Cell lines and treatments

Authenticated mycoplasma-free cell lines were drawn from the DSMZ Cell Lines Bank (Braunschweig, Germany) and cultivated as described previously [18]. Expression constructs and small interfering (si)RNAs were transfected into the cells by electroporation using the EPI-2500 impulse generator (Fischer, Heidelberg, Germany) at 350 V for 10 ms and subsequently incubated for 16 h. Expression constructs for HEY1 and HES1 were obtained from Origene (Wiesbaden, Germany). SiRNA oligonucleotides and AllStars negative Control siRNA (termed here siCTR) were obtained from Qiagen (Hilden, Germany). Cell stimulations were performed by treatment with recombinant human Bone morphogenetic protein (BMP) 4, Interleukin (IL)4, IL10, Transforming growth factor beta (TGFb), Tumor necrosis factor alpha (TNFa), or WNT5B for 16 h at concentrations of 20 ng/ml (R & D Systems, Wiesbaden, Germany). Treatments with γ-secretase inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) (Sigma, Taufkirchen, Germany) were performed at concentrations of 1 µM for 16 h, with cAMP-derivate (8-4-Chlorophenylthio-adenosine 3′,5′-cyclic monophosphate, Sigma) and cGMP-derivate (8-bromoguanosine 3′,5′-cyclic monophosphate, Sigma) at concentrations of 100 µM, with Sildenafil (Tocris Bioscience, Bristol, UK) at concentrations of 100 µM and with the NFkB-inhibitor (Calbiochem, Darmstadt, Germany) as indicated in the text. Treatments with anti-TGF beta-1 (clone 9016.2, Thermo Scientific, Germany) were performed with 1 µg/ml for 16 h. DZNep was kindly provided by Dr. Marquez (NIH, Frederick, MD, USA) and applied as described recently [19].

Expression profiling

For quantification of gene expression via profiling, we used data obtained by gene chips HG U133 Plus 2.0 from Affymetrix (High Wycombe, UK). The datasets were generated at the University of Würzburg and generously provided by Prof. Andreas Rosenwald (Institute of Pathology, University of Würzburg, Germany) or obtained from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) (www.ncbi.nlm.nih.gov/gds) or from the National Cancer Institute (NCI). The NCI microarray data for SU-DHL-5 are available through the accession numbers SU-DHL-5_SS392729_HG-U133_Plus_2_HCHP-201545_.CEL, SU-DHL-5 _SS392730_HG-U133_Plus_2_HCHP-201546_.CEL, and SU-DHL-5_SS392731_HG-U133_Plus_2_HCHP-201547_.CEL which were combined in this study. Analyses of expression data were performed using Microsoft Excel and online programs. For creation of heat maps we used CLUSTER version 2.11 and TREEVIEW version 1.60 (http://rana.lbl.gov/EisenSoftware.htm). Expression data of 203 DLBCL patient samples were obtained from the NCBI GEO database (accession number GSE11318) as published recently [20]. Statistical analyses of NKX2-1 (dataset 211024_s_at) expression were performed using R-software.

Genomic array analysis

Genome-wide copy number analysis was performed using the Affymetrix Genotyping Console GTC Software version 4.0 (Affymetrix) and visualized by the Affymetrix GTC-Browser program. The 500K-array dataset for SU-DHL-5 was obtained from the National Cancer Institute (Bethesda, MD, USA), GSK Cancer Cell Line Genomic Profiling Data (https://cabig.nci.nih.gov/community/caArray_GSKdata/).

Chromosomal analyses

Fluorescent in-situ hybridization (FISH) and spectral karyotyping (SKY) analyses were performed as described previously [21], [22]. RP11-BAC-clones were obtained from BacPac Resources, Childreńs Hospital Oakland Research Institute (CA, USA), prepared using the Big BAC DNA Kit (Princeton Separations, Adelphia, NJ, USA) and directly labelled by nick translation with dUTP-fluors (Dyomics, Jena, Germany). For analysis of the NKX2-1 locus BACs RP11-676A19, RP11-945C4, and RP11-74D5 were labelled with Dy-590, Dy-547 and Dy-495, respectively. For analysis of the HEY1 locus we used RP11-300M12 (Dy-590), RP11-89I14 (Dy-547) and RP11-24P11 (Dy-495), for that of MLL we used RP11-347D24 (Dy-547), RP11-91A14 (Dy-590) and RP11-770K18 (Dy-495), and for HIST1 RP11-958P15 (Dy-495), RP11-846O7 (Dy-547) and RP11-6N6 (Dy-590). Fluorescence images were captured and analyzed with an Axio-Imager microscope (Zeiss, Göttingen, Germany) configured to a dual Spectral Imaging FISH and SKY system (Applied Spectral Imaging, Neckarhausen,Germany).

Polymerase chain-reaction (PCR) analyses

Total RNA from cell lines was extracted using TRIzol reagent (Invitrogen, Karlsruhe, Germany). Total human RNA isolated from peripheral blood mononuclear cells (PBMC), bone marrow (BM), lymph nodes (LN), thymus, lung and thyroid was obtained from Clontech (Saint-Germain-en-Laye, France), and RNA isolated from CD3-positive T-cells and CD19-positive B-cells from Miltany Biotec (Bergisch Gladbach, Germany). cDNA was subsequently synthesized from 5 µg RNA by random priming, using Superscript II (Invitrogen).

Real-time quantitative expression analysis (RQ-PCR) was performed by the 7500 Fast Real-time System, using commercial buffer and primer sets (Applied Biosystems, Darmstadt, Germany). For normalization of expression levels we used TATA box binding protein (TBP). Quantitative analyses were performed in triplicate and repeated twice. The standard deviations are indicated in the figures as bars.

Reverse transcription (RT) PCR was performed using taqpol (Qiagen), oligonucleotides as listed in Table S1 (MWG Eurofins, Martinsried, Germany) and the thermocycler TGradient (Biometra, Göttingen, Germany).

Protein analysis

Sodium dodecyl sulphate polyacrylamid gel electrophoresis (SDS-PAGE) was performed in the Mini-Protean 3 system (Bio-Rad, München, Germany). Gels were either stained with PageBlue (Fermentas, Vilnius, Lithuania) or processed for Western blot analysis via the semi-dry method. Proteins obtained from cell lysates were transferred onto nitrocellulose membranes (Bio-Rad) and blocked with 5% dry milk powder dissolved in phosphate-buffered-saline buffer (PBS). The following antibodies were used: anti-NKX2-1 (EP1584Y, Abgent, Heidelberg, Germany), anti-NKX3.1 (clone 3–9, Invitrogen), anti-ERK1 (K-23, Santa Cruz Biotechnology, Heidelberg, Germany). The secondary antibodies were labelled with peroxidase and detected by Western Lightning-ECL (Perkin Elmer, Waltham, MA, USA).

Chromatin immuno-precipitation (ChIP)

ChIP analysis was performed with the ChIP Assay Kit (Millipore-Upstate, Schwalbach, Germany) as described by the manufacturer, isolating genomic DNA fragments generated by sonication, using antibodies anti-NKX2-1 (EP1584Y, Abgent), anti-H2B (53H3, Cell Signaling, Danvers, MA, USA), anti-H2Bub1 (D11, Cell Signaling), anti-H3K4me3 (mAbcam1012, Abcam, Cambridge, UK) and anti-H3K27me3 (mAbcam6002, Abcam). The subsequent ChIP-PCR analysis was performed using taqpol (Qiagen) oligonucleotides as listed in Table S1 (MWG Eurofins) and thermocycler TGradient (Biometra).

Immunostaining

To determine intracellular localizations of proteins we performed immunostaining as described previously [23]. We used antibodies anti-NKX2-1 (EP1584Y, Abgent) and anti-SMAD3 (7F3, Abnova, Heidelberg, Germany). Secondary antibodies were purchased from Cell Signaling. For analysis cells were mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA) containing the counterstain DAPI.

Analysis of DNA methylation

SU-DHL-5 cells were treated with 10 µg/ml trichostatin A (TSA, Sigma) or 10 mM 5-Aza-2′-deoxycytidine (Sigma) for 20 h before being examined by RQ-PCR. To identify particular methylated cytidines within the CpG island at the HOPX locus, genomic DNA of SU-DHL-5 and SU-DHL-4 (for control) was subjected to bisulfite-conversion and analyzed as described recently [24]. The converted DNA was amplified by PCR using oligonucleotides as listed in Table S1. The PCR products were subcloned into pGEMT-easy (Promega, Madison, WI, USA) and the inserts sequenced (MWG Eurofins). Sequences of 10 clones were displayed and compared using BiQ Analyzer (http://biq-analyzer.bioinf.mpg.de).

Ectopic expression of NKL homeobox gene NKX2-1 in DLBCL cell line

Here, we screened by expression profiling 20 leukemia/lymphoma T- and B-cell lines for aberrant activities of NKL-family homeobox genes which are implicated in T-cell leukemia [11]. Our findings confirmed expression of NKL family members in T-ALL cell lines, namely: TLX1 in ALL-SIL, TLX3 in HPB-ALL, NKX2-5 in PEER and CCRF-CEM, and NKX3-1 in JURKAT, PER-117 and RPMI-8402 (data not shown). In addition, we identified conspicuous expression of NKX2-1 and NKX3-1 in DLBCL cell line SU-DHL-5. Understanding the unexpected activation of NKL genes in a malignant B-cell line was the main focus of this enquiry.

Quantitative expression analysis of NKX2-1 and NKX3-1 by RQ-PCR confirmed their activation in SU-DHL-5 at the RNA level. While NKX3-1 was also expressed in Hodgkin lymphoma (HL), multiple myeloma (MM), B-cell lymphoma (BCL) and additional DLBCL cell lines, NKX2-1 was nearly undetectable in the same set of 25 lymphoma cell lines (Fig. 1A), prompting analysis of NKX2-1 and NKX3-1 in primary cells, including thyroid, lung and particular hematopoietic samples to chart physiological tissue specificity in the blood system. The results confirmed physiological NKX2-1 expression in tissues of the thyroid and the lung, while in all hematopoietic samples analyzed NKX2-1 transcription was undetectable (Fig. 1B). On the other hand, NKX3-1 expression was confirmed in the cell lines SU-DHL-5, JURKAT, PER-117 and LNCAP (prostate) and physiologically in tissues of the thyroid and the lung, while the hematopoietic samples of B-cells and BM showed just weak activity (Fig. 1C).

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Figure 1. Expression and karyotyping.

(A) RQ-PCR analysis of NKX2-1 (left) and NKX3-1 (right) in SU-DHL-5 (expression level was set to unity) and in leukemia/lymphoma control cell lines. (B) RQ-PCR analysis of NKX2-1 in primary cells, demonstrating physiological expression in thyroid and lung and ectopical expression in SU-DHL-5 cells. Western blot analysis (insert) confirms NKX2-1 expression in SU-DHL-5 cells while control DLBCL cell lines RC-K8, OCI-LY3 and SU-DHL-16 are negative. ERK1 was used as loading control. (C) RQ-PCR analysis of NKX3-1 in primary cells and selected cell lines, demonstrating physiological expression in thyroid, lung and prostate and ectopic expression in SU-DHL-5 and T-ALL cell lines JURKAT and PER-117. Western blot analysis (insert) shows NKX3-1 expression in prostate LNCAP cells while SU-DHL-5 lacks detectable NKX3-1 protein. (D) In silico expression analysis of NKX2-1 was performed in 204 DLBCL patient samples (NCBI GEO accession number GSE11318). Median centred gene expressions were illustrated in a barplot (left), showing 11 patients with enhanced expression of NKX2-1 (red bracket). Illustration of these datasets by a boxplot (right) and a Q-Q-plot (below) confirms significantly enhanced expression of NKX2-1 in these 11 patient samples (red circles). (E) Copy number analysis by genomic profiling indicates absence of aberrations at the locus of NKX2-1 at 14q13. The insert shows an enlargement of chromosome 14 obtained by SKY karyotyping. (F) SKY karyotyping of SU-DHL-5 shows absence of rearrangements at chromosome 14 while chromosome 6 demonstrates presence of aberrations.

https://doi.org/10.1371/journal.pone.0061447.g001

Protein expression of NKX2-1 and NKX3-1 was analyzed by Western blot. While NKX2-1 protein was clearly displayed in SU-DHL-5, NKX3-1 protein was not detectable in that cell line (Fig. 1B,C), consistent with post-transcriptional inhibition which was described recently [25]. For this reason we focused our work on regulation and function of homeobox gene NKX2-1, showing ectopic expression at the RNA and protein level in DLBCL cell line SU-DHL-5. To examine the expression of NKX2-1 in primary material we have checked 204 datasets of untreated DLBCL cases deposited in the GEO database of the NCBI belonging to the study of Lenz and coworkers [20]. This screening revealed statistically significant enhanced NKX2-1 activity in 11 (5%) DLBCL patients (Fig. 1D), supporting the relevance of this oncogenic homeobox gene expression in this malignancy. Of note, the NKX2-1 overexpressing DLBCL patients showed no clear correlation with known disease subsets.

Since deregulated expression of NKL homeobox genes in T-ALL is primarily caused by chromosomal aberrations, we analyzed the karyotype of SU-DHL-5 by SKY, FISH and in a genomic array with respect to the NKX2-1 gene which is located at 14q13 (36.9 Mb). However, copy number data (Fig. 1E), gene-specific FISH results (not shown) and SKY results (Fig. 1F) all returned wild type configurations at this locus, discounting a chromosomal mechanism behind the deregulated transcription.

Turning to potential transcriptional regulators which might induce aberrant NKX2-1 activity, we compared expression array data of SU-DHL-5 with 3 control DLBCL cell lines - SU-DHL-4, SU-DHL-10, and SU-DHL-16. After inspection of the top 1000 up- and downregulated genes in SU-DHL-5, potential candidates were shortlisted and functionally categorized as shown in Table 1. This exercise revealed conspicuous involvement of TFs, chromatin and signaling genes which were then subjected to more detailed consideration.

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Table 1. Gene data of SU-DHL-5.

https://doi.org/10.1371/journal.pone.0061447.t001

Transcription factors HEY1 and NKX2-1 are mutually exciting

Among TFs bHLH factor HEY1 was particularly intriguing (one of the top 10 upregulated genes). RQ-PCR analysis of a panel of cell lines confirmed high expression levels in SU-DHL-5 (Fig. 2A). However, MM and HL cell lines also showed moderate transcription of HEY1. RQ-PCR analysis of SU-DHL-5 in comparison to primary cells revealed prominent HEY1 expression in lung, thyroid, and in BM, LN and thymus. These data suggest a functional role of HEY1 in early lymphopoiesis. Peripheral B-cells and PBMCs lacked HEY1 transcripts (Fig. 2A), indicating downregulation in differentiated B-cells and other leukocytes and thus aberrant upregulation in SU-DHL-5.

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Figure 2. HEY1 and ZHX2.

(A) RQ-PCR analysis of HEY1 in SU-DHL-5 (expression level was set to 1) and in leukemia/lymphoma control cell lines (left) and primary cells (right). (B) RQ-PCR analysis of SU-DHL-5 cells treated for siRNA-mediated knockdown of HEY1 (left) and for overexpression of HEY1 and HES1 (right). The results indicate activation of NKX2-1 by HEY1 (and HES1). (C) The promoter sequence of HEY1 contains binding sites for NKX2-1, SMAD and NFkB (left) which are highlighted in bold letters. SiRNA-mediated knockdown of NKX2-1 in SU-DHL-5 cells resulted in reduced expression of HEY1 as shown by RQ-PCR (right, above). ChIP analysis of the HEY1 promoter in SU-DHL-5 and SU-DHL-4 (for control) demonstrated direct binding of NKX2-1 as shown by PCR amplification of genomic fragments (left, below). Untreated genomic DNA served as positive control, NTC: no template control. (D) RQ-PCR analysis of HEY1 and NKX2-1 in SU-DHL-5 cells treated with NOTCH-inhibitor DAPT (left) or with siRNA directed against SPEN (right). (E) RQ-PCR analysis of ZHX2 in cell lines and primary cells (left), and of ZHX2 and HEY1 in siRNA-treated SU-DHL-5 cells (middle and right).

https://doi.org/10.1371/journal.pone.0061447.g002

To analyze the regulatory impact of HEY1 on NKX2-1 expression we treated SU-DHL-5 cells with siRNA directed against HEY1. Subsequent quantification of HEY1 and NKX2-1 expression demonstrated reduction of both transcripts as compared to cell samples treated with control siRNA (Fig. 2B). Furthermore, overexpression of HEY1 or the related TF HES1 was followed by increased NKX2-1 expression (Fig. 2B). Together, these results show that HEY1 contributes to NKX2-1 expression in DLBCL cell line SU-DHL-5. That HEY1 acts as a transcriptional repressor suggests indirect activation of NKX2-1 probably via inhibition of negative regulators as shown below [26].

Sequence analysis of the promoter region of HEY1 identified 3 potential binding sites for NKX2-1 (Fig. 2C), indicating direct regulatory impact of this homeoprotein in HEY1 expression. Subsequent siRNA-mediated knockdown of NKX2-1 inhibited transcription of NKX2-1 and HEY1, confirming regulation by NKX2-1 (Fig. 2C). ChIP analysis using anti-NKX2-1 confirmed direct binding of NKX2-1 to the promoter region of HEY1 (Fig. 2C). These data show that NKX2-1 activates HEY1 transcription directly. Genomic copy number data and SKY analyses excluded genomic aberrations at the HEY1 locus at 8q21 (Fig. S1), highlighting the role of NKX2-1 in HEY1 regulation.

HEY1 and HES1 are prominent targets of the NOTCH-pathway in lymphopoiesis [4]. To analyze the potential impact of NOTCH on HEY1 expression we treated SU-DHL-5 cells with the γ-secretase inhibitor DAPT. Subsequent RQ-PCR analysis showed reduced HEY1 levels in treated samples (Fig. 2D), confirming NOTCH regulation. Accordingly, siRNA-mediated knockdown of NOTCH corepressor SPEN enhanced HEY1 expression more than twofold (Fig. 2D). Additional findings using DAPT discounted regulation of NKX2-1 by NOTCH-signaling (Fig. 2D).

Expression of Zn-finger homeobox gene 2 (ZHX2) showed elevated levels in SU-DHL-5 as well (Table 1). Comparative RQ-PCR analysis confirmed high transcript levels in SU-DHL-5, even surpassing primary B-cells (Fig. 2E). Of note, we recently showed reduced expression of ZHX2 in HL cell line L-1236 and an activating input of homeobox gene MSX1 [27]. Accordingly, siRNA-mediated knockdown of MSX1 in SU-DHL-5 reduced transcription of ZHX2 but not of HEY1 (Fig. 2E), contrasting with stimulation of HEY1 by the closely related homeobox gene MSX2 in T-ALL cells [28]. SiRNA-mediated knockdown of homeobox gene NKX2-1 reduced transcript levels of HEY1 as shown above while sparing ZHX2 (Fig. 2E), thus discounting direct regulation of ZHX2 by NKX2-1. However, the precise mechanism of ZHX2 enhancement remains unclear.

Aberrant chromatin structures mediate NKX2-1 expression

The catalogue of upregulated genes in SU-DHL-5 encoding chromatin components included prominently MLL which is frequently deregulated in leukemia where it activates homeobox genes of the clustered type via H3K4-trimethylation [16]. Quantification of MLL expression in cell lines confirmed elevated RNA levels in SU-DHL-5 in addition to HL cells (Fig. 3A). SiRNA-mediated knockdown of MLL inhibited expression of NKX2-1 but not of HEY1 (Fig. 3B), showing that MLL supports NKX2-1 expression. To analyze corresponding histone modifications at the promoter-regions of NKX2-1 and HEY1 we performed ChIP using antibodies for activatory H3K4me3 (mediated by MLL) and inhibitory H3K27me3 (mediated by EZH2). Analysis of the NKX2-1 promoter showed presence of H3K27me3 in both SU-DHL-4 and SU-DHL-5, while H3K4me3 was restricted to SU-DHL-5 (Fig. 3B). The HEY1 promoter exclusively bore H3K27me3 in both cell lines (Fig. 3B). The presence of both types of analyzed H3-trimethylations at the NKX2-1 locus in SU-DHL-5 indicates aberrant bivalent chromatin configuration known to prime developmental genes for activation and thus likely to favor NKX2-1 expression in this cell line [29]. Moreover, profiling data indicated overexpression of the MLL complex component AF9 and reduced expression of ASB2 which mediates degradation of MLL (Table 1) [17], [30].

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Figure 3. MLL and HIST1.

(A) RQ-PCR analysis of MLL in SU-DHL-5 (expression level was set to unity) and in leukemia/lymphoma control cell lines. (B) RQ-PCR analysis of MLL, NKX2-1 and HEY1 in siRNA-treated SU-DHL-5 cells (left). ChIP analysis of the NKX2-1 and HEY1 promoters in SU-DHL-5 and SU-DHL-4 (for control) showed representation of particular histone H3 modifications, as shown by PCR amplification of genomic fragments (right). Untreated genomic DNA served as positive control, NTC: no template control. (C) Copy number analysis by genomic profiling indicates presence of aberrations at MLL at 11q23. Inserts show an enlargement of chromosome 11 obtained by SKY karyotyping and an enlargement of the MLL locus obtained by genomic profiling. (D) FISH analysis of the MLL locus in SU-DHL-5 (below) using BAC probes as indicated above. The results show one wild type allele and one amplified MLL locus. (E) RT-PCR analysis of MLL fusion genes in SU-DHL-5 and particular positive and negative control cell lines. TEL expression served as control, NTC: no template control. (F) Copy number analysis by genomic profiling of chromosome 6 indicates extended deletions at both arms. The HIST1 locus maps to the breakpoint region at 6p22. Genes located in deleted regions include JARID2, SOX4, HMGN3, AKAP7 and MAP3K4. Insert shows chromosomes 6 analyzed by SKY karyotyping indicating rearrangements at both chromosomes. (G) FISH analysis of the histone gene cluster HIST1 at 6p22 (below) using painting probe and BACs as indicated above. (H) RQ-PCR analysis of selected histone genes in SU-DHL-5 and SU-DHL-4 for control (left). PAGE analysis of histone proteins in three DLBCL cell lines (middle) demonstrates elevated levels in SU-DHL-5. Western blot analysis of H2B, H2Bub1 and ERK (for control) in four DLBCL cell lines (right) demonstrates elevated levels in SU-DHL-5.

https://doi.org/10.1371/journal.pone.0061447.g003

In leukemia MLL is frequently activated by chromosomal aberrations at 11q23 resulting in amplifications or diverse fusion genes [31]. We looked for chromosomal rearrangements in SU-DHL-5 by genomic profiling, finding duplication of MLL accompanied by deletion of its immediately telomeric region (Fig. 3C). FISH analysis confirmed that MLL gain was coupled with the downstream deletion (Fig. 3D). SKY results excluded chromosomal translocations at 11q23 (Fig. 1D), and RT-PCR analysis of the most prolific MLL-fusion transcripts excluded cryptic fusions with AF4, AF6, AF9, AFX and ENL (Fig. 3E). Collectively, our results show that genomic copy number gain of wild type MLL underlies overexpression of MLL in SU-DHL-5 cells.

Furthermore, overexpression of histone H3E (Table 1) correlated with rearrangements of chromosome 6 targeting histone gene cluster 1 (HIST1) at 6p22 as indicated by genomic array data (Fig. 3F). FISH analysis using probes covering HIST1 combined with a painting probe for chromosome 6 confirmed the breakpoint nearby, lying nevertheless outwith the gene cluster (Fig. 3G). The FISH results are consistent with the SKY data showing two derivative chromosomes 6 harboring deletions at 6p22 and 6q13, respectively (Fig. 1D, 3F). Quantification of several histone RNA species demonstrated abundant expression of H1C, H2BB, H3E and H3H in SU-DHL-5 as compared to SU-DHL-4 (Fig. 3H), consistent with coordinate gene activation at the histone locus at 6p22 by nearby chromosomal rearrangement. Accordingly, SU-DHL-5 contained higher levels of core-histone proteins as shown by SDS-PAGE (Fig. 3H). Moreover, histone H2B when analyzed by Western blot showed raised protein expression and enhanced modification with a single ubiquitin at lysine 120, subsequently named H2Bub1 (Fig. 3H). The latter is of special interest because H2Bub1 reportedly promotes trimethylation of H3K4 by MLL [32], suggesting collaborative activities of the chromosomal aberrations targeting MLL and HIST1.

H2B ubiquitinylation levels are regulated by counteracting ubiquitin-transferases (RNF20, RNF40) and ubiquitin-specific proteases (USPs) [33]. Profiling data indicated repression of USP46 in SU-DHL-5 as confirmed by RQ-PCR analysis in comparison to control cell lines (Fig. 4A). However, USP46 expression was not regulated by the transcriptional repressor HEY1, as analyzed by knockdown and overexpression experiments (Fig. 4A). RQ-PCR analysis of RNF20 and RNF40 showed upregulation in SU-DHL-5 relative to control cell lines (Fig. 4B). Knockdown of RNF20 and RNF40 by siRNA treatment inhibited expression of NKX2-1 (Fig. 4C) demonstrating that these H2B ubiquitin-transferases support expression of that homeobox gene. Moreover, siRNA-mediated knockdown of NKX2-1 inhibited expression of RNF40 (Fig. 4C), showing that expression of RNF40 is supported by NKX2-1 and thus presence of reciprocal regulation.

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Figure 4. Signaling pathways.

(A) RQ-PCR analysis of NKX2-1 and HEY1 in SU-DHL-5 cells treated with TNFa for 1 h (left) and 4 h (right). (B) RQPCR analysis of NKX2-1 and HEY1 in SU-DHL-5 cells treated with NFkB-inhibitor for 4 h (left) and 16 h (right). (C) RQ-PCR analysis of PRKCE in DLBCL cell lines (left) and in SU-DHL-5 cells treated for siRNA-mediated knockdown of NKX2-1 (middle). ChIP analysis of the PRKCE promoter in SU-DHL-5 and SU-DHL-4 (for control) demonstrated binding of NKX2-1 as shown by PCR amplification of genomic fragments (right, below). Untreated genomic DNA served as positive control, NTC: no template control. (D) RQ-PCR analysis of NKX2-1 and HEY1 in SU-DHL-5 cells treated with TGFb for 1 h (left) and 16 h (right). (E) RQ-PCR analysis of NKX2-1 and HEY1 in SU-DHL-5 cells treated with inhibitory antibody directed against TFGb for 16 h showed no significant differences. (F) RQ-PCR analysis of NKX2-1 and HEY1 in SU-DHL-5 cells treated with siRNA directed against SMAD3 and SMAD9 (above). Immuno-fluorescence analysis of SMAD3 (green) and NKX2-1 (red) in SU-DHL-5 demonstrates nuclear colocalization as compared to the DAPI-stained (blue) nucleus (below). (G) RQ-PCR analysis of NKX2-1 (left) and HEY1 (middle) in SU-DHL-5 cells treated with IL4, BMP4, IL10 and WNT5B for 16 h. RQPCR analysis of NKX2-1 and HEY1 in SU-DHL-5 cells treated for siRNA-mediated knockdown of STAT3 (right). (H) Heatmap of PDE genes in 6 DLBCL cell lines. PDE3B, PDE4A, PDE4B, PDE6D and PDE9A are highlighted. Red indicates high expression levels, green low, and black medium levels. (I) RQPCR analysis of NKX2-1 and HEY1 in SU-DHL-5 cells treated with cAMP, cGMP and sildenafil for 4 h and 16 h. (K) RQ-PCR analysis of NKX2-1 and HEY1 in SU-DHL-5 cells treated for siRNA-mediated knockdown of PDE6D (left). RQ-PCR analysis of NOS1 in DLBCL cell lines (middle) and in SU-DHL-5 cells treated for siRNA-mediated knockdown of NKX2-1 (right). (L) RQ-PCR analysis of PDE4A and PDE6D in SU-DHL-5 cells treated for siRNA-mediated knockdown of HEY1.

https://doi.org/10.1371/journal.pone.0061447.g004

These results prompted us to look for additional deregulated histone modifiers which may contribute to the aberrant chromatin structure at NKX2-1. Inhibitory trimethylation of H3K27 is conducted by polycomb repressor complex (PRC) 2 (containing EZH2, JARID2, HOPX, E2F6), and is removed by histone methylase JMJD3 [34], [35]. The expression level of JMJD3 was elevated in SU-DHL-5 as shown by RQ-PCR analysis (Fig. 4D). However, JMJD3 was not regulated by NKX2-1 (Fig. 4D). The expression of EZH2 was not significantly altered in SU-DHL-5 as compared to control cell lines (Fig. 4E). Nevertheless, treatment of SU-DHL-5 with EZH2/PRC2 inhibitor DZNep resulted in enhanced transcription of both NKX2-1 and HEY1 (Fig. 4E). These results are consistent with our ChIP data, showing presence of EZH2-mediated H3K27me3 at the promoter regions of both genes (Fig. 3B). Although microarray expression of the gene encoding PRC2 component JARID2 was normal, genomic array data showed monoallelic deletion at 6p22 (Fig. 3F). Sequence data of SU-DHL-5 cells (provided by the BROAD Institute, Table 1) showed mutation of JARID2. Thus SU-DHL-5 is hemizygous for mutated JARID2. Homeobox only protein (HOPX) is associated with PRC2, regulating its capacity for repression [36], [19]. SU-DHL-5 showed remarkably high levels of HOPX expression as indicated by profiling data (Table 1) and confirmed by RQ-PCR results of lymphoma cell lines (Fig. 4F). SiRNA-mediated knockdown of HOPX resulted in reduced expression of NKX2-1 (Fig. 4G), showing that HOPX activates NKX2-1 transcription. But this activation was not reciprocal, since reduction of NKX2-1 was unaccompanied by altered HOPX levels (Fig. 4G). However, genomic copy number data and SKY results excluded genomic aberrations at the HOPX locus (Fig. S2). Moreover, examination of DNA methylation of a conspicuous CpG island at the HOPX locus (CpG 109) excluded an abnormal configuration (Fig. S3), leaving the mechanism of this striking overexpression elusive. HOPX is associated with E2F6 which showed reduced expression levels in SU-DHL-5 (Fig. 4H). Interestingly, this reduction was mediated by HEY1 as shown by knockdown experiments which led to overexpression of E2F6 (Fig. 4H). Together, our results demonstrate a significant role for several chromatin modifiers underlying NKX2-1 activation in SU-DHL-5. These regulatory interactions are partly reciprocal and constitute feedback-loops, probably resulting in enhanced and stabilized gene activities.

Signaling pathways differentially regulate expression of NKX2-1 and HEY1

The results of comparative expression analysis in SU-DHL-5 indicated deregulation of several signaling components belonging to a variety of pathways, including TNFa/NFkB/PRKC, TGFb/BMP/SMAD, IL4/STAT3 and NOS1/cAMP/cGMP/phosphodiesterases (PDEs) which plausibly contribute to NKX2-1 and/or HEY1 expression (Table 1). These pathways were analyzed in subsequent experiments.

Treatment of SU-DHL-5 cells with TNFa for 1 h or 4 h resulted in activated transcription of both NKX2-1 and HEY1 (Fig. 5A). In accordance with this result, treatment with NFkB inhibitor reduced expression of both genes (Fig. 5B), demonstrating a positive role for TNFa/NFkB-signaling in transcriptional regulation. Furthermore, expression of protein kinase C variant E (PRKCE) was enhanced in SU-DHL-5 when compared to control cell lines (Table 1, Fig. 5C). This may be of interest because the activity of NFkB is regulated by PRKC. SiRNA-mediated reduction of NKX2-1 was accompanied by downregulation of PRKCE (Fig. 5C), indicating a positive regulatory role for this NKL homeobox gene. Sequence analysis of the upstream region of PRKCE (UCSC genome browser, release GRCh37/hg19) revealed a binding site for NKX2-1 at -25.832 bp. ChIP analysis of this site confirmed binding of NKX2-1 antibody, demonstrating direct activation of PRKCE by NKX2-1 (Fig. 5C). This kind of gene regulation represents positive feedback activation which activates involved genes, demonstrating a complex gene regulatory network contributing to ectopic NKX2-1 expression.

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Figure 5. Signaling pathways.

(A) RQ-PCR analysis of NKX2-1 and HEY1 in SU-DHL-5 cells treated with TNFa for 1 h (left) and 4 h (right). (B) RQ-PCR analysis of NKX2-1 and HEY1 in SU-DHL-5 cells treated with NFkB-inhibitor for 4 h (left) and 16 h (right). (C) RQ-PCR analysis of PRKCE in DLBCL cell lines (left) and in SU-DHL-5 cells treated for siRNA-mediated knockdown of NKX2-1 (middle). ChIP analysis of the PRKCE promoter in SU-DHL-5 and SU-DHL-4 (for control) demonstrated binding of NKX2-1 as shown by PCR amplification of genomic fragments (right, below). Untreated genomic DNA served as positive control, NTC: no template control. (D) RQ-PCR analysis of NKX2-1 and HEY1 in SU-DHL-5 cells treated with TGFb for 1 h (left) and 16 h (right). (E) RQ-PCR analysis of NKX2-1 and HEY1 in SU-DHL-5 cells treated with inhibitory antibody directed against TFGb for 16 h showed no significant differences. (F) RQ-PCR analysis of NKX2-1 and HEY1 in SU-DHL-5 cells treated with siRNA directed against SMAD3 and SMAD9 (above). Immuno-fluorescence analysis of SMAD3 (green) and NKX2-1 (red) in SU-DHL-5 demonstrates nuclear colocalization as compared to the DAPI-stained (blue) nucleus (below). (G) RQ-PCR analysis of NKX2-1 (left) and HEY1 (middle) in SU-DHL-5 cells treated with IL4, BMP4, IL10 and WNT5B for 16 h. RQ-PCR analysis of NKX2-1 and HEY1 in SU-DHL-5 cells treated for siRNA-mediated knockdown of STAT3 (right). (H) Heatmap of PDE genes in 6 DLBCL cell lines. PDE3B, PDE4A, PDE4B, PDE6D and PDE9A are highlighted. Red indicates high expression levels, green low, and black medium levels. (I) RQ-PCR analysis of NKX2-1 and HEY1 in SU-DHL-5 cells treated with cAMP, cGMP and sildenafil for 4 h and 16 h. (K) RQ-PCR analysis of NKX2-1 and HEY1 in SU-DHL-5 cells treated for siRNA-mediated knockdown of PDE6D (left). RQ-PCR analysis of NOS1 in DLBCL cell lines (middle) and in SU-DHL-5 cells treated for siRNA-mediated knockdown of NKX2-1 (right). (L) RQ-PCR analysis of PDE4A and PDE6D in SU-DHL-5 cells treated for siRNA-mediated knockdown of HEY1.

https://doi.org/10.1371/journal.pone.0061447.g005

Treatment of SU-DHL-5 cells with TGFb resulted in enhanced transcription of both NKX2-1 and HEY1 after 16 h (Fig. 5D). Interestingly, after 1 h of treatment the expression of HEY1 raised concentration-dependend while NKX2-1 showed no change in transcript levels at this time point (Fig. 5D). These results may indicate that HEY1 is a direct target of TGFb-signaling, contrasting with the delayed and thus indirect mechanism of NKX2-1 regulation. However, treatment of SU-DHL-5 with inhibitory anti-TGFB showed no effect, excluding autocrine activation (Fig. 5E). SiRNA-mediated knockdown of SMAD3 or SMAD9 reduced transcription of HEY1 while that of NKX2-1 remained unperturbed (Fig. 5F), supporting the direct regulation of HEY1 by TGFb/SMAD-signaling. Of note, according to the UCSC genome browser (release GRCh37/hg19) the promoter-region of HEY1 contains binding-sites for SMAD proteins which are colocated with those of NKX2-1 (Fig. 2C): a significant observation, since SMAD3 protein has been shown to interact with NKX2-1 [37]. Immunostaining of NKX2-1 and SMAD3 in SU-DHL-5 cells consistently revealed colocalization of both TFs (Fig. 5F). Therefore, our results indicate that in SU-DHL-5 cells NKX2-1 and SMAD3 coactivate HEY1 by protein-protein interaction and direct binding to the promoter region.

The impact of additional pathways in expression of NKX2-1 and HEY1 was analyzed by treatment of SU-DHL-5 cells with IL4, BMP4, IL10 and WNT5B for 16 h (Fig. 5G). The most significant effects on NKX2-1 were observed after stimulation with IL4 and for HEY1 with BMP4. BMP4 signaling is mediated by SMAD proteins just like TGFb, showing consistent results for HEY1 regulation. IL4-signaling is mediated by STAT3 which is upregulated in SU-DHL-5 (Table 1). Accordingly, siRNA-mediated knockdown of STAT3 reduced expression of NKX2-1 while HEY1 transcription was not significantly affected (Fig. 5G). These results support an activating role for IL4/STAT3-signaling on NKX2-1 and for BMP4/SMAD-signaling on HEY1 in SU-DHL-5.

Finally, we analyzed the impact of cAMP/cGMP-signaling in expression of NKX2-1 and HEY1. This pathway stood out due to overexpression of NOS1 (synthesizes nitric oxide activating guanylate cyclase) and reduced expression levels of several PDEs as indicated by comparative profiling data (Table 1) and illustrated by a heatmap for PDE-expression (Fig. 5H). Furthermore, overexpressed DDAH1 (Table 1) encodes an inhibitor of the negative regulator ADMA for NOS1. Therefore, SU-DHL-5 cells were treated with cAMP, cGMP and cGMP-specific PDE-inhibitor sildenafil and subsequently quantified for NKX2-1 and HEY1 transcription (Fig. 5I). After 4 h expression of NKX2-1 rose significantly after treatment with sildenafil, while HEY1 rose with cAMP. After 16 h expression of NKX2-1 peaked in response to treatment with sildenafil and cGMP, while HEY1 responded maximally to treatment with cAMP and to a lesser extent with cGMP and sildenafil (Fig. 5I). These data suggest that expression of NKX2-1 and HEY1 is primarily regulated via cGMP and cAMP, respectively. Accordingly, siRNA-mediated knockdown of cGMP-specific PDE6D resulted in enhanced expression of NKX2-1 while HEY1 remained unchanged (Fig. 5K). The enhanced expression of NOS1 in SU-DHL-5 as compared to control cell lines was confirmed by RQ-PCR analysis (Fig. 5K). SiRNA-mediated knockdown of NKX2-1 resulted in strong reduction of NOS1, indicating an activatory role for NKX2-1 (Fig. 5K). However, ChIP analysis excluded direct binding of NKX2-1 to the promoter region of NOS1 (data not shown), suggesting an indirect activation mechanism. Finally, the transcriptional repressor HEY1 was found to underly PDE4A repression, while non-participant in PDE6D regulation, as shown by overexpression experiments (Fig. 5L). Thus, deregulated expression of PDEs (PDE6D, PDE4A) and NOS1 via HEY1 and NKX2-1 indicate feedback regulation of both TFs.