Limits...
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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Changes of gene expression in the forebrain following genomic inversion.Quantification by RT-qPCR of the relative expression levels of the Tfap2c and Bmp7 mRNAs in the lateral and medial forebrain for INV-L1 (A), INV-L2 (B) and INV-M (D), normalized as in Fig. 2. 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. (C) Absence of LacZ staining in the forebrain of SB/INV-L1 and SB/INV-L2 E11.5 embryos. (E) LacZ staining of SB-A2 (up) and INV-M (bottom) E11.5 embryos.
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pgen-1004897-g006: Changes of gene expression in the forebrain following genomic inversion.Quantification by RT-qPCR of the relative expression levels of the Tfap2c and Bmp7 mRNAs in the lateral and medial forebrain for INV-L1 (A), INV-L2 (B) and INV-M (D), normalized as in Fig. 2. 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. (C) Absence of LacZ staining in the forebrain of SB/INV-L1 and SB/INV-L2 E11.5 embryos. (E) LacZ staining of SB-A2 (up) and INV-M (bottom) E11.5 embryos.

Mentions: The expression and structural changes observed in the heart suggested that the TZ behaved as a simple insulator region. In INV-L1 and INV-L2, the Tfap2c domain was fully maintained and unaffected by the genomic rearrangements. Therefore one would expect little impact on Tfap2c. However, we observed an up-regulation of Tfap2c in the medial telencephalon in both alleles (Fig. 6A–B). This up-regulation is unlikely to be caused by the juxtaposition of new forebrain enhancers, as the regulatory sensor did not detect any forebrain activity in L1 and L2 position, in either the inverted or non-inverted configurations (Fig. 6C). We noted that in INV-L1 and –L2, Bmp7, which is strongly expressed in the medial forebrain, was relocated away from Tfap2c and its forebrain enhancer. This rearrangement had no effect on Bmp7 expression in the forebrain, suggesting that it was the presence of Bmp7 in cis that negatively influenced Tfap2c. Supporting this hypothesis, we did not observe any up-regulation of Tfap2c in the medial forebrain of INV-M embryos (Fig. 6D), where Bmp7 remained adjacent to the Tfap2c. These observations prompted us to re-examine the 4C profiles. As stated before, the intensity of the 4C signals diminished strongly beyond the TZ region. However, we observed that the 4C contacts made by the Bmp7 promoter, albeit weak, were stronger over the Tfap2c domain than over the region located symmetrically from the viewpoint (S9 Fig., green boxes). Reciprocally, Tfap2c showed weak but consistent interactions with the Bmp7 region in WT and INV-M (S9 Fig., blue boxes), interactions which are not observed with a symmetrically located region, or with the region at the equivalent place in INV-L2. To further test if the INV-L1 and –L2 up-regulation of Tfap2c depended on the removal of Bmp7, we produced INV-Bmp7 which consists in a simple inversion of the gene itself. Consequently, Bmp7 remained adjacent to the Tfap2c domain, and separated from it by the TZ (S11A Fig.). In this configuration, we did not observe significant changes of Bmp7 or Tfap2c expression, with the exception of a small reduction of Bmp7 expression in the lateral forebrain. Altogether, these results supported that the simple presence of an active Bmp7 in cis, despite the presence of the TZ region, can affect Tfap2c expression in the medial forebrain.


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)

Changes of gene expression in the forebrain following genomic inversion.Quantification by RT-qPCR of the relative expression levels of the Tfap2c and Bmp7 mRNAs in the lateral and medial forebrain for INV-L1 (A), INV-L2 (B) and INV-M (D), normalized as in Fig. 2. 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. (C) Absence of LacZ staining in the forebrain of SB/INV-L1 and SB/INV-L2 E11.5 embryos. (E) LacZ staining of SB-A2 (up) and INV-M (bottom) E11.5 embryos.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004897-g006: Changes of gene expression in the forebrain following genomic inversion.Quantification by RT-qPCR of the relative expression levels of the Tfap2c and Bmp7 mRNAs in the lateral and medial forebrain for INV-L1 (A), INV-L2 (B) and INV-M (D), normalized as in Fig. 2. 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. (C) Absence of LacZ staining in the forebrain of SB/INV-L1 and SB/INV-L2 E11.5 embryos. (E) LacZ staining of SB-A2 (up) and INV-M (bottom) E11.5 embryos.
Mentions: The expression and structural changes observed in the heart suggested that the TZ behaved as a simple insulator region. In INV-L1 and INV-L2, the Tfap2c domain was fully maintained and unaffected by the genomic rearrangements. Therefore one would expect little impact on Tfap2c. However, we observed an up-regulation of Tfap2c in the medial telencephalon in both alleles (Fig. 6A–B). This up-regulation is unlikely to be caused by the juxtaposition of new forebrain enhancers, as the regulatory sensor did not detect any forebrain activity in L1 and L2 position, in either the inverted or non-inverted configurations (Fig. 6C). We noted that in INV-L1 and –L2, Bmp7, which is strongly expressed in the medial forebrain, was relocated away from Tfap2c and its forebrain enhancer. This rearrangement had no effect on Bmp7 expression in the forebrain, suggesting that it was the presence of Bmp7 in cis that negatively influenced Tfap2c. Supporting this hypothesis, we did not observe any up-regulation of Tfap2c in the medial forebrain of INV-M embryos (Fig. 6D), where Bmp7 remained adjacent to the Tfap2c. These observations prompted us to re-examine the 4C profiles. As stated before, the intensity of the 4C signals diminished strongly beyond the TZ region. However, we observed that the 4C contacts made by the Bmp7 promoter, albeit weak, were stronger over the Tfap2c domain than over the region located symmetrically from the viewpoint (S9 Fig., green boxes). Reciprocally, Tfap2c showed weak but consistent interactions with the Bmp7 region in WT and INV-M (S9 Fig., blue boxes), interactions which are not observed with a symmetrically located region, or with the region at the equivalent place in INV-L2. To further test if the INV-L1 and –L2 up-regulation of Tfap2c depended on the removal of Bmp7, we produced INV-Bmp7 which consists in a simple inversion of the gene itself. Consequently, Bmp7 remained adjacent to the Tfap2c domain, and separated from it by the TZ (S11A Fig.). In this configuration, we did not observe significant changes of Bmp7 or Tfap2c expression, with the exception of a small reduction of Bmp7 expression in the lateral forebrain. Altogether, these results supported that the simple presence of an active Bmp7 in cis, despite the presence of the TZ region, can affect Tfap2c expression in the medial forebrain.

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