Activating RNAs associate with Mediator to enhance chromatin architecture and transcription

Abstract

Recent advances in genomic research have revealed the existence of a large number of transcripts devoid of protein-coding potential in multiple organisms. Although the functional role for long non-coding RNAs (lncRNAs) has been best defined in epigenetic phenomena such as X-chromosome inactivation and imprinting, different classes of lncRNAs may have varied biological functions. We and others have identified a class of lncRNAs, termed ncRNA-activating (ncRNA-a), that function to activate their neighbouring genes using a cis-mediated mechanism. To define the precise mode by which such enhancer-like RNAs function, we depleted factors with known roles in transcriptional activation and assessed their role in RNA-dependent activation. Here we report that depletion of the components of the co-activator complex, Mediator, specifically and potently diminished the ncRNA-induced activation of transcription in a heterologous reporter assay using human HEK293 cells. In vivo, Mediator is recruited to ncRNA-a target genes and regulates their expression. We show that ncRNA-a interact with Mediator to regulate its chromatin localization and kinase activity towards histone H3 serine 10. The Mediator complex harbouring disease- displays diminished ability to associate with activating ncRNAs. Chromosome conformation capture confirmed the presence of DNA looping between the ncRNA-a loci and its targets. Importantly, depletion of Mediator subunits or ncRNA-a reduced the chromatin looping between the two loci. Our results identify the human Mediator complex as the transducer of activating ncRNAs and highlight the importance of Mediator and activating ncRNA association in human disease.

Figure 1. Mediator confers the ncRNA-as dependent activation of a heterologous reporter

a, Schematic representation of genomic control insert (top) or ncRNA-a7 insert (bottom) in luciferase reporter vector driven by a TK-promoter. b, Depletion of ncRNA-a7 reduces the luciferase activity in the ncRNA-a7 luciferase reporter cell lines. c, Depletion of the transcription factors or enhancers in the control or ncRNA-a7 reporter cell lines. The red bar signifies reduced transcription using siRNA against MED12, comparable with siRNA against ncRNA-a7. d, Depletion of different Mediator subunits using the ncRNA-a7 reporter cell lines. e, Depletion of the Mediator subunits in ncRNA-a1 or ncRNA-a3 luciferase reporter cell lines. All data shown are mean ±SEM of three independent experiments. **p<0.01, ***p<0.001 by two-tailed Student’s T-test.

Figure 2. Functional association of Mediator and activating ncRNAs

a, Knockdowns of ncRNA-a7 or Mediator subunits decrease SNAI1, AURKA expression. Depletion of ncRNA-a7 diminished SNAI1 and AURKA expression by Real-time PCR (top red bars). Similarly knockdowns of MED1 (green bars) or MED12 (yellow bars) reduce SNAI1 or AURKA expression. b, Knockdowns of ncRNA-a3 or Mediator subunits decrease TAL1 expression. Expression of TAL1 or control STIL following knockdowns of ncRNA-a3 (red bars), MED1 (green bars) and MED12 (yellow bars) are shown. c-d, Knockdowns of ncRNA-a7 (c) or ncRNA-a3 (d) reduce the genomic occupancy of MED1, MED12 (top panels) or RNAPII (bottom panels) on SNAI1, AURKA (c) or TAL1 (d) in A549 cells. e-f, Activating ncRNAs specifically stimulate Mediator kinase activity toward histone H3.1 substrate in vitro. Quantification of kinase assay following addition of ncRNA-a7 (e) or ncRNA-a3 (f). Error bars represent ±SEM (n=3), p<0.01 by two-tailed Student’s T-test. The mean ±SEM for all results represent three independent experiments. *p<0.05, **p<0.01, ***p<0.001 by two-tailed Student’s T-test.

Figure 3. Interaction of Mediator and activating ncRNAs is disrupted by FG syndrome mutations of MED12

a-b, ncRNA-as associate with Mediator complex in vitro. RNA immune-precipitation (RIP) was performed using protein A dynabeads coupled with IgG, MED1 or MED12 antibodies. Identical results were obtained in three independent experiments. c, Purification of Mediator protein complex and associated RNAs. The purified FLAG-MED12 affinity elution was subjected to Superose 6 size-exclusion chromatography and the Mediator subunits were detected by Western blot. The Superose 6 fractions (14-38) are shown on top and molecular mass markers (kilodaltons) are at the bottom. The RNAs in each fraction was extracted and was followed by RT-PCR using specific primers. d, UV cross-link followed by RIP using HEK293T whole cell lysates. The associated RNAs were analyzed by RT-qPCR with individual specific RNA primers. The mean ±SEM are from three independent experiments. ***p<0.001 by two-tailed Student’s T-test. e, Protein sequences of MED12 mutants causing FG syndrome (Opitz-Kaveggia syndrome). Part of the protein sequence of MED12 exon 21 (top panel). The mutated sites indicate the G958E mutant (red) and R961W mutant (blue). f, HEK293T cells were transfected with FLAG-MED12 or mutant constructs. After UV cross-linking, total cell extracts were immunoprecipitated and analyzed by Western blotting. g, Following UV-RIP of wild type and mutant MED12 samples shown in (f), RT-qPCR was performed using transcript specific primers from the lncRNAs shown. The data is a representative of three independent experiments. The two-tailed Student’s T-test, indicated a p<0.001 for ncRNA-a1 and ncRNA-a3, while there was no significant difference for HOTAIR and HOTTIP.

Figure 4. Mediator complex and ncRNA-as promote chromatin looping

The schematic diagrams represent the genomic locus between the ncRNA-a7, SNAI1 and AURKA loci (a), the ncRNA-a3 and TAL1 (d). The top arrows show the position of primers, the digestion sites are shown in the middle and the s1-8, A-G or 1-8 fragments are presented below. The looping events between ncRNAs and its targets were detected between ncRNA-a7 and SNAI1 (b), ncRNA-a7 and Aurka (c) and ncRNA-a3 and TAL1(e) using chromosome conformation capture (3C). Depletion of MED1 or MED12 abolished the loop interaction (b,c,e, middle panels). Knockdown of ncRNA-a7 or ncRNA-a3 reduced the chromosomal looping events (b,c,e, lower panels). The interaction frequency between the anchoring points and distal fragments were determined by Real-time PCR and normalized to BAC templates and control anchors. Each error bar represents ±SEM from three independent experiments, p<0.01 by two-tailed Student’s T-test. Representative gel images of the 3C experiments for AURKA are presented in Supplementary Figure 3.