Long noncoding RNA as modular scaffold of histone modification complexes

Abstract

Long intergenic noncoding RNAs (lincRNAs) regulate chromatin states and epigenetic inheritance. Here, we show that the lincRNA HOTAIR serves as a scaffold for at least two distinct histone modification complexes. A 5' domain of HOTAIR binds polycomb repressive complex 2 (PRC2), whereas a 3' domain of HOTAIR binds the LSD1/CoREST/REST complex. The ability to tether two distinct complexes enables RNA-mediated assembly of PRC2 and LSD1 and coordinates targeting of PRC2 and LSD1 to chromatin for coupled histone H3 lysine 27 methylation and lysine 4 demethylation. Our results suggest that lincRNAs may serve as scaffolds by providing binding surfaces to assemble select histone modification enzymes, thereby specifying the pattern of histone modifications on target genes.

Fig. 1

5′ domain of HOTAIR binds PRC2 and 3′ domain of HOTAIR binds LSD1. (A) LSD1 IP specifically retrieves HOTAIR RNA. Data (mean ± SD, n = 3) is relative to mock-IP (IgG or FLAG). ND, not-detectable. (B) In vitro transcribed (IVT), biotinylated HOTAIR retrieves EZH2, LSD1, CoREST, and REST, but not G9a, CDYL, or YY1. (C) IVT biotinylated HOTAIR binds to purified PRC2 and LSD1 complexes. PRC2_3m: recombinant purified core PRC2 complex with 3 members (EZH2, SUZ12, EED). PRC2_5m: recombinant purified PRC2 complex with 5 members (+RbAP48, AEBP2). LSD1.com: Tandem affinity purified protein complex associated with FLAG-HA-LSD1 from HeLa cells. Composition of protein complexes are shown in fig. S1C. (D)The first 300 bp (lined boxes) of HOTAIR is necessary and sufficient to bind PRC2; the last 646 bp (meshed boxes) is necessary and sufficient to bind LSD1 complex. The profiles are established by RNA pull-down of HeLa extract; retrieved proteins are detected by immunoblotting.

Fig. 2

HOTAIR is necessary and sufficient for interaction between EZH2 and LSD1. (A) In foreskin fibroblasts, EZH2 interacts with LSD1 (lanes 1, 4). Knockdown of HOTAIR (lanes 3, 6), but not GFP (lanes 2, 5), abolishes this interaction. HOTAIR levels (mean ± SD) are shown on the right. (B) HOTAIR expression in FLAG-LSD1 HeLa cells induces EZH2 and LSD1 interaction (lanes 3 and 6). (C) Full length HOTAIR induces EZH2 and LSD1 interaction (lanes 3, 10) but not HOTAIR mutants lacking either 5′ or 3′ domain (lanes 4 to 7 and 11 to 14). Presence of indicated RNA domains is confirmed by RT-PCR (bottom panel).

Fig. 3

HOTAIR coordinates localization of PRC2 and LSD1 genome-wide. (A) Changes in mRNA and occupancy of H3K4me2, H3K27me3, LSD1, and SUZ12 across HOXD locus after RNAi of HOTAIR in foreskin fibroblasts. Yellow boxes indicate regions of notable correlation between gain of H3K4me2 and concordant loss of LSD1, H3K27me3, and SUZ12. (B) The patterns of change in LSD1 (x axis) and SUZ12 occupancy (y axis) upon HOTAIR knockdown across the HOXD locus are significantly correlated (Pearson correlation, R = 0.59, p < 10⁻⁹, t-test). This correlation is concentrated in proximal promoters of HOXD genes (R = 0.86). (C) Positive correlation of changes in SUZ12 (x axis) and H3K27me3 occupancy (y axis) and negative correlation of LSD1 (x axis) and H3K4me2 occupancy (y axis). (D) Venn diagram shows the genes occupied by SUZ12 (4740 genes), LSD1 (2116 genes), or both (721 genes). (E) Heatmap of SUZ12 and LSD1 co-occupied genes (721 genes). Each column is an experiment; each row is a gene. HOTAIR knockdown led to concordant loss of SUZ12 and LSD1 occupancy. Chromatin occupancy is indicated in blue per the scale bar. (F) HOTAIR knockdown leads to transcription de-repression of target genes. Mean ± SD of qRT-PCR data are shown.

Fig. 4

HOTAIR-dependent SUZ12 and LSD1 binding motifs. (A) SUZ12 occupancy sites lost upon HOTAIR knockdown (HOT-S sites) are enriched for a DNA motif very similar to the right half of canonical REST motif. (B) LSD1 occupancy sites lost upon HOTAIR knockdown (HOT-L sites) are enriched for a CG-rich motif. (C) A nearly identical CG-rich motif is enriched in LSD1/SUZ12 binding sites gained upon HOTAIR overexpression, suggesting that this motif is involved in HOTAIR target selection.