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.

The 1S-Luc construct was created by amplifying a 500-base pair (bp) sequence of the GATA-2 1S promoter region using the appropriate primer pair (S1 Table). This sequenced region was subsequently digested with MluI and BglII, and the digested products were purified and ligated into the pGL3 basic vector, which had been previously digested and purified using the same pair of restriction enzymes. The (−1)Luc and (−1.8)Luc plasmids were created using the NheI and BglII restriction enzymes by the same protocol used for 1SLuc. (−3.4)Luc, (−4.6) Luc, and (−4.6)Luc2 and using GeneArt SeamLess cloning (Invitrogen, Carlsbad, CA, USA). The GATA-2 +9.9 kb site was inserted upstream of the 1SLuc, (−3.4)Luc, or (−4.6)Luc sequence. GATA and/or E-box mutated constructs were generated using a QuickChange™ Site-Directed Mutagenesis Kit (Agilent technologies, Santa Clara, CA, USA).

Promoter assay
To evaluate the GATA-2 transcriptional activity, aliquots of YN-1 cells were transfected with 1 μg of GATA-2 promoter construct and 100 ng of the pRL or the pGL4.74 [hRluc/TK] vector (Promega) via FuGene HD (Promega). The cells were harvested 24 h after plasmid transfection, and firefly and Renilla luciferase activity levels in the cell extracts were determined using a Dual-Luciferase Reporter Assay System (Promega) or a ONE-Glo Luciferase Assay System, with GloMax 20/20 Luminometer (Promega).

siRNA screening
For siRNA screening, we targeted 995 genes encoding transcription factors from the siPerfect transcription factor library (RNAi, Tokyo, Japan). The library was arranged in 13 96-well plates (Thermo Scientific, Yokohama, Japan), with each well containing 5 pmol of a pool of 21-mer siRNA duplexes. Lamin A siRNA and nontarget siRNA were used as the positive and negative controls, respectively. Next, 1 × 10 4 stable YN-1 cells expressing pGL4.20 (GATA-2 +9.9/1S) in 100 μl of RPMI containing puromycin were transferred to each of the 96 wells in each plate (Berthold, Land Baden-Württemberg, Germany). We added the transfection reagent GenomO-NE-Si (Ishihara Sangyo, Osaka, Japan) to the siRNA library plates according to the manufacturer's protocol. Subsequently, we added 0.9 μl of siRNA transfection mixture per well (final concentration in wells, 5nM). siRNA transfection was conducted twice (at 0 and 24 h). At 48 h, we assessed the luciferase activity of each well using a ONE-Glo Luciferase Assay System (Promega) and Berthold Centro LB960 luminometer (Berthold). For the luciferase activity assay, we added 100 μl of ONE-Glo Reagent to each well on the plates and evaluated firefly luciferase activity with a luminometer after incubating the plates for 3 min at room temperature.

CRISPR/Cas9-based genome editing
To knockout endogenous CITED2 in YN-1 cells, the GeneArt CRISPR Nuclease Vector with CD4 enrichment kit (GE Life Sciences/Dharmacon) was used. The target sequence for nuclease recruitment was CATATGATGGCCATGAACCACGG (+25 to +47 from the translation initiation site, according to NCBI accession number NM_001168389). The plasmid was transfected using an Amaxa Nucleofector (Nucleofector solution V, Nucleofector program T-016; Lonza Group), and CD4-positive cells were subsequently cloned.

Real-time quantitative reverse transcriptase (RT)-polymerase chain reaction (PCR)
Real-time quantitative RT-PCR was conducted using the SYBR Green master mix (Qiagen, Venlo, The Netherlands) according to a previously described protocol [32]. All primer sequences are listed in S1 Table. Western blotting analysis Whole-cell lysates were prepared by boiling 1 × 10 7 cells/ml in sodium dodecyl sulfate (SDS) buffer [32]. Samples (5 μl) were resolved using SDS-polyacrylamide gel electrophoresis and analyzed with specific antibodies.

Statistics
Statistical significance was assessed with a two-sided Student's t test.

GATA-2 1S transcript is highly expressed in the YN-1 cell line
To conduct high-throughput siRNA screening, the selection of a hematopoietic cell line that expresses high levels of the target gene, in this case GATA-2, is desirable. In this study, we selected six hematopoietic cell lines, YN-1, K562, KG1a, Jurkat, U937, and NALM6, as candidate lines. Real-time quantitative RT-PCR and western blot analyses revealed that GATA-2 was abundantly expressed on both the gene and protein levels in YN-1, K562, and KG1a cell lines, but was almost undetectable in Jurkat, U937, and Nalm6 cells (Fig 1A and 1B). The validity of the GATA-2 antibody was confirmed by both GATA-2 overexpression and knockdown in K562 cells (S1 Fig). As previously described, GATA-2 transcription involves two different exons, a distal (1S) and a proximal (1G) promoter; the former is considered to be particularly important in hematopoietic cells [5,12,13], although it remained unknown regarding the contribution of 1S and 1G transcripts to the absolute level of GATA-2 expression in hematopoietic cells. Therefore, we conducted a quantitative RT-PCR analysis of each transcript. The GATA-2 1G transcript was expressed in YN-1, K562, and KG1a cells ( Fig 1C); noticeably, GATA-2 1S was the most abundantly expressed in YN-1 cells (Fig 1C). KG1a cells were derived from a human patient who presented with acute myelogenous leukemia characterized by an undifferentiated blast cell phenotype [29]. Therefore, the cells might have retained the characteristics of HSCs and thereby expressed GATA-2. On the other hand, the K562 and YN-1 lines were derived from a patient with chronic myelogenous leukemia [27,28]. Although GATA-2 expression has already been confirmed in K562 cells [28,32], the relative expression levels of 1S and 1G transcript had not yet been characterized. In addition, GATA-2 expression had not been evaluated in YN-1 cells, and the cause of the abundant expression of GATA-2 1S in this cell line remained unknown. In the current study, we selected the YN-1 cell line for siRNA screening. siRNA Screening to Identify Upstream Gene of GATA-2 GATA-2 1S promoter fused to +9.9 kb enhancer induced high promoter activity We conducted a transient luciferase promoter analysis to determine the optimal configuration for siRNA screening. Based on the importance of 1S promoter for GATA-2 expression in hematopoietic cells [12,13], we selected 1S promoter as a potential promising tool to identify novel upstream factor of GATA-2 in hematopoietic cells. As shown in Fig 2A, the 1S promoter exhibited a modest promoter activity, and the addition of a +9.9 kb enhancer to this promoter (+9.9/1S) resulted in a higher luciferase activity. On the other hand, addition of -4.6 to -0.5 kb fragment to the 1S promoter led to lower promoter activity compared with that of the 1S promoter alone (Fig 2A). The reason for lower promoter activity following addition of −4.6 to −1 kb sequence could be partially explained by the repressive effect of CEBPA (CCAAT/enhancer binding protein, alpha) through its binding site at −1.2 and −2.4 kb sites (Fig 2A) [33]. Low promoter activity based on a sequence alteration at −1 kb, which does not include CEBPA binding sites (Fig 2A), might imply the presence of other unrecognized repressive elements at this position. Regarding the functional validation of the sequence +9.9/1S, we introduced a mutation within the GATA-binding element and/or E-box motifs and demonstrated that the GATA-binding domain plays an important role in luciferase activity induced by the +9.9/1S construct ( Fig 2B). Wozniak et al. [34] analyzed the murine +9.5/1S construct in detail; these authors demonstrated that the +9.5/1S construct showed a high luciferase activity, and the disruption of the GATA binding element within +9.5 kb eliminated its enhancer activity. Therefore, we selected the +9.9 kb/1S-luciferase construct for the screening.

Establishment of YN-1 cells with stable luciferase activity under the control of +9.9 kb/1S
A luciferase plasmid was introduced into the YN-1 cells, which were subsequently selected using puromycin to ensure stable expression. We used an empty luciferase vector (Luc2) and a vector containing a +9.9/1S or −4.6 kb sequence. Similar to the results of the transient luciferase assay (Fig 2), the luciferase activity was highest in the YN-1 cells expressing the +9.9 kb/1S construct ( Fig 3A). Next, to test whether an already known GATA-2 regulator might affect the luciferase activity, we knocked down BRG1 [15]. Previous reports have shown that Brg1 knockdown in G1E cells repressed the expression of GATA-2, through its effect on the +9.5 enhancer [15]. As shown in Fig 3B-3E, knockdown of BRG1 resulted in a significant downregulation of GATA-2 expression and luciferase activity.

High-throughput siRNA screening identified GATA-2 transcription regulators
To discover novel regulators of GATA-2 expression, we conducted siRNA-based highthroughput 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).
Following the screening analysis, the top 10% of the genes that significantly (p < 0.05) repressed luciferase activity after their knockdown were selected from each plate (n = 83: Table 1, S2 Table), which included potential GATA-2 regulators of GATA-1 and GFI1B. On the other hand, however, the analysis did not identify known GATA-2 regulators, such as TAL1 [32,35], presumably due to the limited screening sensitivity. Subsequently, we narrowed down the genes, based on their potential importance in hematopoiesis. In the present study, we focused on the gene CITED2 (Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2). 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.
doi:10.1371/journal.pone.0137079.g005 CITED2 is known as CBP/p300-dependent transcriptional co-activator [36][37][38]. In a previous study, conditional deletion of Cited2 in adult mice resulted a loss of HSCs, leading to bone marrow failure and increased lethality [36]. Noticeably, in Cited2 −/− Lin − c-Kit + cells exhibited reduced Bmi1, Notch1, Lef1, Mcl1, and Gata2 expression [37]. Furthermore, a previous report demonstrated that BMI1 could induce Gata2 expression [39]. Based on an analysis of clinical samples from patients with AML, Korthuis et al. recently demonstrated that CITED2 might play an important role in maintenance of this disease [40]. These data support our finding that CITED2 is an upstream regulator of GATA-2 in humans.
Aplastic anemia (AA) is characterized by a decrease in HSCs and fatty marrow replacement. We, along with other research groups, have demonstrated reduced GATA-2 expression in CD34-positive cells from patients with AA [24,25]. On the other hand, decreased GATA-2 expression in mesenchymal stem cells has been shown to lead to accelerated adipocyte differentiation and an impaired ability to support hematopoiesis [41]. Therefore, in the bone marrow, GATA-2 not only generates and/or maintains HSCs, but also plays a role in the regulating the hematopoietic microenvironment. Thus, our system potentially represents a powerful tool with which to identify the regulatory mechanisms of GATA-2 and might lead to the development of novel therapeutic approaches for bone marrow failure syndrome. (CITED2) affects both 1S and +9.9 kb/1S luciferase activity. Luciferase analysis was performed with empty or contained the GATA-2 1S promoter and/or +9.9 kb site in both YN-1 cells and CITED2-knockout YN-1 clone (Fig 5). Luciferase activity was measured 24 h after the first transfection. Results are presented as ratios of luciferase activities (means of Firefly/ Renilla ± standard error, n = 3). Ã p<0.05. (EPS) S1