The long noncoding RNAs NEAT1 and MALAT1 bind active chromatin sites

Abstract

Mechanistic roles for many lncRNAs are poorly understood, in part because their direct interactions with genomic loci and proteins are difficult to assess. Using a method to purify endogenous RNAs and their associated factors, we mapped the genomic binding sites for two highly expressed human lncRNAs, NEAT1 and MALAT1. We show that NEAT1 and MALAT1 localize to hundreds of genomic sites in human cells, primarily over active genes. NEAT1 and MALAT1 exhibit colocalization to many of these loci, but display distinct gene body binding patterns at these sites, suggesting independent but complementary functions for these RNAs. We also identified numerous proteins enriched by both lncRNAs, supporting complementary binding and function, in addition to unique associated proteins. Transcriptional inhibition or stimulation alters localization of NEAT1 on active chromatin sites, implying that underlying DNA sequence does not target NEAT1 to chromatin, and that localization responds to cues involved in the transcription process.

Figure 1

CHART enriches the lncRNAs NEAT1 and MALAT1. (A and B) NEAT1 CHART enrichment of NEAT1 and MALAT1 RNA (panel A) and DNA (panel B) assessed by qPCR from MCF-7 cells. n=3 (C and D) MALAT1 CHART enrichment of NEAT1 and MALAT1 RNA (panel C) and DNA (panel D) assessed by qPCR from MCF-7 cells. n=3 (E) CHART-seq of DNA and PSF ChIP-seq over chromosome 11. Peaks extending beyond the y-axis are depicted with hatch marks and annotated with the actual peak height. (F) Co-enrichment of NEAT1 and MALAT1 RNA and PSF at the NEAT1 and MALAT1 genomic loci. All error bars in A-D represent s.e.m. See also Figure S1 and Table S1.

Figure 2

NEAT1 and MALAT1 localize to actively-transcribed genomic loci. (A) Venn diagram depicting number of NEAT1-specific, MALAT1-specific, and co-enriched CHART-seq sites. (B) Overlap between gene bodies and CHART-seq binding sites. (C and D) Probability density distributions of expression levels (RPKM; reads per kb per million) of genes overlapping MALAT1 peaks (panel C), NEAT1 peaks (panel D), or both (panels C and D). (E-G) Probability density distributions of input-normalized coverage density for H3K4me3 (panel E), H3K36me3 (panel F), and H3K27me3 (panel G) over a 500 bp window around NEAT1 and MALAT1 peak maxima. Dotted lines in panels E-G clarify separation between peak maxima that are enriched (coverage density > 1) or depleted (coverage density < 1) for a particular modification. (H) Probability density distributions of NEAT1 and MALAT1 peak maxima positions along length-normalized gene bodies. (I) CHART-seq enrichment of NEAT1 and MALAT1 and ChIP-seq enrichment of PSF at the SAP18 genomic locus. See also Figure S2, Table S2, and Table S3.

Figure 3

NEAT1 and MALAT1 localize to genomic sites in trans. (A) NEAT1 and MALAT1 DNA CHART-seq and PSF ChIP-seq over chromosome 17. The HEXIM1 locus is boxed. (B-D) DNA CHART-seq and PSF ChIP-seq depicting the NEAT1-specific target SP3 (panel B), colocalization of NEAT1 and MALAT1 RNA at the HEXIM1 locus (panel C), and the MALAT1-specific target CALR/RAD23A (panel D). (E and F) qPCR validation of trans sites enriched by NEAT1 (panel E) and MALAT1 (panel F) DNA CHART in MCF-7 cells. Error bars represent s.e.m. n=3. See also Figure S3.

Figure 4

Validation of NEAT1 trans site localization by RNA/DNA co-FISH. (A) Representative images of NEAT1 RNA colocalization with MALAT1 and MAPK15 DNA, but not a gene-poor region, determined by RNA/DNA co-FISH. (B and C) Quantification of NEAT1 RNA colocalization with the MALAT1 locus (panel B) and each allele for trans genomic loci (panel C). “n” indicates number of DNA loci analyzed. Asterisks indicate a significant (p < 0.05) increase in NEAT1 colocalization with the particular DNA locus compared to the gene-poor region, determined by a chi-squared test. See also Figure S4.

Figure 5

NEAT1 and MALAT1 colocalize with the paraspeckle component, PSF. (A) Metagene profile of PSF ChIP-seq density over active (RPKM > 1) and inactive (RPKM <= 1) genes. (B) PSF ChIP-seq coverage centered over NEAT1-specific and MALAT1-specific peak maxima (“Unique”) and maxima of NEAT1 and MALAT1 peaks that overlap with MALAT1 and NEAT1 binding sites, respectively (“Shared”). See also Figure S5.

Figure 6

NEAT1 localization is responsive to changes in transcription. (A and B) NEAT1 DNA CHART-seq over the RPS24 locus (panel A) and KMT2E locus (panel B) in cells treated with DMSO (“Vehicle”) or flavopiridol. (C-D) Probability density distributions of NEAT1 CO1 (panel C) or MALAT1 CO1 (panel D) peak midpoint positions along nearby length-normalized gene bodies in vehicle or flavopiridol treated MCF-7 cells. (E) NEAT1 DNA CHART-seq over the estrogen receptor-regulated gene GREB1 in cells treated with ethanol (“Vehicle”) or 17β-estradiol (“E2”). (F) Table describing number of CHART-seq peaks identified upon vehicle treatment and E2 treatment. (G-H) Scatterplots of transcript abundance in cells treated with vehicle or E2, as measured by GRO-seq (see Extended Experimental Procedures), for genes nearby vehicle-specific (black) or E2-specific (red) CHART-seq peaks for NEAT1 CO1 (panel G) or MALAT1 CO1 (panel H). Indicated p-values are calculated using a Wilcoxon signed rank test. See also Figure S6, Table S2, Table S4, and Table S5.

Figure 7

CHART enriches proteins associated with NEAT1 and MALAT1 and reveals mechanistic insights into lncRNA function. (A) Western blot of NEAT1 and MALAT1 CHART-enriched material for the paraspeckle components PSPC1 and PSF, the nuclear speckle component SRSF1, and a chromatin-associated protein, histone H3. (B) Table indicating number of known nuclear speckle and paraspeckle proteins enriched ten-fold over input for the indicated CHART conditions. (C) Venn diagram depicting overlap between proteins associated with NEAT1 and MALAT1. (D) Western blot of NEAT1 and MALAT1 CHART-enriched material for PURA and ESRP2 protein. (E) Model of NEAT1 and MALAT1 binding to trans genomic sites. (F) Model depicting preferential localization of NEAT1 RNA at the TSS and TTS and MALAT1 RNA at the TTS. See also Table S6 and Figure S7.