promoter and recruitments of SRC-1 and SRC-3 to the enhancer. The inhibitory effects of knocking down p300 and the chromatin remodeling coactivator, Brm/Brg-1, on EPO transcription were additive, suggesting that p300 and Brm/Brg-1 act independently. p300 was also required for hypoxia induced transcription of the HIF-1alpha target gene, VEGF, but was dispensable for induction of two other HIF-1alpha target genes, PGK and LDHA. Knocking down CBP, a homolog of p300, augmented hypoxic induction of VEGF, LDHA and PGK. Different HIF target genes also exhibited different requirements for members of the p160 coactivator family.


CONCLUSIONS
p300 plays a central coactivator role in hypoxic induction of EPO. The coactivators exhibit different specificities for different HIF target genes and each can behave differently in transcriptional regulation of different target genes mediated by the same transcription factor.
Wang
Feng
F
Department of Pathology and Laboratory Medicine, and Jonsson Comprehensive Cancer Center, University of California Los Angeles, Los Angeles, California, USA.
Zhang
Ruixue
R
Wu
Xiaomeng
X
Hankinson
Oliver
O
D002460:Cell Line; D004742:Enhancer Elements, Genetic; D004921:Erythropoietin / Q000235:genetics*; D051548:Histone Acetyltransferases / Q000378:metabolism; D006801:Humans; D000860:Hypoxia / Q000235:genetics*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics; D056921:Nuclear Receptor Coactivator 3 / Q000235:genetics; D011485:Protein Binding; D021381:Protein Transport; D015533:Transcriptional Activation; D050880:p300-CBP Transcription Factors / Q000235:genetics*
D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D004921:Erythropoietin; D051548:Histone Acetyltransferases; C538866:NCOA3 protein, human; D056921:Nuclear Receptor Coactivator 3; D050880:p300-CBP Transcription Factors