Hepatic Long Intergenic Noncoding RNAs: High Promoter Conservation and Dynamic, Sex-Dependent Transcriptional Regulation by Growth Hormone

Abstract

Long intergenic noncoding RNAs (lincRNAs) are increasingly recognized as key chromatin regulators, yet few studies have characterized lincRNAs in a single tissue under diverse conditions. Here, we analyzed 45 mouse liver RNA sequencing (RNA-Seq) data sets collected under diverse conditions to systematically characterize 4,961 liver lincRNAs, 59% of them novel, with regard to gene structures, species conservation, chromatin accessibility, transcription factor binding, and epigenetic states. To investigate the potential for functionality, we focused on the responses of the liver lincRNAs to growth hormone stimulation, which imparts clinically relevant sex differences to hepatic metabolism and liver disease susceptibility. Sex-biased expression characterized 247 liver lincRNAs, with many being nuclear RNA enriched and regulated by growth hormone. The sex-biased lincRNA genes are enriched for nearby and correspondingly sex-biased accessible chromatin regions, as well as sex-biased binding sites for growth hormone-regulated transcriptional activators (STAT5, hepatocyte nuclear factor 6 [HNF6], FOXA1, and FOXA2) and transcriptional repressors (CUX2 and BCL6). Repression of female-specific lincRNAs in male liver, but not that of male-specific lincRNAs in female liver, was associated with enrichment of H3K27me3-associated inactive states and poised (bivalent) enhancer states. Strikingly, we found that liver-specific lincRNA gene promoters are more highly species conserved and have a significantly higher frequency of proximal binding by liver transcription factors than liver-specific protein-coding gene promoters. Orthologs for many liver lincRNAs were identified in one or more supraprimates, including two rat lincRNAs showing the same growth hormone-regulated, sex-biased expression as their mouse counterparts. This integrative analysis of liver lincRNA chromatin states, transcription factor occupancy, and growth hormone regulation provides novel insights into the expression of sexspecific lincRNAs and their potential for regulation of sex differences in liver physiology and disease. High-throughput sequencing of mammalian transcriptomes has revealed nearly ubiquitous transcription of the genome and the generation of large numbers of noncoding transcripts. Noncoding RNAs (ncRNAs) have drawn much attention as potential chromatin regulators, exemplified by classical ncRNAs, such as Xist (1). Several thousand ncRNAs have been discovered in human (2, 3), mouse (4-7), zebrafish (8,9), and fruit fly (10,11). Individual ncRNAs were shown to play diverse regulatory roles in gene expression (9,(12)(13)(14)(15); however, the vast majority of ncRNAs are poorly characterized, both computationally and experimentally. Many ncRNAs share salient features of protein-coding genes, including transcription by RNA polymerase II, 5= capping, splicing, polyadenylation, and deposition of histone marks associated with transcription, specifically H3K4me3 at the promoter and H3K36me3 across the gene body (4). These ncRNAs are typically Ͼ200 nucleo...

Genome-Wide Association and Replication Study of Hepatotoxicity Induced by Antiretrovirals Alone or with Concomitant Anti-Tuberculosis Drugs

“…Liver-expressed lincRNA promoters show greater enrichment for proximal binding of liver transcription factors than protein-coding gene promoters, which may reflect the higher conservation of liver lincRNA promoters (Melia et al, 2016). In fact, recently, hepatic lncRNAs are reported to be involved in the progression of liver fibrosis (He et al, 2014;Yu et al, 2016;Zheng et al, 2015).…”

Long non-coding RNA Gm15441 attenuates hepatic inflammasome activation in response to metabolic stress

“…A set of 24,197 annotated mouse RefSeq genes (which includes some RefSeq lncRNAs) and a set of 15,558 liver-expressed lncRNA genes (Lodato et al, 2017;Melia and Waxman, 2019) was considered for differential expression analysis. These lncRNAs include intergenic lncRNAs, as well as lncRNAs that are antisense or intragenic with respect to RefSeq genes, and were discovered using a computational pipeline for lncRNA discovery described elsewhere (Melia et al, 2016) based on 186 mouse liver RNA-seq datasets representing 30 different biological conditions. RefSeq and lncRNA genes that showed significant differential expression following exposure to WY-14643 were identified by EdgeR as outlined elsewhere (Melia et al, 2016).…”

“…PPARA-dependent lncRNAs identified by RNA-seq were selected and mRNA levels monitored over a 24 hour period after WY-14643 treatment, revealing that lncRNA expression profiles paralleled those of known protein-coding PPARA target genes (Figure S1A-D). As lncRNA expression is often highly tissue specific (Iwakiri et al, 2017;Melia et al, 2016;Perron et al, 2017), the relative basal levels were measured for PPARA-responsive lncRNAs expressed in oxidative tissues that utilize fatty acids as an energy source and express PPARA at the highest levels. Ppara mRNA and its classic target gene Cyp4a14 served as controls.…”

“…Down-regulation of lncRNA lncOb reduces leptin, leading to a leptin responsive form of obesity (Dallner et al, 2019). Other studies found that lncRNAs are regulated during adipocyte differentiation (Yuan et al, 2019a;Yuan et al, 2019b), are expressed in liver in a sex-dependent manner when regulated by growth hormone (Melia et al, 2016;Melia and Waxman, 2019), and can be strongly induced by xenobiotic exposure (Lodato et al, 2017). A compartment-specific transcriptional profiling approach revealed that lncRNA PAXIP1-AS1 regulates pulmonary arterial hypertension by modulating smooth muscle cell function (Jandl et al, 2019).…”

Impact of 3-dimensional genome organization, guided by cohesin and CTCF looping, on sex-biased chromatin interactions and gene expression in mouse liver

“…The health impacts of sex differences in liver include a higher incidence of aggressive liver cancer in males [5], increased susceptibility to autoimmune hepatitis in females [6], and sex differences in metabolism of some pharmaceuticals and environmental chemicals [7]. Transcriptomic and epigenetic sex-bias is best characterized in mouse liver, where ~1,000 genes [8], including many lncRNA genes [9,10] and miRNAs [11], exhibit sex-biased expression that may be regulated by thousands of sex-specific DNase I hypersensitive sites (DHS) [12,13]. This prior work has shown that the majority of sex-biased genes do not have geneproximal sex-biased chromatin marks or DHS, suggesting most epigenetic sex bias occurs at distal elements such as enhancers [12,13].…”