Table 1.
Oligonucleotides used for PCR and cloning.
Figure 1.
(A) Expression analyses of human (left) and murine (right) NKX3-1 transcripts in primary tissues including bone marrow (BM), lymph node (LN), thymus (Th), peripheral blood mononuclear cells (PBMC), T-cells, B-cells, prostate (Pr), retina (Re), spleen (Sp) and eye (Ey) were performed by RQ-PCR. Note the data demonstrate exclusive NKX3-1 expression in prostate cells. (B) RQ-PCR analysis of NKX3-1 transcripts in 24 T-ALL cell lines revealed 7 positive cell lines indicated in red letters (left). Western blot analysis shows NKX3-1 protein expression in JURKAT, PER-117 and RPMI-8402 but not in LOUCY and PEER (right). Prostate cell line LNCAP served as positive control for NKX3-1 and ERK1/2 as loading control. (C) RQ-PCR analysis of NKX3-1 (left) and NKX2-5 (right) was performed in T-ALL cell lines in addition to primary tissues. The respective expression deficits of NKX3-1 and NKX2-5 in T-ALL versus tissue controls LNCAP (prostate) and heart were similar.
Figure 2.
Analyses of NKX3-1 deregulation in T-ALL.
(A) FISH analysis of NKX3-1 positive cell lines PER-117 and JURKAT was performed by two NKX3-1 flanking and one straddling probe (inserts). Genomic array analysis of T-ALL cell lines PER-117 and JURKAT shows no change of copy numbers along the chromosome 8 including locus NKX3-1 at 8p21 (marked by a red line). Together, these data demonstrate absence of rearrangements or copy number changes at NKX3-1. (B) RQ-PCR analyses of T-ALL cell lines treated with siRNAs directed against FOXA1, ETS1 and SOX4 demonstrate reduced expression of the targeted genes while sparing NKX3-1. (C) RQ-PCR analysis of NKX3-1 in JURKAT cells treated with several ligands for particular signalling pathways. Data show reduced NKX3-1 expression in cells treated with BMP4 or ATRA.
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.
Table 2.
Expression results for GATA/TAL1/LYL1/LMO transcription factors.
Figure 4.
Identification of additional NKX3-1 regulators.
(A) Heat map of 209 genes which displayed a significant differential expression between two groups of T-ALL cell lines concerning NKX3-1 expression, as analysed by the LIMMA package. Four T-ALL cell lines (SUP-T1, LOUCY, ALL-SIL, PEER) are NKX3-1 negative while five others (PER-117, MOLT-4, JURKAT, MOLT-14, RPMI-8402) are NKX3-1 positive. Red indicates strong expression levels, green low levels and black medium levels. Sixteen conspicuously expressed genes are highlighted. (B) RQ-PCR analysis of JURKAT cells treated with activating anti-CD3 antibody demonstrates concomitantly upregulation of NKX3-1, GATA3, LMO1 and TAL1. (C) Treatment of PER-117 cells with specific ligands demonstrates increased expression of NKX3-1 after stimulation with IL13 or IGF2. (D) Treatment of PER-117 cells with IL13 resulted in strong activation of LYL1 and slight activation of GATA2 expression, while that of LMO2 and GATA3 remained unperturbed. (E) Treatment of PER-117 cells with IGF2 resulted in differentially altered expression levels of LMO2, LYL1 and GATA3 while GATA2 remained unchanged. (F) Treatment of JURKAT cells with specific ligands demonstrates decreased expression of NKX3-1 after stimulation with IL7, while the expression increased after stimulation with IGF2. (G) Treatment of JURKAT cells with IL7 resulted in decreased expression of GATA3, LMO1 and TAL1 (left). Lentiviral overexpressed STAT5A in JURKAT cells resulted in decreased expression of both NKX3-1 and GATA3 (right). (H) Treatment of JURKAT cells with IGF2 resulted in marginally increased expression of GATA3 and TAL1 while no change was visible for LMO1.
Table 3.
Selected genes differentially expressed in NKX3-1 positive cell lines.
Figure 5.
MSX2 activates NKX3-1 expression.
(A) JURKAT and MOLT-4 cells were lentivirally transfected for overexpression of MSX2. Additionally, JURKAT were treated for knockdown of MSX2. Collectively, the data demonstrate the positive impact of MSX2 on NKX3-1 expression. (B) Overexpression of MSX2 in JURKAT cells showed no significant alteration in analyzed gene expression (left), while the same treatment in MOLT-4 cells indicated decreased expression of GATA3 and LMO2 but not of TAL1 (right). (C) Reporter gene assay of a genomic NKX3-1 upstream fragment (left) demonstrates stimulation by MSX2 as analyzed by RQ-PCR in PEER cells. The oligonucleotide sequences used for cloning of the reporter gene construct are underlined and the binding site for MSX2 is shown in bold. (D) IGF2 treatment resulted in increased expression of MSX2 in JURKAT cells (left). SiRNA mediated knockdown of IGF2BP1 in JURKAT cells resulted in reduced expression of NKX3-1, indicating a stimulatory input. SiRNA mediated knockdown of IGF2BP1 in PER-117 resulted in reduced expression of MSX2, indicating an activatory input of IGF2BP1 in IGF2-signalling.
Figure 6.
NKX3-1 activates SIX6 expression.
(A) Reporter gene assay of a genomic SIX6 fragment located in exon 2 (left) demonstrates an activatory input of NKX3-1 as analyzed by RQ-PCR in PEER cells. The oligonucleotide sequences used for cloning of the reporter gene construct are underlined and the binding site for NKX3-1 is emboldened. (B) RQ-PCR analysis of SIX6 expression in T-ALL cell lines (left) and primary cells (right) demonstrates SIX6 transcripts in 5 cell lines and primary retina cells but not in hematopoietic cells. NKX3-1 positive cell lines are in red, coexpressing cell lines are marked with an asterix.
Figure 7.
The figure summarizes the results of this study, highlighting the major activators of NKX3-1 in bold, comprising TAL1, LYL1 and MSX2, and their potential association to the T-ALL subtypes TAL1-positive and immature. T-ALL cell line JURKAT expresses TAL1, GATA3 and the TCR-complex while cell line PER-117 expresses LYL1 and GATA2. Signalling pathways BMP4 and IGF2 regulate expression of MSX2 and are associated to early stages of T-cell development.