Systematic Characterization of Long Noncoding RNAs Reveals the Contrasting Coordination of Cis- and Trans-Molecular Regulation in Human Fetal and Adult Hearts

Abstract

Background: The molecular regulation of heart development is regulated by cis- and trans-factors acting on the genome and epigenome. As a class of important regulatory RNAs, the role of long noncoding RNAs (lncRNAs) in human heart development is still poorly understood. Furthermore, factors that interact with lncRNAs in this process are not well characterized.

Methods and results: Using RNA sequencing, we systematically define the contrasting lncRNA expression patterns between fetal and adult hearts. We report that lncRNAs upregulated in adult versus fetal heart have different sequence features and distributions. For example, the adult heart expresses more sense lncRNAs compared with fetal heart. We also report the coexpression of lncRNAs and neighboring coding genes that have important functions in heart development. Importantly, the regulation of lncRNA expression during fetal to adult heart development seems to be due, in part, to the coordination of specific developmental epigenetic modifications, such as H3K4me1 and H3k4me3. The expression of promoter-associated lncRNAs in adult and fetal hearts also seems to be related to these epigenetic states. Finally, transcription factor-binding analysis suggests that lncRNAs are directly regulating cardiac gene expression during development.

Conclusions: We provide a systematic analysis of lncRNA control of heart development that gives clues to the roles that specific lncRNAs play in fetal and adult hearts.

Keywords: fetal heart; gene expression; genomics; long noncoding RNAs; transcription factor.

Figure 1

Transcriptome patterns in fetal and adult heart. (A) Principal component analyses of 4 fetal (blue) and 3 adult (green) heart samples based on transcriptome expression. Fetus_1 (GSM1059494), Fetus_2 (GSM1059495), Fetus_3 and Fetus_4 (both in-house acquired fetal heart samples); Adult_1 (GSM1698563), Adult_2 (GSM1698564), and Adult_3 (GSM1101970). (B) Hierarchical clustering of differentially expressed transcripts in fetal and adult heart shows a distinct expression pattern that is unique to each developmental stage.

Figure 2

Characterization of lncRNAs up-regulated in fetal and adult heart. (A) The distribution of exon number in fetal and adult heart up-regulated lncRNAs. The x-axis represents the exon number of detected lncRNAs and y-axis represents the total percentage for the indicated group. (B) LncRNA length distribution in fetal and adult heart. The x-axis represents length of detected lncRNAs. (C) Schematic showing the four categories of lncRNAs based on their relative genomic locations to neighboring coding genes (mRNAs): Sense, Antisense, Bidirectional, and Intergenic. (D) Expression differences of the four types of lncRNAs in fetal and adult heart.

Figure 3

Co-expression of lncRNA-mRNA pairs between human fetal and adult heart. (A) Expression level of 3 pairs of lncRNA-mRNA from RNA-seq datasets. RPKM is used to define the expression level. Left y-axis represents lncRNA expression and right y-axis represents neighboring mRNA (protein-coding) gene expression. (B) qPCR validation of the co-expression of lncRNA-mRNA pairs. *P<0.05, **P<0.01, and ***P<0.001. Blue bars represent lncRNA expression and red bars represent neighboring protein-coding gene expression.

Figure 4

Functional clustering of lncRNAs in fetal and adult heart based on protein-coding gene co-expression. (A) IPA pathway analysis of diseases and biological functions of protein coding genes exhibiting co-expression with lncRNAs. (B) IPA Pathway analysis of toxicity functions. Pathways are ranked based on significance (−log P-value).

Figure 5

Identification of enhancer- and promoter-asociated lncRNAs in adult and fetal heart. (A) Heatmap of chromatin modifications in putative lncRNA regulatory regions. ChIP signals are shown for H3k4me1 (enhancer) and H3k4me3 (promoter) histone modifications within 2 kb of the TSS for each lncRNA. (B) Distribution of elncRNAs and plncRNAs between adult and fetal heart. Regions of overlap in Venn diagrams indicate lncRNAs that are up-regulated in both adult and fetal hearts.

Figure 6

Expression patterns of enhancer- and promoter- associated lncRNAs in adult and fetus. (A) The total percentage of plncRNAs expressed in adult and fetal hearts. (B) The total percentage of elncRNAs expressed in adult and fetal hearts. (C) Percentage of co-expression of plncRNAs and associated coding genes that share the same putative promoter.

Figure 7

Conserved transcription factor binding sites in putative cardiac development lncRNA regulatory regions. Each row represents one potential TFBS for the indicated transcription factor in fetal and adult hearts, and colors indicate the relative downstream lncRNA expression (within 2 kb) of this putative regulatory region. Eighty-one human conserved transcription factors were used in this analysis. The range represents the log2 percentage of TFBS.