Gene expression profiling identifies HOXB4 as a direct downstream target of GATA-2 in human CD34+ hematopoietic cells

Abstract

Aplastic anemia is characterized by a reduced hematopoietic stem cell number. Although GATA-2 expression was reported to be decreased in CD34-positive cells in aplastic anemia, many questions remain regarding the intrinsic characteristics of hematopoietic stem cells in this disease. In this study, we identified HOXB4 as a downstream target of GATA-2 based on expression profiling with human cord blood-derived CD34-positive cells infected with control or GATA-2 lentiviral shRNA. To confirm the functional link between GATA-2 and HOXB4, we conducted GATA-2 gain-of-function and loss-of-function experiments, and HOXB4 promoter analysis, including luciferase assay, in vitro DNA binding analysis and quantitative ChIP analysis, using K562 and CD34-positive cells. The analyses suggested that GATA-2 directly regulates HOXB4 expression through the GATA sequence in the promoter region. Furthermore, we assessed GATA-2 and HOXB4 expression in CD34-positive cells from patients with aplastic anemia (n = 10) and idiopathic thrombocytopenic purpura (n = 13), and demonstrated that the expression levels of HOXB4 and GATA-2 were correlated in these populations (r = 0.6573, p<0.01). Our results suggested that GATA-2 directly regulates HOXB4 expression in hematopoietic stem cells, which may play an important role in the development and/or progression of aplastic anemia.

Figure 1. Expression profiling of GATA-2-regulated genes in CD34-positive cells.

(A) GATA-2 knockdown in human cord blood-derived CD34-positive cells. Anti-GATA-2 Western blotting analysis of whole-cell extract from cord blood-derived CD34-positive cells, infected with control or GATA-2 shRNA, respectively.Alpha-Tubulin was used as a loading control. (B) Quantitative RT-PCR validation of array results (mean ± SE, n = 3). 28S mRNA was quantified as a control. (C) Gene Ontology analysis. Genes showing greater than or equal to 1.4-fold change based on microarray analysis and greater than or equal to 20 normalized expression values were included in the analysis. A Z-value of 2.0 was used as the standard cut-off value.

Figure 2. GATA-2 regulates HOXB4 expression in K562 cells and CD34-positive cells.

(A, B) Transient overexpression of GATA-2 in K562 cells. (A) Expression of Myc/His-tagged GATA-2 was confirmed by Western blotting. As a control, empty vector was independently transfected into K562 cells. Alpha-Tubulin was used as a loading control. (B) Quantitative RT-PCR analysis of HOXB4 mRNA in K562 cells overexpressing GATA-2 (mean ± SD, n = 4). mRNA levels were normalized relative to GAPDH. *p<0.05. (C, D) Stable GATA-2 overexpression in K562 cells. GATA-2 expression vector was transduced into K562 cells and 28 clones were established after selection with G418 (Sigma). (C) Western blotting to detect stable expression of exogenous Myc/His-tagged GATA-2 in K562 cells. The results of 3 representative clones (A1, A4, and A6) and 2 control clones are shown. Alpha-Tubulin was used as a loading control. (D) Correlation between GATA-2 and HOXB4 mRNA in each clone. mRNA expression was normalized relative to that of GAPDH, and the correlation of these genes was assessed by Spearman's rank correlation method. (E–F) GATA-2 knockdown in K562 cells. (E) Anti-GATA-2 Western blotting analysis of whole-cell extracts and (F) Quantitative RT-PCR analysis of GATA-2 and HOXB4 (mean ± SE, n = 3), from K562 cells transfected with siRNA against human GATA-2 or control siRNA. Alpha-Tubulin was used as a loading control. mRNA levels were normalized relative to GAPDH. *p<0.05. (G) MSCV retroviral vector-mediated GATA-2 overexpression in cord blood-derived CD34-positive cells. Quantitative RT-PCR analysis was performed to detect GATA-2 and HOXB4 expression (mean ± SE, n = 3). mRNA levels were normalized relative to 28S. *p<0.05.

Figure 3. GATA-2 regulates HOXB4 promoter activity.

(A) GATA-1 and GATA-2 ChIP-seq profile at HOXB4 in K562 cells. GATA-1 and GATA-2 signal map for HOXB4 was shown. Arrows, ChIP-seq peak locations relative to the start site of the respective GATA-1 target gene (kb). The ChIP-seq data were mined from data described by Fujiwara et al and were analyzed with the integrated genome browser (IGB) (Affymetrix). (B) The 5′ upstream region of the human HOXB4 gene. The putative GATA-binding element is boxed. The translational initiation site is underlined. Two transcriptional start sites are indicated by arrows. (C) Impact of GATA site deletion on HOXB4 promoter activity. We generated wild-type and −160/−157 GATA-deleted constructs fused to a luciferase reporter gene, and transient transfection assay was performed in K562 cells (mean ± SD, n = 3). *p<0.05. (D) GATA-2 knockdown in K562 cells. Anti-GATA-2 Western blotting analysis of whole-cell extract (left) and quantitative RT-PCR analysis of GATA-2 (right) from K562 cells, infected with control or GATA-2 shRNA, respectively. Alpha-Tubulin was used as a loading control, and GAPDH mRNA was quantified as a control. (E) Impact of GATA-2 deletion on HOXB4 promoter activity. HOXB4 promoter assay was conducted with K562 cells expressing control or GATA-2 shRNA (mean ± SD, n = 3). *p<0.05.

Figure 4. GATA-2 binds to HOXB4 promoter.

(A) Electrophoretic mobility shift assays. Aliquots of 3 ug of nuclear extracts from K562 (lane 2) or GATA-2 expression vector-transfected (lanes 3–6) K562 cells were incubated with FITC-labeled oligomers including the GATA site at −160/−157 within the HOXB4 promoter in the absence (lanes 1–4) or presence (lanes 5–6) of a 100-fold molar excess of the indicated oligonucleotides. For GATA-2 overexpression, pcDNA(−)/Mys-His expression vector was used (Figures 2A, 2B). (B) Quantitative ChIP analysis of GATA-2 chromatin occupancy at HOXB4 promoter, GATA-2 −1.8 kb, and NECDIN promoter, with K562 cells (upper) and cord blood-derived CD34-positive cells (lower) (mean ± SD, n = 3).

Figure 5. Significant correlation between GATA-2 and HOXB4 expression in CD34-positive cells in patients with AA and ITP.

Bone marrow cells were collected from 10 patients with AA and 13 patients with ITP after obtaining informed consent . Mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation. CD34-positive cells were then collected using the MACS system. GATA-2 and HOXB4 mRNA levels were examined by quantitative RT-PCR analysis, and were normalized relative to GAPDH mRNA expression. The correlation of HOXB4 and GATA-2 mRNA expression in each sample was calculated with Spearman's rank correlation test.