Enhancer-driven chromatin interactions during development promote escape from silencing by a long non-coding RNA

Abstract

Background: Gene regulation in eukaryotes is a complex process entailing the establishment of transcriptionally silent chromatin domains interspersed with regions of active transcription. Imprinted domains consist of clusters of genes, some of which exhibit parent-of-origin dependent monoallelic expression, while others are biallelic. The Kcnq1 imprinted domain illustrates the complexities of long-range regulation that coexists with local exceptions. A paternally expressed repressive non-coding RNA, Kcnq1ot1, regulates a domain of up to 750 kb, encompassing 14 genes. We study how the Kcnq1 gene, initially silenced by Kcnq1ot1, undergoes tissue-specific escape from imprinting during development. Specifically, we uncover the role of chromosome conformation during these events.

Results: We show that Kcnq1 transitions from monoallelic to biallelic expression during mid gestation in the developing heart. This transition is not associated with the loss of methylation on the Kcnq1 promoter. However, by exploiting chromosome conformation capture (3C) technology, we find tissue-specific and stage-specific chromatin loops between the Kcnq1 promoter and newly identified DNA regulatory elements. These regulatory elements showed in vitro activity in a luciferase assay and in vivo activity in transgenic embryos.

Conclusions: By exploring the spatial organization of the Kcnq1 locus, our results reveal a novel mechanism by which local activation of genes can override the regional silencing effects of non-coding RNAs.

Figure 1

Schematic of the Kcnq1 imprinted domain on mouse chromosome 7. The imprinting pattern in the embryo is shown. Arrows indicate direction of transcription. Arrows above the genes represent maternal transcription, arrows below the line are paternal transcription and genes with two arrows have biallelic expression.

Figure 2

(A) Developmental imprinting pattern of Kcnq1. Allele-specific expression of Kcnq1 as assayed by reverse transcription (RT)-PCR and restriction digest with NlaIII on E10.5, 11.5, 12.5, 13.5, 14.5, 16.5 and neonatal heart (nnH) from F1 hybrid B6(CAST7) × C57BL/6J crosses. Digestion products specific for B6(CAST7) (maternal) and C57BL/6J (paternal) alleles are indicated. Positive signs (+) denote addition of NlaIII to the RT-PCR product. (B) Quantification of relative paternal-specific and maternal-specific expression during development. (C) Kcnq1 RNA abundance during stages of development in which the imprinting pattern switches from monoallelic to biallelic, as assayed by real-time PCR. (D) Methylated DNA immunoprecipitation (MeDIP) analysis of the Kcnq1 and Kcnq1ot1 promoter regions in sperm and 7.5 days post coitum (dpc) embryos. 5meC lane = DNA precipitated by antibody against methylated cytosine; IgG = non-specific immunoprecipitation; Input = DNA before immunoprecipitation; - = no antibody control. Specific bands for Kcnq1 and Kcnq1ot1 are indicated; NS = non-specific amplification product. The Kcnq1ot1 promoter is methylated maternally in 7.5 dpc embryos and unmethylated in sperm, thus serving as a positive control for immunoprecipitation of methylated DNA in E7.5 DNA and a negative control in sperm DNA.

Figure 3

Chromosome conformation capture (3C) mapping of long-range chromatin interactions in the Kcnq1 region in neonatal tissues. (A) Genomic organization of the Kcnq1 region scanned in this study, spanning 387 kb. Dark boxes in the Kcnq1 gene are exons. Arrows show direction of transcription; maternal-specific and paternal-specific expression is indicated above and below the line. Reactivated Kcnq1 expression is depicted as a red arrow. Vertical bars indicate only the AflIII/NcoI restriction sites analyzed in this study, with the fragments assayed by the variable primers for contact with the Kcnq1 promoter numbered. White arrowhead indicates the anchor primer at the Kcnq1 promoter. Graphical representation is not to scale. (B,C) Relative crosslinking frequency of regions interacting with the Kcnq1 promoter, with association values plotted on the y-axis. Distances in kb relative to the Kcnq1 promoter are plotted along the x-axis (not to scale). Each value is derived from three independent samples and the standard error is indicated. Hatched vertical line represents the anchor position. High crosslinking frequencies with the anchor fragment indicate close proximity. (B) 3C map of neonatal heart and brain. (C) Reciprocal 3C analysis of heart-specific interactions in the Kcnq1 region by anchoring the PCR reactions at fragment 12 (intron 1).

Figure 4

Reciprocal chromosome conformation capture (3C) scans anchoring at evolutionarily conserved regions. (A) Genomic organization of the Kcnq1 region scanned in this study, spanning 387 kb. Dark boxes in the Kcnq1 gene are exons. Arrows show direction of transcription; maternal-specific and paternal-specific expression is indicated above and below the line. Reactivated Kcnq1 expression is depicted as a red arrow. Vertical bars indicate the AflIII/NcoI restriction sites analyzed in this study, with the fragments assayed with the variable primers for contact with the tissue-specific evolutionarily conserved regions (ECRs) numbered. White arrowhead, anchor primer for neonatal heart; gray arrowhead, anchor primer for neonatal brain. Graphical representation is not to scale. (B) Relative crosslinking frequency of regions interacting with ECR 4. The peaks correspond to the Kcnq1 promoter and fragment 12 within intron 1. (C) Relative crosslinking frequency of regions interacting with ECR 2. The peaks correspond to the Kcnq1 promoter and fragment 12 within intron 1. Hatched vertical lines represent the anchor position.

Figure 5

Developmental profile of chromatin interactions in the Kcnq1 region. (A) Genomic organization of the Kcnq1 region scanned in this study, spanning 387 kb. Dark boxes in the Kcnq1 gene are exons. Arrows show direction of transcription; maternal-specific and paternal-specific expression is indicated above and below the line. Reactivated Kcnq1 expression is depicted as a red arrow. Vertical bars indicate only the AflIII/NcoI restriction sites analyzed in this study, with the fragments assayed with the variable primers for contact with the Kcnq1 promoter numbered. White arrowhead indicates the anchor primer at the Kcnq1 promoter. Graphical representation is not to scale. (B) Developmental profile of interactions of the Kcnq1 promoter in ES cells, 11.5 days post coitum (dpc) heart, and neonatal heart. Relative crosslinking frequency of regions interacting with the Kcnq1 promoter, with association values plotted on the y-axis. Distances in kb relative to the Kcnq1 promoter are plotted along the x-axis (not to scale). Each value is derived from three independent samples and the standard error is indicated. Hatched vertical line represents the anchor position. High crosslinking frequencies with the anchor fragment indicate close proximity.

Figure 6

Identification and functional validation of candidate regulatory elements. (A) Representation of the upstream region of Kcnq1, indicating the fragments tested in the chromosome conformation capture (3C) assays. (B) Graphic display of the conservation profiles for the upstream region of Kcnq1. The base genome is mouse. Evolutionarily conserved regions (ECRs) of a minimum of 100 bp conserved above 70% sequence identity are displayed as red (intergenic) peaks, with the x-axis representing positions in the base genome and the y-axis representing percentage identity between the base and the aligned genomes. Annotated genes are depicted in blue. Numbered peaks represent the fragments tested for enhancer activity. (C) DNA sequences were inserted in pGL3-promoter vector upstream of the luciferase reporter. Luciferase activity was normalized to Renilla activity. All transfections were performed in triplicate. The asterisk denotes the interaction observed in neonatal heart by 3C. (D) Representative LacZ-stained embryos with in vivo enhancer activity. ECR4 and ECR2 exhibit tissue-specific activity, with numbers showing the reproducibility of LacZ reporter staining.

Figure 7

Model for escape from silencing by the Kcnq1 gene. Enhancer candidates, depicted as purple circles, become active upon binding tissue-specific transcription factors (TF); ncRNA, wavy lines. (A) Appearance of enhancer-specific transcription factors promotes a conformational change that sequesters the Kcnq1 promoter from the effects of Kcnq1ot1. (B) Factors binding in intron 1 of Kcnq1 act as a boundary for the Kcnq1ot1 ncRNA on the paternal allele, leaving it available for tissue-specific activation by enhancer-binding regulatory proteins.