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Chromatin-enriched lncRNAs can act as cell-type specific activators of proximal gene transcription

Nature Structural & Molecular Biology volume 24, pages 596–603 (2017)Cite this article

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Abstract

We recently described a new class of long noncoding RNAs (lncRNAs) that are distinguished by especially tight chromatin association and whose presence is strongly correlated to expression of nearby genes. Here, we examine the cis-enhancer mechanism of this class of chromatin-enriched RNA (cheRNA) across multiple human cell lines. cheRNAs are largely cell type specific and provide the most reliable chromatin signature to predict cis-gene transcription in every human cell type examined. Targeted depletion of three cheRNAs decreases expression of their neighboring genes, indicating potential co-activator function, and single-molecule fluorescence in situ hybridization (smFISH) of one cheRNA-distal target gene pair suggests a spatial overlap consistent with a role in chromosome looping. Additionally, the cheRNA HIDALGO stimulates the fetal hemoglobin subunit gamma 1 (HBG1) gene during erythroid differentiation by promoting contacts to a downstream enhancer. Our results suggest that multiple cheRNAs activate proximal lineage-specific gene transcription.

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Figure 1: Tissue specificity and cis-gene activity of cheRNAs.
Figure 2: cheRNA PVT1 bridges the site of its production to its target gene, MYC.
Figure 3: Examination of cis-enhancer activity of cheRNA–gene pairs.
Figure 4: HIDALGO exhibits hallmarks of a promoter and is induced with HBG1 during erythrogenesis.
Figure 5: HIDALGO promotes HBG1 expression.
Figure 6: HIDALGO couples an enhancer at its TSS to the HBG1 promoter.

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Acknowledgements

We are grateful for ChIP-seq data from ENCODE, in particular that deposited by the Snyder and Bernstein labs. We thank the following University of Chicago Core Facility personnel: P. Faber and H. Whitehurst in Functional Genomics for Illumina sequencing and D. Leclerc in Flow Cytometry for FACs training. K562 cells with dCas9-KRAB stably integrated into the genome were generously provided by L. Gilbert and J. Weissman. We would also like to thank V. Lynch for helpful discussions regarding cheRNAs and transposable elements. These studies were supported by Stanley Manne Children's Research Institute's Grant to V.G. We thank Y. Galat for technical help in ESC culture. M.S.W. is supported by the Chicago Biotechnology Consortium with support from The Searle Funds at The Chicago Community Trust; A.J.R. is supported by the Ellison Medical Foundation (AG-NS-1118-13), NIH (R01-GM115945), and American Cancer Society (130230-RSG-16-248-01-DMC).

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Authors and Affiliations

  1. Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois, USA

    Michael S Werner, Matthew A Sullivan, Rohan N Shah, Adrian T Grzybowski & Alexander J Ruthenburg

  2. Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois, USA

    Matthew A Sullivan, Rohan N Shah & Alexander J Ruthenburg

  3. Department of Pediatrics and Pathology, The University of Chicago, Chicago, Illinois, USA

    Rangarajan D Nadadur & Ivan P Moskowitz

  4. Department of Pathology, Stanley Manne Children's Research Institute, Ann and Robert H. Lurie Children's Hospital of Chicago, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA

    Vasiliy Galat

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Contributions

A.J.R. and M.S.W. designed the study and wrote the paper with valuable input from all of the other authors. M.S.W. performed the experiments for Figures 1 and 2 and the initial experiments for Figures 3, 4, and 5. M.A.S. repeated most of the Figures 3, 4, and 5 experiments in higher replicate with assistance from M.S.W., and performed 3C with ASO-treated cells. R.N.S. and A.T.G. performed ICeChIP experiments. R.D.N. performed the luciferase assay to examine enhancer activity of the HIDALGO TSS with oversight from I.P.M. V.G. cultured H1 ESCs used.

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Integrated supplementary information

Supplementary Figure 1 In vitro transcribed RNA standards establish a calibration curve for quantitation of nuclear RNAs.

a, Agarose gel of spike-in standards demonstrates purity of transcripts. b, Scatter plot of spike-in standards demonstrates a linear relationship with molecules added and abundance measured by RT-qPCR. c, Scatter plot of RNA-seq reads (y-axis) versus molecules spiked-in per cell (x-axis) generally recapitulates a linear relationship in all libraries, the slopes of these curves were used to compute molecules/cell of each cheRNA. As a comparison the highest read value in each library was back calculated by the fold-dilution to a theoretical read value (green lines).

Supplementary Figure 2 Properties of cheRNAs in K562 and H1 ESCs.

a, A tabular summary of cheRNA properties as compared to lncRNAs59 and eRNAs29 by cell type (see methods). Percent overlaps are calculated as overlap of features in columns (‘-a’ in bedtools intersect) to features in rows (‘-b’). Cell-type specific lncRNAs and eRNAs were determined if they exhibited coverage from the respective cell types in our RNA-seq data. b, Coding Potential Assessment Tool (CPAT) of H1 and K562 cheRNAs compared to all Gencode (v23) lincRNAs and mRNAs (not defined by cell-type). c, Same as in Fig. 1e but with more comparisons, including categorization by location of cheRNAs relative to neighboring genes, and TADs containing cheRNAs, and the expression of all genes within TADs. Gene-gene pair analysis in TADs is representative of nearest gene expression of randomly chosen 3,293 genes (same as number of cheRNAs) within TADs. P values calculated relative to all cheRNAs, ****P<1x10-16, ***P<1x10-5, **P<0.001, *P<0.05, by Mann-Whitney U test. d, Fraction of our cheRNA set that overlap with CAGE peaks from existing data from FANTOM81. e, Gene ontology (GO) performed with GREAT on the closest neighboring gene for each cheRNA suggests lineage-specific functions for H1 and K562 cheRNAs. The categories with significant hypergeometric enrichment relative to the whole genome background are shown for each cell line measured with raw P values and annotation categories comprising 50-150 genes. See Supplementary Bioinformatics Note for details.

Supplementary Figure 3 Assessment of specificity in CRISPRi experiments and Hi-C data in K562 cells demonstrate cheRNA-gene pairs lie within the boundaries of topological domains.

a, RT-qPCR of GAPDH from CRISPRi experiments in Figure 2d-f demonstrates unchanged or inconsistent changes in GAPDH, in contrast to nearby genes. b, CRISPRi in HEK293 cells at cheRNAs targets BONIFACIO (b) and PAINE c, that are not expressed does not affect neighboring gene expression (n=3 biological replicates). IL6-ILYCH gene-cheRNA pair could not be queried because there is no detectable IL6 in HEK293 cells. d-f, Three-dimensional chromosome contacts measured by Hi-C in K562 cells from Rao et al., indicated by red color-scale33. X and y axes are the same chromosome span (indicated), and off diagonal contact enrichment represents a contact domain. CheRNA-gene pairs from figure are indicated with dotted lines.

Supplementary Figure 4 Specificity of cheRNAs through differentiation and characterization of HIDALGO transcription.

a, Overlap of cheRNAs in K562 cells in un-induced and hemin-induced states demonstrate a majority overlap, in contrast to between cell lines in general (Fig. 1d). b, RNA-seq of chromatin pellet (CPE) (purple shades) and soluble nuclear extract (SNE) (green shades) from K562 cells in +/- hemin conditions, and H1 hESC overlaid at the HBG1-HIDALGO locus. c, Schematic of HIDALGO 5’/3’ RACE products with indicated genetic features (PAS = polyadenylation sequence, TATA = TATA box, TFBS = transcription factor binding site). Red stripes indicate in-frame stop codons. Primer sets for the deconvolution of relative amounts of each isoform indicated in cyan on the gene structure are available in Supplementary Table 2. Note that the TATA just upstream of HIDALGO transcript #4 is “TATAAG”. d, 3’ RACE of HBG1. Inset represents densitometry of HBG1 3’ RACE run on an agarose gel stained with ethidium bromide. e, 5’ RACE of HBG1. Agarose gel stained with ethidium bromide depicts different HIDALGO transcripts during a hemin time-course. 3′ and 5′ RACE were performed with SMARTER 5’/3’ RACE kit (Clontech). f, RT-qPCR of HIDALGO with a panel of qPCR primers designed to assess individual transcript isoforms before and after two hours of hemin induction. Although isoform #4 cannot be uniquely probed, its several-fold induction can be inferred from it comprising little of the resting HIDALGO pool, but accounting for ~1/3 of the #1-4 increase upon induction. (n = 3, error presented is S.E.M.). g, Melting temperatures for amplicons derived from qPCR primers (Supplementary Table 2) designed to differentiate between HBG1 and HBG2 transcription by amplifying unique regions within each intron 2 demonstrate specificity.

Supplementary Figure 5 Examination of the interconnection of HIDALGO and HBG1/2 RNA products.

a, RT-qPCR of GAPDH after CRISPRi treatment with two sgRNAs positioned the same distance away from the 3’ end of GAPDH as sgRNA-4 used for targeting HIDALGO (Fig. 5b). b, Comparison of HBG1 and HBG2 mature mRNA, with differences highlighted in red, a primer set that amplifies both genes equally (HBG1/2), and 3’UTR specific primer sets that uniquely amplify HBG1 or HBG2 mRNA. c, Similar to figure 6a, 3C of HIDALGO-HBG1 locus, but +/- CRISPRi (sgRNA #3) transcriptional inhibition instead of anti-sense mediated knockdown. d, Same as Figure 5e but with a primer set targeting HBG1/2 mRNA simultaneously (see above, and Supplementary Table 2) displayed with propagated error bars. e, Similar to figure 6b, calibrated ICeChIP-qPCR targeting H3K4me3 at indicated loci in either anti-sense or CRISPRi-mediated knockdown of HIDALGO compared to negative control anti-sense oligos or negative control sgRNA, or “WT” K562 cells. f, (top) Zoomed out view of the HIDALGO-HBG1-HBG2 locus, (middle) overlay of HBG1 and HBG2 gene structure with sites of primer hybridization indicated, (bottom) intron-specific primer sets in region of intron 2 for both genes HBG1 and HBG2 as well as mismatches for the cross amplification schematically depicted.

Supplementary Figure 6 Examination of early and mature HBG1/2 RNA populations during early erythroid differentiation and in response to HIDALGO depletion in K562.

a, Assessment of HBG1 and HBG2 3’UTR primer set specificity by RT-qPCR of two independent ASO (-) cDNA samples spiked with approximate indicated copies of HBG1 template corresponding to the 500bp region of HBG1 whose 3’ end is the HBG1 3’UTR (purchased from Integrated DNA Technologies). Vertical distance corresponds to log2 transformation of the fold change in 2ΔCt of spiked-in sample relative to 2ΔCt of the same cDNA sample without any spike-in; error bars correspond to standard deviation from technical qPCR reaction replicates and includes the propagated uncertainty in the zero spike-in 2ΔCt value used for fold change calculations. b, Raw mean Ct value (averaged from 3 technical replicates) vs. approximate HBG1 spike-in copy number, for samples and primers in a (linear regression fit R2 = 0.98 and 0.96). c, Same as Figure 4c, but with inclusion of RT-qPCR data from HBG1 3’UTR primer set. d, Same as c, but using the respective HBG2 instead of HBG1 primer sets. e, Same as Figure 5d, but using the respective 3’UTR primer sets instead of intron primer sets.

Supplementary Figure 7 Knockdown of known lncRNA and eRNA-associated protein factors by CRISPRi suggests alternate pathways are involved in HIDALGO regulation of HBG1.

RT-qPCR of mRNA for each target indicated, HBG1, and HIDALGO after knockdown with CRISPRi using multiple sgRNAs. Values represent 2ΔCt relative to 18S rRNA. Error bars represent S.D., n = 4 technical replicates.

Supplementary Figure 8 HIDALGO and other cheRNAs may be derived from transposable element insertion.

a, Phylogenetic comparison of HIDALGO promoter and adjacent region by MultiZ alignment (green histogram) and primate net-alignments (black bars) in the UCSC genome browser (hg38) (http://genome.ucsc.edu). b, Overlap of the indicated genomic elements with cheRNAs that have CAGE support60 (K562, n = 1895; H1, n = 358), and Gencode lncRNAs with CAGE-support compared to average random expectation derived from overlap of 1000 Monte Carlo simulations of the same spans randomly shuffled within the genome. c, phylogenetic tree of primate evolution adapted from Johnson et al. (ref. 57), highlighting the introduction of transposable elements and the relationship of fetal hemoglobin switching to simian/prosimian divergence. d, Schematic of HIDALGO/HBG1 locus and regions cloned into luciferase constructs, and normalized luciferase in K562 cells for each construct (see methods). ****P < 10-4 (Welch’s two tailed t-test).

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Werner, M., Sullivan, M., Shah, R. et al. Chromatin-enriched lncRNAs can act as cell-type specific activators of proximal gene transcription. Nat Struct Mol Biol 24, 596–603 (2017). https://doi.org/10.1038/nsmb.3424

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