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

2015-09-01 17:51:04

PLOS ONE; 1 September 2015: DOI:10.1371/journal.pone.0137079


Yo Saito, Tohru Fujiwara, Keiichi Ohashi, Yoko Okitsu, Noriko Fukuhara, Yasushi Onishi, Kenichi Ishizawa, Hideo Harigae



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.



Introduction


Hematopoiesis is a complex process controlled by numerous transcription factors that regulate and coordinate the expression of lineage-specific genes. Previous baseline studies have suggested that the GATA family of transcription factors, which act in developmental regulation, is directly involved in hematopoiesis. GATA-1, GATA-2, and GATA-3 are known as the hematopoietic GATA factors, given their important roles in this process. Among them, GATA-2 is required for the maintenance and expansion of hematopoietic stem cells (HSCs) and/or multipotent progenitors during early hematopoiesis.



To date, the mechanisms underlying GATA-2 transcription have been extensively analyzed. Two first exons/promoters of the gene, named 1S and 1G, have been identified in both mice and humans. Transcripts involving the 1G promoter are commonly found in tissues expressing GATA-2, whereas 1S transcripts are believed to play an important role in hematopoietic cells. During erythroid differentiation, GATA-2 levels decline concomitantly with an increase in GATA-1 levels. GATA-1 represses Gata-2 transcription by displacing GATA-2 from the sites at -77, -3.9, -2.8, -1.8, and +9.5 kilobase (kb) relative to the 1S promoter, which are known as GATA switch sites [5, 14]. However, despite the compelling evidence supporting the locations and characteristics of these GATA switch sites, targeted individual deletions of the -1.8, -2.8, and -3.9 kb sites lead to minor increases in Gata2 expression in murine hematopoietic precursors. On the other hand, deletion of the +9.5 site leads to delayed embryonic lethality compared with global Gata2 knockout. Noticeably, in humans, the heterozygous mutation of the intronic enhancer at +9.9 kb, which corresponds to +9.5 kb in mice, has been found in patients with GATA-2 deficiency (MonoMAC syndrome), which is characterized by a predisposition to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Thus, the 1S promoter and +9.5 kb enhancer regions could be considered as important regulatory regions for GATA-2 expression.



Several transcription factors involved in various signaling pathways, such as the Wnt and Notch pathways, are known to participate in GATA-2 regulation; however, relatively less is known about how these transcriptional molecular mechanism associate with GATA-2 expression. Given the pathophysiological links between GATA-2 and aplastic anemia, MonoMAC syndrome, and lung cancer, it is extremely important to clarify and comprehensively understand the details regarding the mechanisms behind the upstream transcription of GATA-2. With this in mind, we conducted a high-throughput screening analysis based on a small interfering RNA (siRNA) library to provide a novel insights into the factors involved in the regulation of GATA-2 expression.



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