Transcription of the non-coding RNA upperhand controls Hand2 expression and heart development

Abstract

HAND2 is an ancestral regulator of heart development and one of four transcription factors that control the reprogramming of fibroblasts into cardiomyocytes1,2,3,4. Deletion of Hand2 in mice results in right ventricle hypoplasia and embryonic lethality1,5. Hand2 expression is tightly regulated by upstream enhancers6,7 that reside within a super-enhancer delineated by histone H3 acetyl Lys27 (H3K27ac) modifications8. Here we show that transcription of a Hand2-associated long non-coding RNA, which we named upperhand (Uph), is required to maintain the super-enhancer signature and elongation of RNA polymerase II through the Hand2 enhancer locus. Blockade of Uph transcription, but not knockdown of the mature transcript, abolished Hand2 expression, causing right ventricular hypoplasia and embryonic lethality in mice. Given the substantial number of uncharacterized promoter-associated long non-coding RNAs encoded by the mammalian genome9, the UphHand2 regulatory partnership offers a mechanism by which divergent non-coding transcription can establish a permissive chromatin environment.

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Figure 1: Co-expression of upperhand (Uph) and Hand2.
Figure 2: Cardiac defects of Uph KO embryos.
Figure 3: Loss of cardiac Hand2 expression in Uph KO embryos.
Figure 4: Uph transcription in cis is required for active enhancer marks in the Uph locus and RNAPII elongation at the Hand2 gene.

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Acknowledgements

We thank D. Tennison for technical assistance and J. Cabrera for graphics. We thank the Histopathology Core Facility at University of Texas Southwestern for histology. This work was supported by grants from the National Institutes of Health (HL-077439, AR-067294, HL-130253, DK-099653 and U01-HL-100401), Fondation Leducq Networks of Excellence, Cancer Prevention and Research Institute of Texas and the Robert A. Welch Foundation (grant 1-0025 to E.N.O.), a pre-doctoral fellowship from the American Heart Association (14PRE19830031) to K.M.A. and a Muscular Dystrophy Association Development Grant (MDA377340) to D.M.A.

Author information

K.M.A., D.M.A. and E.N.O. designed experiments and analysed data. J.R.M. generated mutant mice from constructs designed and created by K.M.A. and D.M.A. K.M.A. performed qPCR, phenotypic analysis, dissections, ChIP, RNA immunoprecipitation, cardiomyocyte fractionation, Southern blot, and India ink injections. D.M.A performed northern blot, RACE, in vitro transcription/translation, and GapmeR transfections. J.M.S. and K.M.A. performed in situ hybridization and generated images. K.M.A., D.M.A, E.N.O. and R.B.D. wrote and edited the manuscript.

Correspondence to Eric N. Olson.

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Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks H. Chang and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Sequence alignment of several mammalian Uph transcripts.

a, Sequence alignment of several mammalian Uph transcripts performed using ClustalW. See Methods for source data. b, Diagram of the Hand2 locus in mammals showing the genomic organization and orientation of Uph. The Hand2 branchial arch enhancer (green) and cardiac enhancer (yellow) are shown. c, Diagram of the Uph transcripts expressed in the mouse heart, determined using 5′ and 3′ RACE using primers specific to exon 4 of Uph. AP1, marathon adaptor primer; 3′GSP, 3′ Uph-specific primer from exon 4; 5′GSP, 5′ Uph-specific primer from exon 4. d, Whole mount in situ hybridization of E10.5 mouse embryos. Expression was detected in heart, branchial arches (arrowhead), and limb bud. Scale bars, 1 mm. e, Northern blot analysis of total RNA from adult mouse tissues using a probe specific to the major Uph transcript. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 2 Uph is a cytoplasmic lncRNA.

a, Subcellular fractionation of 18S, Uph and Malat1 lncRNA in mouse neonatal cardiomyocytes (n = 3 biological replicates from 1 of 5 independent experiments; mean ± s.e.m.). b, In vitro transcription and translation of a plasmid encoding the major Uph RNA. A plasmid encoding the myoregulin (MLN) micropeptide was used as a positive control, and myoregulin with a frameshift mutation (MLN RNA-FS) was used as a negative control. In contrast to myoregulin, Uph and the negative control (MLN RNA-FS) did not produce any detectable proteins, indicating that Uph is a bona fide lncRNA. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 3 Targeting strategy for insertion of transcriptional termination or heterologous sequence into exon 2 of Uph.

a, Uph KO targeting strategy. Transcription activator-like effector nucleases (TALENs) were used to insert a triple polyadenylation (tpA) termination sequence into exon 2 (E2) of Uph. b, Using the same TALEN pair as in a, we introduced the coding sequence of tdTO, lacking a polyadenylation sequence, into exon 2 of the Uph locus. Exon 2, which includes the tdTO coding sequence, was spliced out of the mature Uph transcript, preventing expression of tdTO in these mice. c, Southern blot analysis of wild-type, heterozygous Uph+/− (Het) and Uph KO genomic DNA. BamHI-digested DNA hybridized with a 5′-specific probe and NdeI-digested DNA hybridized with a 3′-specific probe. For gel source data, see Supplementary Fig. 1. d, Southern blot analysis of wild-type, UphtdTO/+ heterozygous and UphtdTO/tdTO knock-in (KI) genomic DNA verified the correct targeting of the tdTO sequence into the Uph locus. DNA was digested with BamHI and hybridized with a 5′-specific probe or digested with NdeI and hybridized with a 3′-specific probe. For gel source data, see Supplementary Fig. 1. e, f, Expression of Uph (e) and Hand2 (f) in wild-type and UphtdTO/tdTO homozygous mice at E10.0 was not changed by the insertion of the tdTO sequence into exon 2 of the Uph locus (n = 3, representative of 3 independent experiments; mean ± s.e.m.). g, qPCR shows Uph transcripts decreased by ~97% in E10.5 hearts (n = 3 mice of each genotype from 1 of 3 independent experiments; mean ± s.e.m.).

Extended Data Figure 4 Aortic arch arteries are normal in Uph KO embryos.

a, qPCR quantification of gene expression at E10.0 showed robust downregulation of Uph and Hand2 expression in Uph KO hearts, with normal expression of other cardiac transcription factors (n = 3 mice of each genotype from 1 of 3 independent experiments; mean ± s.e.m.). b, India ink was injected into either the left ventricle of wild-type embryos or the single ventricle of Uph KO embryos at E10.5, to visualize the aortic arch arteries and circulation, which appeared normal in Uph KO embryos. aa, aortic arch arteries; as, aortic sac; da, dorsal aorta. Scale bars, 1 mm.

Extended Data Figure 5 Uph+/− Hand2+/− compound heterozygotes recapitulate Uph KO phenotype.

a, Uph+/− Hand2+/− double heterozygote embryos developed a single ventricle, pericardial effusion, and were severely growth restricted by E11.5. Scale bars, 1 mm. b, Uph expression is normal in double heterozygotes, whereas Hand2 was reduced by ~90%, relative to wild-type embryos (n = 5 mice for wild type, Hand2 het and Uph het, n = 3 for double het, n = 2 for Hand2 KO, from 1 of 2 independent experiments; mean ± s.e.m.). c, Immunoprecipitation of RNA using either IgG or WDR5 in HL-1 cells followed by qPCR for Uph revealed no binding to WDR5. The WDR5-interacting lncRNA HOTTIP was used as a positive control (n = 3 biological replicates from 1 of 3 independent experiments; mean ± s.e.m.).

Extended Data Figure 6 Knockdown of mature Uph transcripts in HL-1 or Neuro2a cells does not affect Hand2 expression.

a, Expression of Uph (red) and Hand2 (blue) in various tissues and cell lines. Uph and Hand2 are robustly expressed in the heart, and the HL-1 and Neuro2a cell lines. Uph and Hand2 transcripts are not expressed in the liver, 10T1/2 fibroblasts or skeletal muscle C2C12 myotubes (n = 3 technical replicates; mean ± s.e.m.). b, c, Uph transcripts were reduced by ~90% in HL-1 (b) and Neuro2a (c) cells when transfected with two independent GapmeR antisense oligonucleotide probes (ASO A and B) against Uph. Expression of Hand2 was not changed (n = 2 for ASO A and B, n = 3 for control, from 1 of 2 independent experiments; mean ± s.e.m.). d, Uph transcripts were similarly downregulated across each exon–exon junction, measured using qPCR (n = 2 for ASO A and B, n = 3 for control, from 1 of 2 independent experiments; mean ± s.e.m.). e, Fractionation of HL-1 cells transfected with control or Uph-specific ASOs. The nuclear fraction of antisense-oligonucleotide-treated HL-1 cells showed a similar downregulation of Uph transcripts (n = 3 biological replicates from 1 of 3 independent experiments; mean ± s.e.m.). f, Overexpression of enhanced green fluorescent protein (eGFP; blue), the major Uph RNA (red) or Hand2 RNA (green) in HL-1 cells revealed that the mature Uph transcript did not alter Hand2 expression relative to wild type, and that Hand2 does not influence the expression of Uph in these cells (n = 2 biological replicates from 1 of 2 independent experiments; mean ± s.e.m.).

Extended Data Figure 7 ChIP and qPCR analyses of active cardiac enhancers regulating Nkx2-5 expression.

a, Diagram of the Nkx2-5 locus with numbers (1 and 2) indicating the region analysed by qPCR following ChIP. Shown in red are the ENCODE/LICR H3K4me1 active enhancer marks in the heart at E14.5. See Methods for source data. b, ChIP coupled with qPCR analysis of H3K4me1 marks, normalized to total histone H3, showed that the H3K4me1 marks bound to the Nkx2-5 promoter region are unchanged between wild-type, Uph KO and UphtdTO/tdTO homozygous hearts at E10.0. c, ChIP coupled with qPCR analysis of H3K27ac marks, normalized to total histone H3, showed that the H3K27ac marks bound to the Nkx2-5 promoter region are unchanged between wild-type, Uph KO and UphtdTO/tdTO homozygous hearts at E10.0. d, GATA4 binding to a GATA4 site in the Nkx2-5 promoter is unchanged between wild-type and Uph KO hearts at E10.0. e, Diagram of the Uph–Hand2 locus, with black bars indicating the regions analysed by qPCR following ChIP. Shown in red is the ENCODE/LICR H3K27me3 track for mouse heart. See Methods for source data. The branchial arch enhancer (green) and cardiac enhancer (yellow) are shown. f, ChIP coupled with qPCR analysis of the polycomb repressive marker H3K27me3, normalized to total histone H3, showed no differences between genotypes at each locus (1–3) indicated in the diagram in e. g, qPCR revealed no difference in the levels of Ser2-phosphorylated RNAPII between genotypes at the Nkx2-5 gene body. Each point is one of 3 technical replicates of 5 pooled hearts for each genotype in each ChIP experiment, from 1 of 2 independent experiments; mean ± s.e.m.

Extended Data Table 1 Relevant sequences

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This file contains the Source data for Extended Data Figures 1e, 2d, 3c, 3d. (PDF 1082 kb)

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Anderson, K., Anderson, D., McAnally, J. et al. Transcription of the non-coding RNA upperhand controls Hand2 expression and heart development. Nature 539, 433–436 (2016). https://doi.org/10.1038/nature20128

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