Transcriptional Activation of Prostate Specific Homeobox Gene NKX3-1 in Subsets of T-Cell Lymphoblastic Leukemia (T-ALL)
Figure 3
Analyses of LMO-, bHLH-, and GATA-factors in NKX3-1 deregulation.
Quantification of gene expression by RQ-PCR in 24 T-ALL cell lines for (A) LMO1 (left) and LMO2 (right), (B) TAL1 (left) and LYL1 (right), (C) GATA3 (left) and GATA2 (right). NKX3-1 expressing cell lines are indicated in red letters. Additionally, Western blot analyses were performed for LYL1 (B), GATA3 and GATA2 (C). ERK1/2 served as loading control. The results of these expression analyses are given in Table 2. (D) RQ-PCR analyses of T-ALL cell lines treated with siRNAs directed against LMO1 or LMO2 demonstrate reduced expression of the targeted genes. The expression levels of NKX3-1 were decreased significantly in JURKAT and MOLT-14 but not in PER-117. Lentiviral mediated overexpression of LMO2 in JURKAT resulted in increased NKX3-1 expression. (E) JURKAT cells were treated for overexpression of TAL1 or LYL1 by lentiviral gene transfer and for knockdown of TAL1 by siRNA. NKX3-1 expression data indicate activation by TAL1 and inhibition by LYL1. In contrast, siRNA mediated knockdown of LYL1 in PER-117 indicate activation of NKX3-1 expression. Knockdowns of both TAL1 and LYL1 in JURKAT and PER-117, respectively, were confirmed by RQ-PCR. (F) JURKAT cells were treated for knockdown of GATA3 by siRNA, indicating activation of NKX3-1 expression by GATA3. Overexpression of GATA3 or GATA2 by electroporation of expression constructs showed no significant effect for GATA2 in JURKAT or in PER-117. However, GATA3 enhanced NKX3-1 expression in JURKAT while it was inhibited in PER-117, indicating differences in regulation. (G) PER-117 cells were treated for knockdown (left) and for overexpression of GATA2 (right). Collectively, the data demonstrate activation by GATA2 in LYL1 as well as in GATA3. (H) ChIP analysis was performed using anti-GATA2 for precipitation of genomic DNA fragments. Subsequent PCR analysis indicates direct binding of GATA2 at the LYL1 but not NKX3-1 promoter. NTC: no template control. (I) JURKAT cells were treated for siRNA mediated knockdown of MLL. RQ-PCR analyses demonstrate concomitant repression of MLL and enhancement of NKX3-1, TAL1, GATA3 and LMO1 transcription. (K) Stimulation of JURKAT cells with ATRA (left) or BMP4 (right) modulated expression levels of GATA3, LMO1 and TAL1. While ATRA stimulation inhibited expression of both GATA3 and LMO1, BMP4 stimulation enhanced GATA3 and TAL1 but inhibited LMO1.