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. 2015 Aug 11;112(32):E4428-37.
doi: 10.1073/pnas.1507253112. Epub 2015 Jul 27.

MIR retrotransposon sequences provide insulators to the human genome

Jianrong Wang  1 , Cristina Vicente-García  2 , Davide Seruggia  2 , Eduardo Moltó  2 , Ana Fernandez-Miñán  3 , Ana Neto  3 , Elbert Lee  4 , José Luis Gómez-Skarmeta  3 , Lluís Montoliu  2 , Victoria V Lunyak  4 , I King Jordan  5
Affiliations

MIR retrotransposon sequences provide insulators to the human genome

Jianrong Wang et al. Proc Natl Acad Sci U S A. .

Abstract

Insulators are regulatory elements that help to organize eukaryotic chromatin via enhancer-blocking and chromatin barrier activity. Although there are several examples of transposable element (TE)-derived insulators, the contribution of TEs to human insulators has not been systematically explored. Mammalian-wide interspersed repeats (MIRs) are a conserved family of TEs that have substantial regulatory capacity and share sequence characteristics with tRNA-related insulators. We sought to evaluate whether MIRs can serve as insulators in the human genome. We applied a bioinformatic screen using genome sequence and functional genomic data from CD4(+) T cells to identify a set of 1,178 predicted MIR insulators genome-wide. These predicted MIR insulators were computationally tested to serve as chromatin barriers and regulators of gene expression in CD4(+) T cells. The activity of predicted MIR insulators was experimentally validated using in vitro and in vivo enhancer-blocking assays. MIR insulators are enriched around genes of the T-cell receptor pathway and reside at T-cell-specific boundaries of repressive and active chromatin. A total of 58% of the MIR insulators predicted here show evidence of T-cell-specific chromatin barrier and gene regulatory activity. MIR insulators appear to be CCCTC-binding factor (CTCF) independent and show a distinct local chromatin environment with marked peaks for RNA Pol III and a number of histone modifications, suggesting that MIR insulators recruit transcriptional complexes and chromatin modifying enzymes in situ to help establish chromatin and regulatory domains in the human genome. The provisioning of insulators by MIRs across the human genome suggests a specific mechanism by which TE sequences can be used to modulate gene regulatory networks.

Keywords: chromatin; gene regulation; genomics; insulators; transposable elements.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Bioinformatic screen and validation of MIR insulators. (A) Scheme of bioinformatic screen used to predict MIR insulators. Predicted MIR insulators contain intact B-box promoter sequences, are bound by RNA Pol III, segregate active (green) versus repressive (red) chromatin, segregate expressed versus silent genomic regions, and are located in intergenic genomic regions and distant (>10 kb) from each other. (B) Relative distances (normalized by domain sizes) between MIR insulators and lamina-associated domain boundaries (gray). Randomly selected B-box–containing MIR sequences are shown as controls (white). (C) Relative distances between MIR insulators and topologically associated domain boundaries in hESC. (D) Relative distances between MIR insulators and topologically associated domain boundaries in IMR90 cells. (E) Local ChIA–PET interactions flanking MIR insulators classified into one-side interactions (orange) and cross-interactions (blue). (F) Depletion of cross-interactions around MIR insulators. The observed fold between cross versus one-side interactions (red line) is compared with the distribution of folds from locally shuffled ChIA–PET interactions. (G) Sequence conservation of MIR insulators (divided into B-box and the remaining parts). Average (±SE) conservation levels of MIR insulators and 100-bp upstream/downstream sequences (red bars) are compared with randomly selected B-box–containing MIR sequences (gray bars). (H) Examples of predicted MIR insulators (black boxes) on a locus of chromosome 1. Repressive histone marks (blue), active histone marks (red), lamina domain (black bar), RNA-seq signals (purple), ChIA–PET interactions (orange) and genes are shown.
Fig. 2.
Fig. 2.
Enhancer-blocking assays (EBAs) for predicted MIR insulators. (A) Human EBA. Enhancer-blocking activity levels (fold enrichment) are normalized relative to the empty vector. Average enhancer-blocking activity levels (±SE) for positive (5′ HS4 and II/III) and negative (II/III mutated) controls along with results for three predicted MIR insulators MIR1, MIR2, and MIR3 (located on chromosomes 1, 2, and 11, respectively) are shown. For each sequence analyzed, inserts were cloned upstream of the enhancer (negative control site, gray bars) and between the enhancer and promoter (test site, black bars). (B) Enhancer-blocking activity in zebrafish. Negative (empty vector, white) control sequences along with predicted MIR insulators (purple) were inserted between the CNS enhancer and the somite promoter. The ratio of GFP expression in somites versus CNS indicates relative enhancer-blocking activity. The observed ratio for MIR2 is significantly different from control zebrafish (P = 0.011), whereas control versus MIR1 (P = 1.000) and versus MIR3 (P = 0.934) are not. (C) Zebrafish EBA. Representative pictures of GFP expression in zebrafish somites and CNS generated from negative and positive (5′ HS4) controls and the MIR insulator located on chromosome 2. (D) The MIR insulator active in zebrafish EBA. Histone marks, lamina domains, RNA-seq, and ChIA–PET interactions are shown as in Fig. 1H. (E) B-box mutation of MIR insulator. B-box mutated MIR insulator is tested in zebrafish EBA (gray) and compared with the wild-type MIR insulator (purple) and the negative control (white). The observed ratio for MIR2 is significantly different from control zebrafish (P = 0.0002), whereas the equivalent ratio for the mutated-MIR2 versus control (P = 0.069) is not.
Fig. 3.
Fig. 3.
Specific enrichment signature of chromatin features around predicted MIR insulators. The 8-kb windows centered on predicted MIR insulators were evaluated for the fold enrichment (compared with genomic background) of (A) RNA Pol III binding, (B) RNA Pol II binding, and (C) levels of five histone modifications. For each enrichment curve, a corresponding negative control (lower lines marked with crosses) is shown based on a randomly selected set of B-box–containing MIR sequences of the same size.
Fig. 4.
Fig. 4.
T-cell–specific functions of predicted MIR insulators. (A) Results of gene ontology (GO) and pathway (KEGG) analysis of proximal genes on the active chromatin side of MIR insulators. P values (−log10 normalized) are shown for the KEGG (red) and GO biological process (orange) analyses; the gray line corresponds to P = 0.05. (B) List of 21 T-cell receptor signaling pathway genes located on the active domain side proximal to MIR insulators. (C) Portion of the T-cell receptor pathway showing membrane receptors that mediate T-cell stimulation via antigen presenting cells. (D) Expression levels and the chromatin environment across a genomic cluster of three T-cell receptor genes—CD28, CTLA4, and ICOS (blue gene models)—and their colocated MIR insulators (purple bars) are shown for CD4+ T cells, GM12878 and K562. Relative gene expression levels (high-red to low-green) are shown coincident with the gene models. Genomic distributions of three histone modifications are shown as H3K4me3 (red), H3K36me3 (orange), and H3K27me3 (blue).
Fig. 5.
Fig. 5.
Cell-type–specific chromatin barrier activity and gene regulation by MIR insulators. ChIP-seq fold enrichment levels around tissue-specific MIR insulators are shown for (A) H3K4me3, (B) H3K36me3, and (C) H3K27me3 in CD4+ T cells (black), GM12878 cells (red), and K562 (orange) cells. Insets show the average differences (±SE) between the active versus repressive domains surrounding MIR insulators for the marks and cells. (D) Average gene expression levels (±SE) are shown for genes located in the active domain side proximal to MIR insulators. Gene expression levels are z transformed within each cell type. (E) Average (±SE) differences in the gene expression levels for genes located on the opposite sides of individual MIR insulators. Gene expression difference values are z transformed within each cell type. For all bar plots, significance of the differences between CD4+ T cells and other cells are indicated as *P < 0.05, **P < 0.01, and ***P < 0.001.
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