Hypoxia-inducible factor 1-mediated human GATA1 induction promotes erythroid differentiation under hypoxic conditions.
Bottom Line: Enforced HIF1α expression increased GATA1 expression, while HIF1α knockdown by RNA interference decreased GATA1 expression.These results demonstrate that the up-regulation of GATA1 during hypoxia is directly mediated by HIF1.The mRNA expression of some erythroid differentiation markers was increased under hypoxic conditions, but decreased with RNA interference of HIF1α or GATA1.Flow cytometry analysis also indicated that hypoxia, desferrioxamine or CoCl(2) induced expression of erythroid surface markers CD71 and CD235a, while expression repression of HIF1α or GATA1 by RNA interference led to a decreased expression of CD235a.
Affiliation: National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.Show MeSH
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Mentions: To determine whether HIF1 involves in the hypoxia-induced expression of GATA1, we constructed a HIF1α expression plasmid, pcDNA6V5HisB/HIF1α (pHIF1α), and a plasmid expressing a HIF1α-specific interference RNA sequence, psilencer 2.1/U6-HIF1α-RNAi (pSiHIF1α). These plasmids or their corresponding empty vectors were, respectively, transfected into K562 cells. The mRNA levels of the HIF1α gene (HIF1A) and GATA1 were determined by real-time PCR, and their protein levels were determined by Western blotting. As expected, there was a dramatic rise in HIF1A mRNA and protein in K562 cells transfected with pHIF1α, and this HIF1α over-expression induced expression of GATA1 mRNA and protein (Fig. 2A and B). In contrast, there was a sharp decrease in HIF1A mRNA and protein in K562 cells transfected with pSiHIF1α, accompanied by a significant decrease in GATA1 mRNA and protein levels (Fig. 2C and D). Furthermore, K562 cells were also transfected with siRNA that specifically targeted HIF1A (siHIF1α). Comparing with control siRNA (siCON), RNA interference-mediated knockdown of HIF1α resulted in a decreased expression of GATA1 (Fig. 2E and F). Over-expression of HIF1α increased GATA1 expression, while knockdown of HIF1α decreased GATA1 expression. Moreover, transfection of K562 cells with the dominant-negative form of HIF1α (pDN) that competes with endogenous HIF1α for dimerization with HIF1β but forms an inactive transcription heterodimer, resulted in a significant decrease of GATA1 expression under hypoxic conditions (Fig. 2G and H). These results established that HIF1 regulates the expression of GATA1 under hypoxia.
Affiliation: National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.