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A discrete transition zone organizes the topological and regulatory autonomy of the adjacent tfap2c and bmp7 genes.

Tsujimura T, Klein FA, Langenfeld K, Glaser J, Huber W, Spitz F - PLoS Genet. (2015)

Bottom Line: The impact of engineered chromosomal rearrangements on the topology of the locus and the resultant gene expression changes indicate that this transition zone functionally organizes the structural partition of the locus, thereby defining enhancer-target gene allocation.This partition is, however, not absolute: we show that it allows competing interactions across it that may be non-productive for the competing gene, but modulate expression of the competed one.Altogether, these data highlight the prime role of the topological organization of the genome in long-distance regulation of gene expression.

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.

ABSTRACT
Despite the well-documented role of remote enhancers in controlling developmental gene expression, the mechanisms that allocate enhancers to genes are poorly characterized. Here, we investigate the cis-regulatory organization of the locus containing the Tfap2c and Bmp7 genes in vivo, using a series of engineered chromosomal rearrangements. While these genes lie adjacent to one another, we demonstrate that they are independently regulated by distinct sets of enhancers, which in turn define non-overlapping regulatory domains. Chromosome conformation capture experiments reveal a corresponding partition of the locus in two distinct structural entities, demarcated by a discrete transition zone. The impact of engineered chromosomal rearrangements on the topology of the locus and the resultant gene expression changes indicate that this transition zone functionally organizes the structural partition of the locus, thereby defining enhancer-target gene allocation. This partition is, however, not absolute: we show that it allows competing interactions across it that may be non-productive for the competing gene, but modulate expression of the competed one. Altogether, these data highlight the prime role of the topological organization of the genome in long-distance regulation of gene expression.

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Inversion alleles reallocate the target of the heart enhancer.(A) A schematic representation of the three different inversions obtained in this study, with loxP sites as triangles, genes as plain boxes, and enhancers as ovals (FB1 or mm75) or grouped in rectangles for the ones predicted by chromatin marks (FB1/forebrain H3K27ac: blue, mm75/heart H3K27ac: pink). The TZ is represented by a whiskered red bar. In the generated inversion lines, the heart enhancer(s) were brought next to the LacZ reporter. (B) LacZ staining of the three inversion lines in E11.5 heart. Quantification by mRNA RT-qPCR of expression levels of Tfap2c (C), Bmp7 (D), Ptgis (E) and Dok5 (F) in the inversion alleles. Expression levels in wild type (wt) were normalized as 1. The error bars represent the s.d. of three biological replicates. The statistical significance was assessed by a two-sided Student's t-test. *p<0.05; **p<0.01; ***p<0.001; n.s.: non-significant.
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pgen-1004897-g004: Inversion alleles reallocate the target of the heart enhancer.(A) A schematic representation of the three different inversions obtained in this study, with loxP sites as triangles, genes as plain boxes, and enhancers as ovals (FB1 or mm75) or grouped in rectangles for the ones predicted by chromatin marks (FB1/forebrain H3K27ac: blue, mm75/heart H3K27ac: pink). The TZ is represented by a whiskered red bar. In the generated inversion lines, the heart enhancer(s) were brought next to the LacZ reporter. (B) LacZ staining of the three inversion lines in E11.5 heart. Quantification by mRNA RT-qPCR of expression levels of Tfap2c (C), Bmp7 (D), Ptgis (E) and Dok5 (F) in the inversion alleles. Expression levels in wild type (wt) were normalized as 1. The error bars represent the s.d. of three biological replicates. The statistical significance was assessed by a two-sided Student's t-test. *p<0.05; **p<0.01; ***p<0.001; n.s.: non-significant.

Mentions: We used insertions carrying loxP sites in the opposite orientation to the one left at the SB-B(3end) position in cis to engineer three balanced inversions by CRE-mediated recombination (Fig. 4A, S1 Table). In INV-L1 and -L2, the distance between Bmp7 and the heart enhancer increased to 5.7 and 1.1 Mb, respectively, whereas the relative order and distances between Tfap2c, the enhancers and the TZ region were unchanged (S7 Fig.). In INV-M, the heart enhancer was now equidistant from Bmp7 and Tfap2c (187 and 207 kb, compared to distances of 80 kb and 312 kb in the wild-type allele, with mm75 taken as reference). However, in this allele, the TZ was now located between Bmp7 and the heart enhancer(s). With each inversion, the LacZ reporter remained adjacent to the heart enhancer region and displayed its normal heart expression (Fig. 4B, S7 Fig.), demonstrating that these rearrangements did not disrupt heart enhancer activity. In the three inversions, Bmp7 expression was strongly reduced in the heart, comparable to levels observed with del1 (Fig. 4C). In contrast, Tfap2c expression was enhanced by a thousand-fold in the heart of INV-M animals (Fig. 4D), implying that in this genomic configuration, the heart enhancers now activated Tfap2c instead of Bmp7. This complete switch of the heart enhancer(s) from Bmp7 to Tfap2c coincided with the new relative position of the TZ. The importance of the position of the TZ was further supported by a lack of up-regulation of Tfap2c in INV-L1 and INV-L2 (Fig. 4D), where its location with regards to the TZ/heart enhancers remained unchanged. In INV-L1, we instead found an up-regulation of Ptgis (Fig. 4E), which was now located on the other side of the TZ, next to mm75. As Ptgis was closer to the heart enhancer (S7A Fig.) we were unable in this case to fully rule out a possible influence of distance on promoter choice. However, in INV-L2, Dok5, the new gene juxtaposed “next to” the heart enhancer(s) opposite to TZ was much further away than Tfap2c (1.1 Mb versus 0.3 Mb). In this context, neither Dok5 (Fig. 4F) nor Tfap2c were up-regulated in the heart, ruling out the possibility that the heart enhancer(s) act simply by default the nearest gene.


A discrete transition zone organizes the topological and regulatory autonomy of the adjacent tfap2c and bmp7 genes.

Tsujimura T, Klein FA, Langenfeld K, Glaser J, Huber W, Spitz F - PLoS Genet. (2015)

Inversion alleles reallocate the target of the heart enhancer.(A) A schematic representation of the three different inversions obtained in this study, with loxP sites as triangles, genes as plain boxes, and enhancers as ovals (FB1 or mm75) or grouped in rectangles for the ones predicted by chromatin marks (FB1/forebrain H3K27ac: blue, mm75/heart H3K27ac: pink). The TZ is represented by a whiskered red bar. In the generated inversion lines, the heart enhancer(s) were brought next to the LacZ reporter. (B) LacZ staining of the three inversion lines in E11.5 heart. Quantification by mRNA RT-qPCR of expression levels of Tfap2c (C), Bmp7 (D), Ptgis (E) and Dok5 (F) in the inversion alleles. Expression levels in wild type (wt) were normalized as 1. The error bars represent the s.d. of three biological replicates. The statistical significance was assessed by a two-sided Student's t-test. *p<0.05; **p<0.01; ***p<0.001; n.s.: non-significant.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4288730&req=5

pgen-1004897-g004: Inversion alleles reallocate the target of the heart enhancer.(A) A schematic representation of the three different inversions obtained in this study, with loxP sites as triangles, genes as plain boxes, and enhancers as ovals (FB1 or mm75) or grouped in rectangles for the ones predicted by chromatin marks (FB1/forebrain H3K27ac: blue, mm75/heart H3K27ac: pink). The TZ is represented by a whiskered red bar. In the generated inversion lines, the heart enhancer(s) were brought next to the LacZ reporter. (B) LacZ staining of the three inversion lines in E11.5 heart. Quantification by mRNA RT-qPCR of expression levels of Tfap2c (C), Bmp7 (D), Ptgis (E) and Dok5 (F) in the inversion alleles. Expression levels in wild type (wt) were normalized as 1. The error bars represent the s.d. of three biological replicates. The statistical significance was assessed by a two-sided Student's t-test. *p<0.05; **p<0.01; ***p<0.001; n.s.: non-significant.
Mentions: We used insertions carrying loxP sites in the opposite orientation to the one left at the SB-B(3end) position in cis to engineer three balanced inversions by CRE-mediated recombination (Fig. 4A, S1 Table). In INV-L1 and -L2, the distance between Bmp7 and the heart enhancer increased to 5.7 and 1.1 Mb, respectively, whereas the relative order and distances between Tfap2c, the enhancers and the TZ region were unchanged (S7 Fig.). In INV-M, the heart enhancer was now equidistant from Bmp7 and Tfap2c (187 and 207 kb, compared to distances of 80 kb and 312 kb in the wild-type allele, with mm75 taken as reference). However, in this allele, the TZ was now located between Bmp7 and the heart enhancer(s). With each inversion, the LacZ reporter remained adjacent to the heart enhancer region and displayed its normal heart expression (Fig. 4B, S7 Fig.), demonstrating that these rearrangements did not disrupt heart enhancer activity. In the three inversions, Bmp7 expression was strongly reduced in the heart, comparable to levels observed with del1 (Fig. 4C). In contrast, Tfap2c expression was enhanced by a thousand-fold in the heart of INV-M animals (Fig. 4D), implying that in this genomic configuration, the heart enhancers now activated Tfap2c instead of Bmp7. This complete switch of the heart enhancer(s) from Bmp7 to Tfap2c coincided with the new relative position of the TZ. The importance of the position of the TZ was further supported by a lack of up-regulation of Tfap2c in INV-L1 and INV-L2 (Fig. 4D), where its location with regards to the TZ/heart enhancers remained unchanged. In INV-L1, we instead found an up-regulation of Ptgis (Fig. 4E), which was now located on the other side of the TZ, next to mm75. As Ptgis was closer to the heart enhancer (S7A Fig.) we were unable in this case to fully rule out a possible influence of distance on promoter choice. However, in INV-L2, Dok5, the new gene juxtaposed “next to” the heart enhancer(s) opposite to TZ was much further away than Tfap2c (1.1 Mb versus 0.3 Mb). In this context, neither Dok5 (Fig. 4F) nor Tfap2c were up-regulated in the heart, ruling out the possibility that the heart enhancer(s) act simply by default the nearest gene.

Bottom Line: The impact of engineered chromosomal rearrangements on the topology of the locus and the resultant gene expression changes indicate that this transition zone functionally organizes the structural partition of the locus, thereby defining enhancer-target gene allocation.This partition is, however, not absolute: we show that it allows competing interactions across it that may be non-productive for the competing gene, but modulate expression of the competed one.Altogether, these data highlight the prime role of the topological organization of the genome in long-distance regulation of gene expression.

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.

ABSTRACT
Despite the well-documented role of remote enhancers in controlling developmental gene expression, the mechanisms that allocate enhancers to genes are poorly characterized. Here, we investigate the cis-regulatory organization of the locus containing the Tfap2c and Bmp7 genes in vivo, using a series of engineered chromosomal rearrangements. While these genes lie adjacent to one another, we demonstrate that they are independently regulated by distinct sets of enhancers, which in turn define non-overlapping regulatory domains. Chromosome conformation capture experiments reveal a corresponding partition of the locus in two distinct structural entities, demarcated by a discrete transition zone. The impact of engineered chromosomal rearrangements on the topology of the locus and the resultant gene expression changes indicate that this transition zone functionally organizes the structural partition of the locus, thereby defining enhancer-target gene allocation. This partition is, however, not absolute: we show that it allows competing interactions across it that may be non-productive for the competing gene, but modulate expression of the competed one. Altogether, these data highlight the prime role of the topological organization of the genome in long-distance regulation of gene expression.

Show MeSH
Related in: MedlinePlus