Regulation of the ESC transcriptome by nuclear long noncoding RNAs

Abstract

Long noncoding (lnc)RNAs have recently emerged as key regulators of gene expression. Here, we performed high-depth poly(A)(+) RNA sequencing across multiple clonal populations of mouse embryonic stem cells (ESCs) and neural progenitor cells (NPCs) to comprehensively identify differentially regulated lncRNAs. We establish a biologically robust profile of lncRNA expression in these two cell types and further confirm that the majority of these lncRNAs are enriched in the nucleus. Applying weighted gene coexpression network analysis, we define a group of lncRNAs that are tightly associated with the pluripotent state of ESCs. Among these, we show that acute depletion of Platr14 using antisense oligonucleotides impacts the differentiation- and development-associated gene expression program of ESCs. Furthermore, we demonstrate that Firre, a lncRNA highly enriched in the nucleoplasm and previously reported to mediate chromosomal contacts in ESCs, controls a network of genes related to RNA processing. Together, we provide a comprehensive, up-to-date, and high resolution compilation of lncRNA expression in ESCs and NPCs and show that nuclear lncRNAs are tightly integrated into the regulation of ESC gene expression.

Figure 1.

Transcriptome characterization in ESCs and NPCs. (A) Schematic of RNA sources for high-throughput RNA sequencing. (B) Pipeline for ESC and NPC transcriptome analysis. (C) Average linkage clustering dendrogram based on Spearman rank correlation of mRNA and lncRNA expression levels. (D) LncRNAs in the intersection of AB2.2 and Cast/BL6 backgrounds detected in ESCs and NPCs. (E) Analysis of POU5F1, NANOG, SOX2, and POU3F2 binding within 2.5 kb of the transcription start site of expressed lncRNAs (ChIP-seq data reanalyzed from Marson et al. 2008; Lodato et al. 2013). (F) Heatmap (gene-wise Z-score) of lncRNAs in Cast/BL6 clones determined as differentially expressed between ESCs and NPCs (FDR < 0.01).

Figure 2.

Identification of nuclear-enriched lncRNAs. (A) Nuclear to total log₂ FPKM ratio by annotated gene biotype (Ensembl v.76) in AB2.2 ESCs plotted against FPKM values obtained from total cell RNA-seq. Only genes with expression >1 FPKM in either nuclear or total fraction are plotted. (B) Single-molecule RNA FISH in AB2.2 ESCs and NPCs using probe pairs specific for Platr14, Firre, and other indicated lncRNAs (green). Housekeeping Ppib mRNA (red) is used as a control. Images represent maximum intensity projections. Dotted lines demarcate the nuclear outline of individual cells or ESC colonies. (Scale bar) 10 µm. Mitotic images are enlarged twofold.

Figure 3.

Identification of ESC-associated lncRNAs. (A) Heatmap (GeneWise Z-score) of the 50 lncRNAs most specifically (calculated based on Z-score) expressed in ESCs at ≥1 FPKM. ENCODE mouse tissue poly(A)⁺ RNA-seq data represent the average expression of two biological replicates. (B) Expression heatmap of the gene module containing Pou5f1 as determined by weighted gene coexpression analysis. (C) Gene ontology (GO) terms for biological process selected from the top 20 nonredundant terms enriched in the Pou5f1 gene module. Enrichment was ranked based on Fisher's exact test.

Figure 4.

Platr14 lncRNA is functionally integrated in ESC gene expression regulation. (A) Connectivity between genes within the Pou5f1 module (see Fig. 3B) is plotted against the module's first principal component (module gene expression). Dotted lines correspond to first and third quartiles. (Red) lncRNA genes; (blue) mRNA genes. (B) Schematic of annotated Platr14 isoforms. Targeting sites of ASO#2 and ASO#3 are indicated. Black lines above the transcript model represent targeting sites of RNA FISH probes. For greater detail and cloned Platr14 cDNA, see Supplemental Figure S7. (C) Expression of Platr14 in AB2.2 ESCs and ENCODE tissues. (D) Expression level of Platr14 in ESCs 24 h after transfection with control ASO (Cntr) or two independent ASOs specific against Platr14 lncRNA. Data represent the mean and standard deviation of four biological replicates. (E) Single-molecule RNA FISH for Platr14 lncRNA (green) and Ppib housekeeping mRNA (red) in AB2.2 ESCs transfected with control ASO or Platr14 specific ASO. (Scale bar) 10 µm. (F) Heatmap (gene-wise Z-score) of genes significantly affected within 24 h of transfection of Platr14 specific ASOs (FDR < 0.05). (G) Top 15 nonredundant GO terms enriched in the set of differentially expressed genes for each ASO. Enrichment was ranked based on Fisher's exact test.

Figure 5.

Firre lncRNA is functionally integrated into an RNA processing gene expression module. (A) Single-molecule RNA FISH for Firre (green) and Ppib housekeeping mRNA (red) 24 h after transfection of AB2.2 ESCs with control (ctr) ASO or a Firre-specific ASO. (B) Firre expression level in ESCs transfected as in A with control or two independent Firre-specific ASOs. Data represent mean and standard deviation of four biological replicates. (C) Normalized poly(A)⁺ read coverage across the Firre locus in ESCs transfected as in A and B. RNA FISH target sites and ASO target sites are indicated. Due to the repeat motif content, ASOs are complementary to more than one site of the Firre transcript. (D) Overview of differentially expressed genes 24 h after Firre knockdown with two ASOs in four biological replicates each (FDR < 0.05; see Supplemental Fig. S9 for the full heatmap). (E) GO enrichment for the genes affected by both ASO treatments. Top five biological process terms are shown based on Fisher's exact test and after redundancy trimming. (F) GO terms as in Figure 3C obtained from the weighted gene coexpression analysis-derived gene module containing Firre. (G) Firre module genes (red: lncRNA; blue: mRNA) plotted as in Figure 4A. Labeled genes correspond to 18 of the 31 genes (indicated in D, excluding Firre) significantly down-regulated in both Firre ASO treatments.