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Hypoxia-inducible factor 1-mediated human GATA1 induction promotes erythroid differentiation under hypoxic conditions.

Zhang FL, Shen GM, Liu XL, Wang F, Zhao YZ, Zhang JW - J. Cell. Mol. Med. (2012)

Bottom Line: Enforced HIF1α expression increased GATA1 expression, while HIF1α knockdown by RNA interference decreased GATA1 expression.Chromatin immunoprecipitation (ChIP)-PCR showed that the putative HRE was recognized and bound by HIF1 in vivo.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.

View Article: PubMed Central - PubMed

Affiliation: National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

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Regulation of GATA1 gene expression by HIF1. (A) Real-time PCR analysis of HIF1A and GATA1 mRNA levels in K562 cells transfected with the HIF1α expression plasmid (pHIF1α) or empty plasmid (pcDNA) and subsequently incubated under hypoxia for 24 hrs. (B) Western blotting assay of HIF1α and GATA1 protein levels in K562 cells transfected with the HIF1α expression plasmid or corresponding empty vector and subsequently incubated under hypoxia for 48 hrs. (C) Real-time PCR analysis of HIF1A and GATA1 mRNA levels in K562 cells that were transfected with the HIF1α interference vector (pSiHIF1α) or psilencer control (pSiCON) and subsequently incubated under hypoxia for 24 hrs. (D) Western blotting assay of HIF1α and GATA1 protein levels in K562 cells transfected with the HIF1α interference plasmid or the corresponding empty vector and subsequently incubated under hypoxia for 48 hrs. (E) Real-time PCR analysis of HIF1A and GATA1 mRNA levels in K562 cells transfected with siRNA targeting HIF1A (siHIF1α) or control siRNA (siCON). After 24 hrs of transfection, the transfection medium was replaced with complete medium and the cells were cultured under normoxia (the left two columns) or hypoxia (the right two columns) for an additional 24 hrs. (F) Western blotting assay of HIF1α and GATA1 protein levels in K562 cells transfected with siRNA targeting HIF1A or control siRNA. After 24 hrs of transfection, the transfection medium was replaced with complete medium and the cells were cultured under hypoxia for an additional 24 hrs. The numbers in brackets indicate the mean fluorescent intensities. (G) Real-time PCR analysis of HIF1A and GATA1 mRNA levels in K562 cells transfected with the plasmid. pcDNA or pDN. After 6 hrs of transfection, the transfection medium was replaced with complete medium and the cells were cultured under normoxia (the left two columns) or hypoxia (the right two columns) for 36 hrs. pDN that consists of amino acids 28 through 390 of human HIF1α, contains the basic domain deletion that affects DNA binding, and the carboxyl-terminal truncation that affects transactivation. (H) Western blotting assay of GATA1 protein in K562 cells transfected with the plasmid. pcDNA or pDN. After 6 hrs of transfection, the transfection medium was replaced with complete medium and the cells were cultured under normoxia or hypoxia for 36 hrs. *P < 0.01.
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fig02: Regulation of GATA1 gene expression by HIF1. (A) Real-time PCR analysis of HIF1A and GATA1 mRNA levels in K562 cells transfected with the HIF1α expression plasmid (pHIF1α) or empty plasmid (pcDNA) and subsequently incubated under hypoxia for 24 hrs. (B) Western blotting assay of HIF1α and GATA1 protein levels in K562 cells transfected with the HIF1α expression plasmid or corresponding empty vector and subsequently incubated under hypoxia for 48 hrs. (C) Real-time PCR analysis of HIF1A and GATA1 mRNA levels in K562 cells that were transfected with the HIF1α interference vector (pSiHIF1α) or psilencer control (pSiCON) and subsequently incubated under hypoxia for 24 hrs. (D) Western blotting assay of HIF1α and GATA1 protein levels in K562 cells transfected with the HIF1α interference plasmid or the corresponding empty vector and subsequently incubated under hypoxia for 48 hrs. (E) Real-time PCR analysis of HIF1A and GATA1 mRNA levels in K562 cells transfected with siRNA targeting HIF1A (siHIF1α) or control siRNA (siCON). After 24 hrs of transfection, the transfection medium was replaced with complete medium and the cells were cultured under normoxia (the left two columns) or hypoxia (the right two columns) for an additional 24 hrs. (F) Western blotting assay of HIF1α and GATA1 protein levels in K562 cells transfected with siRNA targeting HIF1A or control siRNA. After 24 hrs of transfection, the transfection medium was replaced with complete medium and the cells were cultured under hypoxia for an additional 24 hrs. The numbers in brackets indicate the mean fluorescent intensities. (G) Real-time PCR analysis of HIF1A and GATA1 mRNA levels in K562 cells transfected with the plasmid. pcDNA or pDN. After 6 hrs of transfection, the transfection medium was replaced with complete medium and the cells were cultured under normoxia (the left two columns) or hypoxia (the right two columns) for 36 hrs. pDN that consists of amino acids 28 through 390 of human HIF1α, contains the basic domain deletion that affects DNA binding, and the carboxyl-terminal truncation that affects transactivation. (H) Western blotting assay of GATA1 protein in K562 cells transfected with the plasmid. pcDNA or pDN. After 6 hrs of transfection, the transfection medium was replaced with complete medium and the cells were cultured under normoxia or hypoxia for 36 hrs. *P < 0.01.

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.


Hypoxia-inducible factor 1-mediated human GATA1 induction promotes erythroid differentiation under hypoxic conditions.

Zhang FL, Shen GM, Liu XL, Wang F, Zhao YZ, Zhang JW - J. Cell. Mol. Med. (2012)

Regulation of GATA1 gene expression by HIF1. (A) Real-time PCR analysis of HIF1A and GATA1 mRNA levels in K562 cells transfected with the HIF1α expression plasmid (pHIF1α) or empty plasmid (pcDNA) and subsequently incubated under hypoxia for 24 hrs. (B) Western blotting assay of HIF1α and GATA1 protein levels in K562 cells transfected with the HIF1α expression plasmid or corresponding empty vector and subsequently incubated under hypoxia for 48 hrs. (C) Real-time PCR analysis of HIF1A and GATA1 mRNA levels in K562 cells that were transfected with the HIF1α interference vector (pSiHIF1α) or psilencer control (pSiCON) and subsequently incubated under hypoxia for 24 hrs. (D) Western blotting assay of HIF1α and GATA1 protein levels in K562 cells transfected with the HIF1α interference plasmid or the corresponding empty vector and subsequently incubated under hypoxia for 48 hrs. (E) Real-time PCR analysis of HIF1A and GATA1 mRNA levels in K562 cells transfected with siRNA targeting HIF1A (siHIF1α) or control siRNA (siCON). After 24 hrs of transfection, the transfection medium was replaced with complete medium and the cells were cultured under normoxia (the left two columns) or hypoxia (the right two columns) for an additional 24 hrs. (F) Western blotting assay of HIF1α and GATA1 protein levels in K562 cells transfected with siRNA targeting HIF1A or control siRNA. After 24 hrs of transfection, the transfection medium was replaced with complete medium and the cells were cultured under hypoxia for an additional 24 hrs. The numbers in brackets indicate the mean fluorescent intensities. (G) Real-time PCR analysis of HIF1A and GATA1 mRNA levels in K562 cells transfected with the plasmid. pcDNA or pDN. After 6 hrs of transfection, the transfection medium was replaced with complete medium and the cells were cultured under normoxia (the left two columns) or hypoxia (the right two columns) for 36 hrs. pDN that consists of amino acids 28 through 390 of human HIF1α, contains the basic domain deletion that affects DNA binding, and the carboxyl-terminal truncation that affects transactivation. (H) Western blotting assay of GATA1 protein in K562 cells transfected with the plasmid. pcDNA or pDN. After 6 hrs of transfection, the transfection medium was replaced with complete medium and the cells were cultured under normoxia or hypoxia for 36 hrs. *P < 0.01.
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fig02: Regulation of GATA1 gene expression by HIF1. (A) Real-time PCR analysis of HIF1A and GATA1 mRNA levels in K562 cells transfected with the HIF1α expression plasmid (pHIF1α) or empty plasmid (pcDNA) and subsequently incubated under hypoxia for 24 hrs. (B) Western blotting assay of HIF1α and GATA1 protein levels in K562 cells transfected with the HIF1α expression plasmid or corresponding empty vector and subsequently incubated under hypoxia for 48 hrs. (C) Real-time PCR analysis of HIF1A and GATA1 mRNA levels in K562 cells that were transfected with the HIF1α interference vector (pSiHIF1α) or psilencer control (pSiCON) and subsequently incubated under hypoxia for 24 hrs. (D) Western blotting assay of HIF1α and GATA1 protein levels in K562 cells transfected with the HIF1α interference plasmid or the corresponding empty vector and subsequently incubated under hypoxia for 48 hrs. (E) Real-time PCR analysis of HIF1A and GATA1 mRNA levels in K562 cells transfected with siRNA targeting HIF1A (siHIF1α) or control siRNA (siCON). After 24 hrs of transfection, the transfection medium was replaced with complete medium and the cells were cultured under normoxia (the left two columns) or hypoxia (the right two columns) for an additional 24 hrs. (F) Western blotting assay of HIF1α and GATA1 protein levels in K562 cells transfected with siRNA targeting HIF1A or control siRNA. After 24 hrs of transfection, the transfection medium was replaced with complete medium and the cells were cultured under hypoxia for an additional 24 hrs. The numbers in brackets indicate the mean fluorescent intensities. (G) Real-time PCR analysis of HIF1A and GATA1 mRNA levels in K562 cells transfected with the plasmid. pcDNA or pDN. After 6 hrs of transfection, the transfection medium was replaced with complete medium and the cells were cultured under normoxia (the left two columns) or hypoxia (the right two columns) for 36 hrs. pDN that consists of amino acids 28 through 390 of human HIF1α, contains the basic domain deletion that affects DNA binding, and the carboxyl-terminal truncation that affects transactivation. (H) Western blotting assay of GATA1 protein in K562 cells transfected with the plasmid. pcDNA or pDN. After 6 hrs of transfection, the transfection medium was replaced with complete medium and the cells were cultured under normoxia or hypoxia for 36 hrs. *P < 0.01.
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.

Bottom Line: Enforced HIF1α expression increased GATA1 expression, while HIF1α knockdown by RNA interference decreased GATA1 expression.Chromatin immunoprecipitation (ChIP)-PCR showed that the putative HRE was recognized and bound by HIF1 in vivo.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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus