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

Abstract

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 DLEU1 and DLEU2 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 DLEU1 and DLEU2 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 in cis 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 miR-15/16 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.

Non-coding RNAs as direct and indirect modulators of epigenetic regulation

“…First, genomic loci producing non-coding transcripts are subject to epigenetic regulation similar to that of protein-coding genes. [38][39][40][41][42] Therefore, as with coding genes, environmental influences can lead to changes in their expression and to functional disturbances. We can include in this category the transcription of lncRNAs designated as "signals," 12 as these are examples of activity-dependent, sequence-independent changes in gene expression.…”

Kruppel-like factor 4 (KLF4) inactivation in chronic lymphocytic leukemia correlates with promoter DNA-methylation and can be reversed by inhibition of NOTCH signaling

“…1 In addition, CLL cells harbor genome-wide aberrant DNA-methylation. 2 Of interest, in CLL, DNA-methylation of single CpGs overall correlated only weakly with gene expression. However, no detailed analysis of DNA-methylation on single gene level was performed that would also take larger regions into account.…”

Long non-coding RNA RP5-833A20.1 inhibits proliferation, metastasis and cell cycle progression by suppressing the expression of NFIA in U251 cells

“…lncRNAs may be classified according to their mode of action and their functions in cells, including genetic imprinting (14,15), modulating the cancer epigenome (9), serving as molecular decoys (16) and post-transcriptional regulation (10). Accumulating evidence has shown that lncRNA expression profiling may facilitate the diagnosis and prognosis of human cancer, including bladder cancer (17), leukemia (18), breast cancer (9) and rectal cancer (19), suggesting that lncRNAs may serve as effective therapeutic targets for intervention. Previous evidence indicates that lncRNAs are important in the pathogenesis of glioma, including HOTAIR (20), H19 (21) and Linc-POU3F3 (8 been annotated, only a few lncRNAs have been functionally characterized in glioma.…”