Abstract IA3: Regulatory networks in onco-lncRNAomics: Cis-regulation and non-conservation
Authors: Lipovich Leonard 
Published: April 2013

Global studies of the transcriptome reveal that approximately half of human transcriptional units (genes) encode solely non-protein-coding RNAs (ncRNAs), which, in addition to microRNAs and other small RNAs, include the far more numerous long non-coding RNAs (lncRNAs). While the lncRNAome (the total set of all lncRNAs) is increasingly recognized as a rich repository of novel regulators, functional information is still lacking for the majority of lncRNAs (Lipovich et al 2010). We used transcript-to-genome alignments to identify and manually curate 4,511 human cis-antisense gene pairs, each comprised of two distinct genes mapping to opposite strands of the same locus with overlapping exons. These pairs contained 3,769 lncRNA genes, most of which (2,818) resided in coding-noncoding (mRNA-lncRNA) pairs, suggesting that antisense lncRNAs may be cis-regulators of protein-coding genes. 803 lncRNAs from cis-antisense loci contained, in addition to nonrepetitive exons, exonic primate-specific Alu repeats, indicating that lncRNAs at cis-antisense loci have undergone gene structure modification specifically in primate evolution. We mapped 410 SNPs to exon-exon, cis-antisense overlaps. Through their simultaneous localization in exons of two overlapping genes, these SNPs may contribute to multigenic or contiguous-gene phenotypes.

1,213 cis-antisense pairs had both sense and antisense transcripts represented by distinct reliable probesets on Affymetrix U133 microarrays. To search for lncRNA regulators of breast cancer gene expression, we first interrogated lncRNA-mRNA co-expression at these loci in a human Affymetrix breast cancer dataset (GSE4922). Co-expression of cis-encoded lncRNAs and mRNAs was observed in half of the cis-antisense pairs. Simultaneous detection of sense and cis-antisense RNAs for each antisense pair in cancer samples opens new avenues for expression-based diagnostic marker development and for functional investigations of cis-antisense regulation involving non-conserved lncRNAs. Four co-expressed lncRNA-mRNA cis-antisense pairs contained lncRNAs which had positional equivalents (Engström et al 2006), but not conserved orthologs, in the mouse genome. BC042008, one such non-conserved lncRNA, was co-expressed in breast cancer with its partner ZEB1, a known repressor of IL-2 and plakophylin, the latter in cancer progression. This implies biomarker and even functional potential for human cis-antisense lncRNAs that are not conserved in mouse.

The abundance of lncRNAs in breast cancer cis-antisense data prompted us to interrogate the complete human lncRNAome, including though not limited to cis-antisense pairs, for breast cancer function. Recently, we generated a catalog of human lncRNAs (Jia et al 2010). By quantitative RTPCR, we identified estrogen-activated (DLEU1, Deleted in Leukemia 1) and estrogen-repressed (BC038211) lncRNAs that are endogenously expressed in human MCF7 cells. Among Affymetrix probesets for 786 genes significantly (p&lt;0.01) associated with estrogen receptor (ER) status of clinical breast tumor samples (Ivshina et al 2006), we observed 10 lncRNAs, including XIST. To directly identify ERα-targeted lncRNAs, we intersected genomewide ChIP-PET ERα TFBS localization (Lin et al 2007) with the MCF7 transcriptome. Results included a novel lncRNA initiating at the TBX2 bidirectional promoter in the BCAS3 (breast carcinoma amplified sequence 3) locus. This non-conserved lncRNA with a nearby ERα binding site has a positional equivalent but not a conserved ortholog in mouse. Having previously used our lncRNA catalog to develop and implement custom human lncRNA expression analysis microarrays in multiple cancer cell lines, post-mortem brain, and surgically resected brain samples, we are currently deploying these arrays to find additional estrogen-responsive lncRNAs in the MCF7 transcriptome.

To understand the regulatory network placement of lncRNAs, including those that lack conservation and reside at cis-antisense gene pairs, we have addressed, in systems beyond breast cancer, two fundamental questions: Which transcription factors directly regulate lncRNAs by binding directly at promoters or intragenic regions of lncRNA genes? And what are the downstream effects of altering the cellular levels of these lncRNAs? To address the first question, we annotated genomewide TFBS datasets for several transcription factors, including mouse Oct4 and Nanog (key regulators of pluripotency in embryonic stem cells) and human REST. By manual identification and protein-coding capacity assessment of the cDNA- or EST-supported transcriptional unit nearest to each TFBS, regardless of whether the unit corresponded to a known gene, we found that 10% of mouse Oct4 and Nanog proximal targets in ES cells are lncRNAs, while 23% of human REST binding sites proximally target lncRNAs. We validated specific lncRNA direct targets of these transcription factors: perturbation of the transcription factors altered the levels of the targeted lncRNAs (Johnson et al 2009).

To examine the second question, we perturbed the lncRNAs Miat, a direct target of Oct4, and AK141205, a direct target of Nanog, in mouse ES cells by RNAi and overexpression. These perturbations caused distinct changes in Oct4 and Nanog mRNA levels, lineage-specific gene expression, and pluripotency, highlighting the lncRNA Miat as a novel Oct4 co-activator in a positive feedback loop of the ES regulatory network (Sheik Mohamed et al 2010). We also used a human cancer cell line system, SH-SY5Y neuroblastoma cells, where we focused on the non-conserved, primate-specific lncRNA BDNFOS, which shares a bidirectional promoter with LIN7C and a cis-antisense overlap with BDNF. Previously, with microarrays and quantitative RTPCR (Lipovich and Loeb, ms in prep), we demonstrated reciprocal patterns of BDNF and BDNFOS transcription in highly active regions of human neocortex removed as a treatment for intractable seizures. RNAi-mediated knockdown of BDNFOS increased BDNF expression, suggesting that BDNFOS directly downregulates BDNF. Therefore, RNAi and overexpression of lncRNAs can help establish both cis- (e.g. BDNF) and trans-regulatory (e.g. Miat) lncRNA network functions.

Finally, to investigate whether the human onco-lncRNAome may function through translation of the lncRNAs, rather than purely at the RNA level, we performed proteogenomic integration of tandem mass spectrometry and RNAseq data from two ENCODE cell lines, GM12878 and the chronic myelogenous leukemia cell line K562 (ENCODE AWG). LncRNAs were ~13 fold less likely to produce detectable peptides than mRNAs expressed at similar levels, indicating that ~92% of Gencode v7 lncRNAs are untranslated in these cell lines. Intersecting 9,640 lncRNA loci with 79,333 peptides yielded 85 unique peptides matching only 69 Gencode lncRNAs, with the majority of these peptides derived from the few rare protein-coding transcripts that were mis-annotated as lncRNAs in Gencode and that were hence absent from our independent, non-Gencode lncRNA catalog. We conclude that ectopic translation and cryptic mRNAs are rare in the lncRNAome of these cells. Summarily, our results, including those from human clinical breast cancer datasets and diverse cancer cell line systems, indicate that numerous specific lncRNAs, including non-conserved and cis-antisense transcripts, exert cis- and trans-regulatory functions in regulatory networks.

Citation Format: Leonard Lipovich. Regulatory networks in onco-lncRNAomics: Cis-regulation and non-conservation [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr IA3.

Notes: A full listing of collaborators, with funding acknowledgments, will be provided during the presentation. Due to time limitations, presentation of all results that are mentioned in this abstract is not guaranteed.