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High-Throughput siRNA Screening to Reveal GATA-2 Upstream Transcriptional Mechanisms in Hematopoietic Cells.

Saito Y, Fujiwara T, Ohashi K, Okitsu Y, Fukuhara N, Onishi Y, Ishizawa K, Harigae H - PLoS ONE (2015)

Bottom Line: GATA-2-dependent disease processes have been extensively analyzed; however, the transcriptional mechanisms upstream of GATA-2 remain less understood.Subsequently, we screened 995 transcription factor genes and revealed that CITED2 acts as a GATA-2 activator in human hematopoietic cells.These results provide novel insights into and further identify the regulatory mechanism of GATA-2.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan.

ABSTRACT
Hematopoietic stem cells can self-renew and differentiate into all blood cell types. The transcription factor GATA-2 is expressed in both hematopoietic stem and progenitor cells and is essential for cell proliferation, survival, and differentiation. Recently, evidence from studies of aplastic anemia, MonoMAC syndrome, and lung cancer has demonstrated a mechanistic link between GATA-2 and human pathophysiology. GATA-2-dependent disease processes have been extensively analyzed; however, the transcriptional mechanisms upstream of GATA-2 remain less understood. Here, we conducted high-throughput small-interfering-RNA (siRNA) library screening and showed that YN-1, a human erythroleukemia cell line, expressed high levels of GATA-2 following the activation of the hematopoietic-specific 1S promoter. As transient luciferase reporter assay in YN-1 cells revealed the highest promoter activity in the 1S promoter fused with GATA-2 intronic enhancer (+9.9 kb/1S); therefore, we established a cell line capable of stably expressing +9.9 kb/1S-Luciferase. Subsequently, we screened 995 transcription factor genes and revealed that CITED2 acts as a GATA-2 activator in human hematopoietic cells. These results provide novel insights into and further identify the regulatory mechanism of GATA-2.

No MeSH data available.


Related in: MedlinePlus

CBP/p300-Interacting Transactivator with Glu/Asp-Rich C-Terminal Domain 2 (CITED2) regulates GATA-2 expression in K562 cells.(A) CITED2 was knocked down with a pool of four synthetic small interfering RNAs (siRNAs) (Dharmacon) in K562 cells. The knockdown process was repeated twice at 24-h intervals. The cells were harvested and assayed 48 h after the first transfection. (A) CITED2 mRNA was significantly repressed after siRNA transfection (mean ± standard error [SE], n = 3, p < 0.001), and this result was confirmed by western blotting. Actin was used as a loading control. (B,C) GATA-2 mRNA expression and GATA-2 1S and 1G transcripts were significantly decreased (mean ± SE, n = 3, p < 0.001). * p<0.05.
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pone.0137079.g006: CBP/p300-Interacting Transactivator with Glu/Asp-Rich C-Terminal Domain 2 (CITED2) regulates GATA-2 expression in K562 cells.(A) CITED2 was knocked down with a pool of four synthetic small interfering RNAs (siRNAs) (Dharmacon) in K562 cells. The knockdown process was repeated twice at 24-h intervals. The cells were harvested and assayed 48 h after the first transfection. (A) CITED2 mRNA was significantly repressed after siRNA transfection (mean ± standard error [SE], n = 3, p < 0.001), and this result was confirmed by western blotting. Actin was used as a loading control. (B,C) GATA-2 mRNA expression and GATA-2 1S and 1G transcripts were significantly decreased (mean ± SE, n = 3, p < 0.001). * p<0.05.

Mentions: We next knocked down CITED2 using a pool of four synthetic siRNAs based on YN-1 cells expressing +9.9 kb/1S-Luc2. As shown in Fig 5A, the expression of CITED2 genes was significantly repressed (by 12%) after siRNA transfection, a result that was confirmed by western blot analysis. Accordingly, we confirmed that GATA-2 mRNA expression, GATA-2 1S transcript, and luciferase activity significantly decreased (by 63%, 55%, and 74%, respectively), under these conditions (Fig 5B). We further established two CITED2-knockout YN-1 cell clones based on CRISPR/Cas9 technique and confirmed these clones by western blotting (Fig 5C). Quantitative RT-PCR analysis confirmed significant downregulation of GATA-2 mRNA and 1S transcript (Fig 5D). To reveal how CITED2 contributes to the GATA-2 expression in general, we also performed siRNA-mediated CITED2 knockdown based on K562 cells, which exhibited relatively lower 1S transcript level than YN-1 cells (Fig 1C). As shown in Fig 6, CITED2 knockdown in K562 cells resulted in significant downregulation of total GATA-2 mRNA as well as both 1S and 1G transcript levels, suggesting that CITED2 might not specifically activate +9.9 kb/1S promoter. Furthermore, to test if CITED2 would specifically affect +9.9 kb enhancer activity, luciferase analysis was conducted based on YN-1 cells and CITED2-knockout YN-1 cell clone (#1), demonstrating that CITED2 depletion significantly reduced both 1S alone and +9.9kb/1S-luciferase activity (S2 Fig). Thus, CITED2 might also regulate GATA-2 expression by impacting upon promoter region. Whereas our results indicated that CITED2 is an upstream regulator of GATA-2 in hematopoietic cells, further analysis would be required to reveal the molecular mechanism by which CITED2 contributes to the GATA-2 expression.


High-Throughput siRNA Screening to Reveal GATA-2 Upstream Transcriptional Mechanisms in Hematopoietic Cells.

Saito Y, Fujiwara T, Ohashi K, Okitsu Y, Fukuhara N, Onishi Y, Ishizawa K, Harigae H - PLoS ONE (2015)

CBP/p300-Interacting Transactivator with Glu/Asp-Rich C-Terminal Domain 2 (CITED2) regulates GATA-2 expression in K562 cells.(A) CITED2 was knocked down with a pool of four synthetic small interfering RNAs (siRNAs) (Dharmacon) in K562 cells. The knockdown process was repeated twice at 24-h intervals. The cells were harvested and assayed 48 h after the first transfection. (A) CITED2 mRNA was significantly repressed after siRNA transfection (mean ± standard error [SE], n = 3, p < 0.001), and this result was confirmed by western blotting. Actin was used as a loading control. (B,C) GATA-2 mRNA expression and GATA-2 1S and 1G transcripts were significantly decreased (mean ± SE, n = 3, p < 0.001). * p<0.05.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4556642&req=5

pone.0137079.g006: CBP/p300-Interacting Transactivator with Glu/Asp-Rich C-Terminal Domain 2 (CITED2) regulates GATA-2 expression in K562 cells.(A) CITED2 was knocked down with a pool of four synthetic small interfering RNAs (siRNAs) (Dharmacon) in K562 cells. The knockdown process was repeated twice at 24-h intervals. The cells were harvested and assayed 48 h after the first transfection. (A) CITED2 mRNA was significantly repressed after siRNA transfection (mean ± standard error [SE], n = 3, p < 0.001), and this result was confirmed by western blotting. Actin was used as a loading control. (B,C) GATA-2 mRNA expression and GATA-2 1S and 1G transcripts were significantly decreased (mean ± SE, n = 3, p < 0.001). * p<0.05.
Mentions: We next knocked down CITED2 using a pool of four synthetic siRNAs based on YN-1 cells expressing +9.9 kb/1S-Luc2. As shown in Fig 5A, the expression of CITED2 genes was significantly repressed (by 12%) after siRNA transfection, a result that was confirmed by western blot analysis. Accordingly, we confirmed that GATA-2 mRNA expression, GATA-2 1S transcript, and luciferase activity significantly decreased (by 63%, 55%, and 74%, respectively), under these conditions (Fig 5B). We further established two CITED2-knockout YN-1 cell clones based on CRISPR/Cas9 technique and confirmed these clones by western blotting (Fig 5C). Quantitative RT-PCR analysis confirmed significant downregulation of GATA-2 mRNA and 1S transcript (Fig 5D). To reveal how CITED2 contributes to the GATA-2 expression in general, we also performed siRNA-mediated CITED2 knockdown based on K562 cells, which exhibited relatively lower 1S transcript level than YN-1 cells (Fig 1C). As shown in Fig 6, CITED2 knockdown in K562 cells resulted in significant downregulation of total GATA-2 mRNA as well as both 1S and 1G transcript levels, suggesting that CITED2 might not specifically activate +9.9 kb/1S promoter. Furthermore, to test if CITED2 would specifically affect +9.9 kb enhancer activity, luciferase analysis was conducted based on YN-1 cells and CITED2-knockout YN-1 cell clone (#1), demonstrating that CITED2 depletion significantly reduced both 1S alone and +9.9kb/1S-luciferase activity (S2 Fig). Thus, CITED2 might also regulate GATA-2 expression by impacting upon promoter region. Whereas our results indicated that CITED2 is an upstream regulator of GATA-2 in hematopoietic cells, further analysis would be required to reveal the molecular mechanism by which CITED2 contributes to the GATA-2 expression.

Bottom Line: GATA-2-dependent disease processes have been extensively analyzed; however, the transcriptional mechanisms upstream of GATA-2 remain less understood.Subsequently, we screened 995 transcription factor genes and revealed that CITED2 acts as a GATA-2 activator in human hematopoietic cells.These results provide novel insights into and further identify the regulatory mechanism of GATA-2.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan.

ABSTRACT
Hematopoietic stem cells can self-renew and differentiate into all blood cell types. The transcription factor GATA-2 is expressed in both hematopoietic stem and progenitor cells and is essential for cell proliferation, survival, and differentiation. Recently, evidence from studies of aplastic anemia, MonoMAC syndrome, and lung cancer has demonstrated a mechanistic link between GATA-2 and human pathophysiology. GATA-2-dependent disease processes have been extensively analyzed; however, the transcriptional mechanisms upstream of GATA-2 remain less understood. Here, we conducted high-throughput small-interfering-RNA (siRNA) library screening and showed that YN-1, a human erythroleukemia cell line, expressed high levels of GATA-2 following the activation of the hematopoietic-specific 1S promoter. As transient luciferase reporter assay in YN-1 cells revealed the highest promoter activity in the 1S promoter fused with GATA-2 intronic enhancer (+9.9 kb/1S); therefore, we established a cell line capable of stably expressing +9.9 kb/1S-Luciferase. Subsequently, we screened 995 transcription factor genes and revealed that CITED2 acts as a GATA-2 activator in human hematopoietic cells. These results provide novel insights into and further identify the regulatory mechanism of GATA-2.

No MeSH data available.


Related in: MedlinePlus