High-Throughput siRNA Screening to Reveal GATA-2 Upstream Transcriptional Mechanisms in Hematopoietic Cells.
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.
Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan.
Yo
Y
Saito
Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan; Molecular Hematology/Oncology, Tohoku University Graduate School of Medicine, Sendai, Japan.
Tohru
T
Fujiwara
Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan.
Keiichi
K
Ohashi
Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan.
Yoko
Y
Okitsu
Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan.
Noriko
N
Fukuhara
Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan.
Yasushi
Y
Onishi
Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan; Department of Hematology and Cell Therapy, Yamagata University Faculty of Medicine, Yamagata, Japan.
Kenichi
K
Ishizawa
Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan; Molecular Hematology/Oncology, Tohoku University Graduate School of Medicine, Sendai, Japan.
Hideo
H
Harigae
D001854:Bone Marrow Cells / Q000378:metabolism*; D045744:Cell Line, Tumor; D050980:GATA Transcription Factors / Q000502:physiology*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D014158:Transcription, Genetic / Q000502:physiology*
D050980:GATA Transcription Factors; D012333:RNA, Messenger; D034741:RNA, Small Interfering