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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

High-throughput small interfering RNA (siRNA) screening to identify regulators of GATA-2 transcription.(A) Schematic representation of the high-throughput siRNA screening. YN-1 cells with stable +9.9/1S-Luc2 expression were selected using puromycin. Subsequently, the cells were transfected twice at 24-h intervals. Luciferase activity was assessed 48 h after the first transfection. We used an siPerfect siRNA library (RNAi) that targeted 995 transcription factor genes. (B) We used Lamin A siRNA to validate the efficiency of the transfection protocol (mean ± standard error, n = 3, p < 0.001). * p<0.05.
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pone.0137079.g004: High-throughput small interfering RNA (siRNA) screening to identify regulators of GATA-2 transcription.(A) Schematic representation of the high-throughput siRNA screening. YN-1 cells with stable +9.9/1S-Luc2 expression were selected using puromycin. Subsequently, the cells were transfected twice at 24-h intervals. Luciferase activity was assessed 48 h after the first transfection. We used an siPerfect siRNA library (RNAi) that targeted 995 transcription factor genes. (B) We used Lamin A siRNA to validate the efficiency of the transfection protocol (mean ± standard error, n = 3, p < 0.001). * p<0.05.

Mentions: To discover novel regulators of GATA-2 expression, we conducted siRNA-based high-throughput screening. As shown in Fig 4A, YN-1 cells expressing +9.9 kb/1S-Luc2 were transfected twice with siRNA at 24-h intervals, and luciferase activity was assessed after 48 h. We used an siRNA library that contained a total 995 genes involved in transcriptional regulation. To test whether our transfection protocol could efficiently knockdown the target genes, siRNA directed against Lamin A was included as a positive control, and knockdown of the encoding gene was confirmed via quantitative RT-PCR (Fig 4B).


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)

High-throughput small interfering RNA (siRNA) screening to identify regulators of GATA-2 transcription.(A) Schematic representation of the high-throughput siRNA screening. YN-1 cells with stable +9.9/1S-Luc2 expression were selected using puromycin. Subsequently, the cells were transfected twice at 24-h intervals. Luciferase activity was assessed 48 h after the first transfection. We used an siPerfect siRNA library (RNAi) that targeted 995 transcription factor genes. (B) We used Lamin A siRNA to validate the efficiency of the transfection protocol (mean ± standard error, n = 3, p < 0.001). * p<0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556642&req=5

pone.0137079.g004: High-throughput small interfering RNA (siRNA) screening to identify regulators of GATA-2 transcription.(A) Schematic representation of the high-throughput siRNA screening. YN-1 cells with stable +9.9/1S-Luc2 expression were selected using puromycin. Subsequently, the cells were transfected twice at 24-h intervals. Luciferase activity was assessed 48 h after the first transfection. We used an siPerfect siRNA library (RNAi) that targeted 995 transcription factor genes. (B) We used Lamin A siRNA to validate the efficiency of the transfection protocol (mean ± standard error, n = 3, p < 0.001). * p<0.05.
Mentions: To discover novel regulators of GATA-2 expression, we conducted siRNA-based high-throughput screening. As shown in Fig 4A, YN-1 cells expressing +9.9 kb/1S-Luc2 were transfected twice with siRNA at 24-h intervals, and luciferase activity was assessed after 48 h. We used an siRNA library that contained a total 995 genes involved in transcriptional regulation. To test whether our transfection protocol could efficiently knockdown the target genes, siRNA directed against Lamin A was included as a positive control, and knockdown of the encoding gene was confirmed via quantitative RT-PCR (Fig 4B).

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