Targeted disruption of Hotair leads to homeotic transformation and gene derepression

Abstract

Long noncoding RNAs (lncRNAs) are thought to be prevalent regulators of gene expression, but the consequences of lncRNA inactivation in vivo are mostly unknown. Here, we show that targeted deletion of mouse Hotair lncRNA leads to derepression of hundreds of genes, resulting in homeotic transformation of the spine and malformation of metacarpal-carpal bones. RNA sequencing and conditional inactivation reveal an ongoing requirement of Hotair to repress HoxD genes and several imprinted loci such as Dlk1-Meg3 and Igf2-H19 without affecting imprinting choice. Hotair binds to both Polycomb repressive complex 2, which methylates histone H3 at lysine 27 (H3K27), and Lsd1 complex, which demethylates histone H3 at lysine 4 (H3K4) in vivo. Hotair inactivation causes H3K4me3 gain and, to a lesser extent, H3K27me3 loss at target genes. These results reveal the function and mechanisms of Hotair lncRNA in enforcing a silent chromatin state at Hox and additional genes.

Figure 1. Hotair knockout causes homeotic transformations

(A) Schematic of Hotair conditional knockout (cKO) allele. Arrowhead, Loxp site. (B) qRT-PCR confirms loss of Hotair expression in KO TTF. (C) Micro-CT scans showing “L6a→S1” homeotic transformation of the lumbar vertebrae, which resulted in losing the 6^th lumbar and having structurally deformed 1^st sacral vertebrae (arrow) in Hotair KO mice. L, lumbar vertebrae; S, sacral vertebrae. (D) Alizarin Red-Alcian Blue staining showing the deformed wrist bones in KO mice. Digits (II-V), carpal elements (1, 2, 3, 4/5), central element (c), radiale (r), ulnare (u), radius (ra) and ulna (ul). Note the fusion of carpal elements c-3, and 1-2-c (circled area), and missing radius (asterisk) in KOs. 4/5 are always naturally fused in WT wrist. (Scale bar: 0.5mm) (E) Summary of skeletal abnormalities in Hotair KO mice. Phenotype penetrance, number (n) of animals examined, and p-values (Fisher’s exact test) are indicated. See also Figure S1.

Figure 2. Hotair knockout de-represses HoxD and imprinted genes

(A) Differential gene expression by RNA-seq in Hotair WT, heterozygous (Het), and KO TTF. Each row is a transcript; each column is a sample. De-repressed HoxD (black) and imprinted genes (maternally expressed, pink; paternally expressed, blue) are indicated. (B) RNA-seq data of HoxD and Dlk1 loci. Distal HoxD genes (D13, D11 and D10, arrow) and Dlk1 were derepressed in KO cells. x-axis showing the genomic coordinate; y-axis showing the normalized RNA-seq signals. Box represents known mRNA exons. (C) qRT-PCR of indicated genes in Hotair KO cells or (D) after acute Hotair deletion in cKO cells. Top: schematic of acute deletion assay. (E) qRT-PCR of indicated genes in Hotair KO embryos. The hind portions of E13.5 embryos from the same litters were analyzed (n>3). Mean± s.d are shown for all panels; *, p<0.05; **, p<0.01 by student's t-test (n>3). NS, not significant. See also Figure S2.

Figure 3. Spatial and temporal gene expression patterns in Hotair KO mice

(A-B) Whole mount in situ hybridization (WISH) of Hoxd10 (A-a/c/d/f) and Hoxd11 (Ba/ c) of E13.5 embryos (n>3 for each genotype). KO embryos showed increased intensity and anterior shift of the expression domains of HoxD genes (highlight with arrows, the dotted lines across hind limbs are used as anatomical limit). Same embryos were co-stained with ethidium bromide, and the somite position of the anterior expression domain were numbered and marked with arrows (A-b/e; B-b/d). Scale bar: 1mm for A-a/b/d/e, B-a/b/c/d; 600µm for A-c/f. (C) WISH of Dlk1 on E12.5 embryos showing ectopic expression in Hotair KO embryos. (WT, n=4; KO, n=5; scale bar: 1mm). (D, E, F) Altered Dlk1 expression and mesenchymal cell fates in Hotair KO wrists. Dlk1 (D), Sox9 (E) and Gdf5 (F) expression in E15.5 wrist sections (n>3 for each genotype). Arrows indicate the joint regions in KO. Dotted circles marked the carpal element 2 and central element c. Arrowhead indicates the intervening Gdf5-positive domain in WT. Note 2-c fusion in KO wrist, showing continuous Dlk1 and Sox9 expression in the junction area of 2 and c; and loss of Gdf5 signal as well. (Scale bar: 300µm for D-a/b, E-a/b, F-a/b; 100µm for D-c/d, E-c/d, F-c/d) See also Figure S3.

Figure 4. Hotair regulates silent chromatin state genome-wide

(A) Hotair binds PRC2 and LSD1 complexes in vivo. RIP of E11.5 embryos with the indicated antibodies was followed by qRT-PCR of Hotair and control RNAs (U1, Malat1) and normalized with 1% input performed in parallel (n=3). Mean±s.d. is shown for all panels; *, p<0.05 student's t-test. (B) ChIP-qPCR in Hotair KO vs. WT cells of H3K27me3 (left panel) and H3K4me3 (right panel) of the indicated genes. Fgf4 was not changed in KO and served as a negative control. (n=3). (C) Average H3K27me3 (left panel) and H3K4me3 (right panel) ChIP-seq signal across the transcription start site (TSS) of upregulated genes in Hotiar KO cells is shown. (D) Relationship of histone modifications to gene de-repression in Hotair WT and KO cells. Heat map zoom-in of ChIP-seq signal in 8-kb bins centered on peak summits after unsupervised hierarchical clustering (left). RNA expression changes (KO/WT) and running sums across clusters are shown (right). Gene activation is seen in cluster i (75 loci) with both H3K27me3 loss and H3K4me3 gain; cluster ii shows 70 loci with only H3K4me3 gain in Hotair KO. See also Figure S4.