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, 477 (7364), 295-300

lincRNAs Act in the Circuitry Controlling Pluripotency and Differentiation

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lincRNAs Act in the Circuitry Controlling Pluripotency and Differentiation

Mitchell Guttman et al. Nature.

Abstract

Although thousands of large intergenic non-coding RNAs (lincRNAs) have been identified in mammals, few have been functionally characterized, leading to debate about their biological role. To address this, we performed loss-of-function studies on most lincRNAs expressed in mouse embryonic stem (ES) cells and characterized the effects on gene expression. Here we show that knockdown of lincRNAs has major consequences on gene expression patterns, comparable to knockdown of well-known ES cell regulators. Notably, lincRNAs primarily affect gene expression in trans. Knockdown of dozens of lincRNAs causes either exit from the pluripotent state or upregulation of lineage commitment programs. We integrate lincRNAs into the molecular circuitry of ES cells and show that lincRNA genes are regulated by key transcription factors and that lincRNA transcripts bind to multiple chromatin regulatory proteins to affect shared gene expression programs. Together, the results demonstrate that lincRNAs have key roles in the circuitry controlling ES cell state.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Functional effects of lincRNAs
(a) A schematic of lincRNA perturbation experiments. ESCs are infected with shRNAs, knockdown level is computed, the best hairpin is selected and profiled on expression arrays, and differential gene expression is computed relative to negative control hairpins. (b) Example of a lincRNA knockdown. Top: Genomic locus containing the lincRNA. Bottom: Heatmap of the 95 genes affected by knockdown of the lincRNA, expression for control hairpins (red line) and expression for lincRNA hairpins (blue line) are shown. (c) Distribution of number of affected genes upon knockdown of 147 lincRNAs (blue) and 40 well-known ESC regulatory proteins (red). Points corresponding to five specific ESC regulatory proteins are marked.
Figure 2
Figure 2. lincRNAs are critical for the maintenance of pluripotency
(a) Activity from a Nanog promoter driving luciferase, following treatment with control hairpins (black) or hairpins targeting luciferase (green), selected protein-coding regulators (red), and lincRNAs (blue). (b) Relative mRNA expression levels of Oct4 following knockdown of selected protein-coding (red) and lincRNA (blue) genes affecting Nanog–luciferase levels. The best hairpin (black line) and second best hairpin (grey line) are shown. All knockdowns are significant with a p-value<0.01. Error bars represent standard error (n=4). (c) Morphology of ESCs and immunofluorescence staining of Oct4 for a negative control hairpin (black line), and hairpins targeting Oct4 (red line), and two lincRNAs (blue line). The first row shows bright field images, the second row shows immunofluorescence staining of the Oct4 protein, and the third row shows DAPI staining of the nuclei.
Figure 3
Figure 3. lincRNAs repress specific differentiation lineages
(a) Expression changes for each lincRNA compared to gene expression of five differentiation patterns. Each box shows significant positive association (red, FDR<0.01) for Oct4 and Nanog (left) and for lincRNAs (right). (b) Expression changes upon knockdown of Oct4 and Nanog (black bars) and representative lincRNAs (grey bars) for five lineage marker genes. The expression changes (FDR<0.05) are displayed on a log scale as the t-statistic compared to a panel of negative control hairpins.
Figure 4
Figure 4. lincRNAs are direct regulatory targets of the ESC transcriptional circuitry
(a) A heatmap representing ChIP-Seq enrichments for 9 transcription factors (columns) at lincRNA promoters (rows). The percentage of bound lincRNAs downregulated upon knock-down of the TF are indicated in boxes (‘na’ were not measured). Right: Examples of lincRNAs from two clusters (‘core regulated’ and ‘myc regulated’) showing their genomic neighbourhood and TF binding. (b) A heatmap representing changes in lincRNA expression (rows) following knockdown of 11 TFs (columns). Middle: Effect of knockdown of Sox2, Oct4 and Nanog on expression levels of linc1405 (gray) and Oct4 (black). Right: Effect of knockdown of Klf2, Klf4, nMyc, and Esrrb on expression levels of linc1428.
Figure 5
Figure 5. lincRNAs physically interact with chromatin regulatory proteins
(a) A schematic of the classes of chromatin regulators profiled: ‘readers’ (blue), ‘writers’ (orange), and ‘erasers’ (green). (b) A heatmap showing the enrichment of 74 lincRNAs (rows) for one of 12 chromatin regulatory complexes (columns). The names are color-coded by chromatin-regulatory mechanism. Major clusters are indicated by vertical lines with a description of the chromatin components.
Figure 6
Figure 6. A model for lincRNA integration into the molecular circuitry of the cell
ESC-specific transcription factors (such as Oct4, Sox2, and Nanog) bind to the promoter of a lincRNA gene and drive its transcription. The lincRNA binds to ubiquitous regulatory proteins, giving rise to cell-type specific RNA–protein complexes. Through different combinations of protein interactions, the lincRNA–protein complex can give rise to unique transcriptional programs. Right: A similar process may also work in other cell types with specific transcription factors regulating lincRNAs, creating cell-type–specific RNA–protein complexes and regulating cell-type–specific expression programs.

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