High-Throughput siRNA Screening to Reveal GATA-2 Upstream Transcriptional Mechanisms in Hematopoietic Cells.
High-Throughput siRNA Screening to Reveal GATA-2 Upstream Transcriptional Mechanisms in Hematopoietic Cells.
复制标题
高通量siRNA筛选以揭示造血细胞中的GATA-2上游转录机制。
DOI：
10.1371/journal.pone.0137079
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Harigae H
中科院分区：
文献类型：
--
作者：
Saito Y;Fujiwara T;Ohashi K;Okitsu Y;Fukuhara N;Onishi Y;Ishizawa K;Harigae H
造血干细胞可以自我更新并分化为所有类型的血细胞。转录因子加塔-2在造血干细胞和祖细胞中表达，并且对于细胞增殖、存活和分化是必需的。最近，再生障碍性贫血、MonoMAC综合征和肺癌的研究证据已经证明了加塔-2与人类病理生理学之间的机制联系。加塔-2依赖性疾病过程已被广泛分析;然而，加塔-2上游的转录机制仍不太清楚。在这里，我们进行了高通量的小干扰RNA（siRNA）文库筛选，并显示，YN-1，一个人红白血病细胞系，表达高水平的加塔-2造血特异性1 S启动子激活后。由于YN-1细胞中的瞬时荧光素酶报告基因测定显示与加塔-2内含子增强子融合的1 S启动子（+9.9 kb/1 S）中的启动子活性最高;因此，我们建立了能够稳定表达+9.9 kb/1 S-荧光素酶的细胞系。随后，我们筛选了995个转录因子基因，并揭示了CITED 2在人类造血细胞中作为加塔-2激活剂。这些结果提供了新的见解，并进一步确定加塔-2的调节机制。
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.