Epigenetic Upregulation of lncRNAs at 13q14.3 in Leukemia Is Linked to the <i>In Cis</i> Downregulation of a Gene Cluster That Targets NF-kB

<div><p>Non-coding RNAs are much more common than previously thought. However, for the vast majority of non-coding RNAs, the cellular function remains enigmatic. The two long non-coding RNA (lncRNA) genes <i>DLEU1</i> and <i>DLEU2</i> map to a critical region at chromosomal band 13q14.3 that is recurrently deleted in solid tumors and hematopoietic malignancies like chronic lymphocytic leukemia (CLL). While no point mutations have been found in the protein coding candidate genes at 13q14.3, they are deregulated in malignant cells, suggesting an epigenetic tumor suppressor mechanism. We therefore characterized the epigenetic makeup of 13q14.3 in CLL cells and found histone modifications by chromatin-immunoprecipitation (ChIP) that are associated with activated transcription and significant DNA-demethylation at the transcriptional start sites of <i>DLEU1</i> and <i>DLEU2</i> using 5 different semi-quantitative and quantitative methods (aPRIMES, BioCOBRA, MCIp, MassARRAY, and bisulfite sequencing). These epigenetic aberrations were correlated with transcriptional deregulation of the neighboring candidate tumor suppressor genes, suggesting a coregulation <i>in cis</i> of this gene cluster. We found that the 13q14.3 genes in addition to their previously known functions regulate NF-kB activity, which we could show after overexpression, siRNA–mediated knockdown, and dominant-negative mutant genes by using Western blots with previously undescribed antibodies, by a customized ELISA as well as by reporter assays. In addition, we performed an unbiased screen of 810 human miRNAs and identified the <i>miR-15/16</i> family of genes at 13q14.3 as the strongest inducers of NF-kB activity. In summary, the tumor suppressor mechanism at 13q14.3 is a cluster of genes controlled by two lncRNA genes that are regulated by DNA-methylation and histone modifications and whose members all regulate NF-kB. Therefore, the tumor suppressor mechanism in 13q14.3 underlines the role both of epigenetic aberrations and of lncRNA genes in human tumorigenesis and is an example of colocalization of a functionally related gene cluster.</p> </div

Hypomethylation at the TSS of the large ncRNAs affects the majority of CLL patients independent of 13q gene dosage and IGHV mutation status.

<p>(A) For validation of DNA-hypomethylation at different CpG islands in 13q14.3 (x-axis; for localization see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003373#pgen-1003373-g001" target="_blank">figure 1</a>), quantitative measurement with BioCOBRA and MassARRAY (“Rb”) technology was performed in B-cells of healthy donors (“h”, n = 15–19, white diamonds) and PBMCs of CLL patients (“p”, n = 47–82, black diamonds; “miR” = 308 bp fragment spanning miR-15a; “Rb” = intragenic CpG island). (B, C) CLL patients with different genomic aberrations show different degree of hypomethylation that is distinct from healthy probands younger (“young”) or older (“old”) than 45 years. DNA-methylation was quantified with BioCOBRA (D6) and MassARRAY (E6). Analyzed were patients with normal karyotype (“nk”), deletion of 13q14.3 (“13q”), deletion of 11q22-q23 (“11q”), deletion of 17p (“17p”) or combinations thereof. Statistics were performed using Wilcoxon rank sum test (*** p<0.001, ** p 0.001 to 0.01, * 0.01 to 0.05 and ns not significant p>0.05). (D) DNA-methylation of D6 or E6 is aberrant in 58/61 CLL patients. Black diamonds: Patient samples with two copies of the critical region in 13q, grey diamonds: samples with deletion of one copy of 13q14.3, white diamonds: healthy donor samples. Dashed lines represent lowest levels of methylation of non-malignant B-cells.</p

The critical region at 13q14.3 displays relaxed chromatin at the transcriptional start site (TSS) of the long non-coding RNA genes <i>DLEU1</i> and <i>DLEU2</i> in CLL cells.

<p>The critical region on 13q14.3 was analyzed for DNA-methylation (lanes 1–3), distribution of histone modifications (lanes 4, 5) and gene expression (lanes 6, 7). DNA-hypomethylation was detected in CLL cells at the transcriptional start sites of the lncRNA genes <i>DLEU1</i> and <i>DLEU2</i> variant <i>Alt1</i>. This finding coincides with enrichment of H3K4me2 and depletion of macroH2A, corroborating relaxation of 13q14.3 in CLL. While expression of the protein coding genes and <i>DLEU2</i> is decreased in CLL cells, lncRNA genes <i>DLEU1</i> and <i>DLEU2/Alt1</i> show upregulation, suggesting a direct regulation by DNA-methylation. Base pair positions on top refer to genome GRCh37 build hg18. The conserved CpG islands (A–E) are shown in green, less stringent CpG islands shown in light green. <i>Lane 1</i>: Methyl-CpG Immunoprecipitation (MCIp) allowed semi-quantitative DNA-methylation analysis in non-malignant B-cells sorted from healthy donors (n = 7) and CLL samples (n = 25). Precipitated DNA was hybridized onto a custom tiling microarray covering the 13q14.3 critical region. Depicted is the median log2 fold enrichment of CLL samples from which the median of log2 fold enrichment of healthy donor B-cell samples has been subtracted. <i>Lane 2</i>: Restriction digest-based analysis of DNA-methylation (aPRIMES) was performed in CpG islands C–E at 13q14.3 at 1 kbp resolution. Shown is the median log2 signal intensity of CLL patients from which the median of non-malignant B cell samples has been subtracted. <i>Lane 3</i>: Hypomethylation at D6 and E6 was validated by BioCOBRA and MassARRAY/Sequenome analyses (for details see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003373#pgen-1003373-g002" target="_blank">Figure 2</a>). Shown is the difference of the median percentage of methylation in CLL cells and non-malignant B cells. <i>Lanes 4,5</i>: ChIP was performed for macroH2A (lane 4) and H3K4me2 (lane 5). Precipitated DNA was quantified using qPCR. Enrichment was normalized to non-specific IgG and two control promoters (CDH12, HK2) that displayed similar enrichment for the two histone marks in CLL samples (n = 7) and peripheral blood mononuclear cells (PBMC) from healthy probands (n = 5). Enrichment was calculated as median log2 fold enrichment in precipitate vs. input for CLL samples after subtraction of enrichment from non-malignant B-cells. <i>Lane 6</i>: Gene expression profiling (GEP) was performed using bead chip arrays (Illumina) in CLL patients (n = 25) and sorted B-cells from healthy donors (n = 9). Plotted is the difference of log fold changes of CLL samples and non-malignant B cells. <i>Lane 7</i>: QRT-PCR for gene expression analysis of 13q14.3 candidate genes (for details see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003373#pgen-1003373-g003" target="_blank">Figure 3</a>).</p

<i>MiR-15/miR-16</i> family is the strongest inducer of NF-kB.

<p>(A) NF-kB induction by miRNA families (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003373#pgen.1003373.s011" target="_blank">Table S5</a> for complete list) was measured using NF-kB luciferase reporter assay. Median z-scores are indicated by black horizontal lines. Each dot represents one miRNA-mimic. (B) Transfection of miRNA-mimics <i>miR-15a</i> and <i>miR-16</i> induces NF-kB activity as compared to unspecific control siRNA. Error bars represent standard deviation of triplicate measurements. (C–E) Upon transfection of miRNA mimic <i>miR-15a</i> and <i>miR-16</i>, expression of NF-kB target genes measured by qRT-PCR is induced in HEK293FT, MCF7 and MDA-MB-231 cells. (F) 3′UTRs of genes involved in the modulation of NF-kB activity and shown to be <i>miR-15a</i> and/or <i>miR-16</i> targets were cloned into luciferase reporter plasmids. After co-transfection with miR-mimics into HEK cells, firefly luciferase activity was measured and normalized to renilla luciferase activity and empty luciferase plasmid as transfection and background control, respectively, and to no-template control (“NTC”). Data is depicted on a logarithmic scale to balance lower and higher activity compared to control, error bars show standard error of the mean of three independent experiments.</p

Model of the tumor suppressor mechanism localized in 13q14.3.

<p>(A) Regions in CpG islands D and E that are DNA-methylated in non-malignant B-cells (left) become demethylated in the vast majority of CLL patients (right). This coincides with relaxed chromatin characterized by absence of macroH2A and enrichment of H3K4me2 at the promoters of the lncRNA genes <i>DLEU1</i> and <i>DLEU2/Alt1</i>. (B) DNA-hypomethylation is correlated with transcriptional upregulation of splicing variants of the two lncRNA genes <i>DLEU1</i> and <i>DLEU2</i> and inversely correlated with the protein-coding genes in 13q14.3. No correlation could be found with levels of mature <i>miR-15a</i> and <i>miR-16</i>, probably because these transcripts are also deregulated by a posttranscriptional processing defect in CLL cells (Allegra, manuscript submitted). (C) Candidate genes localized in the critical region in 13q14.3 are functionally related and all modulate NF-kB signalling, albeit with different impact. Nuclear transporter KPNA3 is binding NF-kB components p65 and p50 and is therefore likely a positive regulator like e.g. RFP2. RFP2 binds to C13ORF1 and induces NF-kB activity via the canonical pathway components p50 and p65, for which its ubiquitin ligase actvity is required. The miRNA genes <i>miR-15a</i> and <i>miR-16-1</i> were identified together with other members of this miR family to be among the strongest activators of NF-kB activity. Previously they have been shown to both modulate cell cycle regulators and inhibit NF-kB via <i>TAB3/TAK1. DLEU7</i> has recently also been reported to inhibit NF-kB by binding and inactivating the <i>TACI/BCMA</i> receptors.</p

Downregulation of 13q14.3 candidate tumor suppressor genes and upregulation of lncRNA genes <i>DLEU1</i> and <i>DLEU2/Alt1</i> correlates with DNA-methylation.

<p>(A–C) Expression of the protein-coding genes <i>KPNA3, C13ORF1</i> and <i>RFP2</i>, the miRNA genes <i>miR-15a</i> and <i>miR-16-1</i> and the lncRNA transcripts from genes <i>DLEU1, DLEU2</i> and <i>alternative transcript Alt1</i> were quantified with qRT-PCR in CLL cells from patients with retention of both copies of 13q14.3 (“p”, n = 34; see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003373#pgen.1003373.s007" target="_blank">Table S1</a>) and compared to B-cells from healthy donors (“h”, n = 20). Mean expression is indicated by a black horizontal line. “HKG” = housekeeping genes, average of <i>PGK, LMNB1</i> and <i>PPIA</i>. (D, E) DNA-methylation levels correlate with transcript levels of genes in 13q14.3: While expression (y-axis) of candidate tumor suppressor genes is directly correlated to DNA-methylation of regions D6 and E6 (x-axis; left panel), lncRNA genes are anti-correlated (right panel). Pearson correlation coefficients are color-coded, blue = negative, yellow = positive correlation. (F) Pearson coefficients of correlation of gene expression (rows) and DNA-methylation at D6 and E6 (columns), colour coded (see legend). DLEU1a = exon1 to exon4; DLEU1b = exon 1 to exon 2; ALT1a/b = exon1, two different amplicons (G) Significance values of Pearson correlation coefficients (t-distribution), values p<0.05 are coded green. (H) The bidirectional promoter of <i>DLEU1</i> and <i>DLEU2</i> and the flanking CpG island was cloned into pCpGL luciferase vector, either including D6 (orange) or excluding D6 (green). (I) Constructs depicted in (H) were either methylated <i>in-vitro</i> using SSsI methylase (m, dark bars) or left unmethylated (u, light bars) and subsequently transfected into Mec1, Granta519 and HeLa cells and luciferase activity quantified. Grey: Luciferase CMV expression vector (V) not containing CpGs was used as negative control, green: excluding D6 region, orange: including D6 region. Error bars signify standard deviation of 2 independent experiments, each performed in duplicate. No changes were detected for E6 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003373#pgen.1003373.s004" target="_blank">Figure S4G</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003373#pgen.1003373.s004" target="_blank">S4J</a>). (J) Binding of the DNA-methylation sensitive chromatin reader CTCF was enriched in CLL samples both in the D6 and E6 regions compared to non-malignant B-cells. CTCF-bound chromatin was immunoprecipitated and quantitated with qPCR. Specific binding was shown by quantification of a sequence not bound by CTCF 2 kb upstream of the DM1 insulator (“ctr”). Statistics were performed using Wilcoxon rank sum test (*** p<0.001, ** p 0.001 to 0.01, * 0.01 to 0.05 and ns not significant p>0.05).</p