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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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Deletion alleles localise enhancers.(A) A schematic representation of the deletions generated. The loxP sites used for CRE-mediated recombination are depicted as filled red triangles for the one carried with the transposon. Open triangles indicate both the position of the static loxP at the 3′end of Bmp7 and the position of the LacZ reporter gene in deletions produced by TAMERE. (B) LacZ staining patterns of the three deletion lines in forebrain (top) and heart (bottom) in E11.5 embryos, in comparison to SB-A1 and SB-B3(3end), respectively. The deletions led to complete loss of the both lateral and medial expression in the forebrain (blue arrows in the SB-A1 embryo). (C) Relative expression levels of the Tfap2c and Bmp7 mRNAs in the heart, lateral and medial forebrains from E11.5 embryos measured by RT-qPCR in del1 homozygous, heterozygous, and control (wt) genotypes. For each gene, expression levels were normalized with Gapdh. Expression of the wild type allele in the lateral forebrain for Tfap2c and in the medial forebrain for Bmp7, respectively, was set as 1. The error bars represent s.d. from three biological replicates. Statistical significance was assessed by a two-sided t-test. *p<0.05; **p<0.01; ***p<0.001. (D) In situ hybridization of the wild type and del1 embryos at E10.5 with the anti-sense RNA probes for Tfap2c and Bmp7. (E) Enhancer activity of FB1 on the LacZ reporter gene. 32 out of 52 transgenic embryos showed broad forebrain expression. (F) The mm75 element drives specific expression in the mouse embryos at E11.5 (from VISTA enhancer browser: http://enhancer.lbl.gov). (G). Regulatory domains along the Tfap2c-Bmp7 interval. The forebrain enhancer (FB1) and the heart enhancer (mm75) are depicted with blue and pink ovals. A light blue (resp. pink) rectangle represents the region encompassing the H3K27ac peaks present in the segment deleted in del1, in the forebrain (resp. heart) (S3 Fig.).
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pgen-1004897-g002: Deletion alleles localise enhancers.(A) A schematic representation of the deletions generated. The loxP sites used for CRE-mediated recombination are depicted as filled red triangles for the one carried with the transposon. Open triangles indicate both the position of the static loxP at the 3′end of Bmp7 and the position of the LacZ reporter gene in deletions produced by TAMERE. (B) LacZ staining patterns of the three deletion lines in forebrain (top) and heart (bottom) in E11.5 embryos, in comparison to SB-A1 and SB-B3(3end), respectively. The deletions led to complete loss of the both lateral and medial expression in the forebrain (blue arrows in the SB-A1 embryo). (C) Relative expression levels of the Tfap2c and Bmp7 mRNAs in the heart, lateral and medial forebrains from E11.5 embryos measured by RT-qPCR in del1 homozygous, heterozygous, and control (wt) genotypes. For each gene, expression levels were normalized with Gapdh. Expression of the wild type allele in the lateral forebrain for Tfap2c and in the medial forebrain for Bmp7, respectively, was set as 1. The error bars represent s.d. from three biological replicates. Statistical significance was assessed by a two-sided t-test. *p<0.05; **p<0.01; ***p<0.001. (D) In situ hybridization of the wild type and del1 embryos at E10.5 with the anti-sense RNA probes for Tfap2c and Bmp7. (E) Enhancer activity of FB1 on the LacZ reporter gene. 32 out of 52 transgenic embryos showed broad forebrain expression. (F) The mm75 element drives specific expression in the mouse embryos at E11.5 (from VISTA enhancer browser: http://enhancer.lbl.gov). (G). Regulatory domains along the Tfap2c-Bmp7 interval. The forebrain enhancer (FB1) and the heart enhancer (mm75) are depicted with blue and pink ovals. A light blue (resp. pink) rectangle represents the region encompassing the H3K27ac peaks present in the segment deleted in del1, in the forebrain (resp. heart) (S3 Fig.).

Mentions: To further characterize the functional relevance of these two domains and associated enhancers, we used in vivo Cre-mediated recombination to engineer chromosomal deletions removing either the telomeric half or the whole of the intergenic region (Fig. 2). Each deletion was produced using a combination of loxP sites in cis and trans[54] in order to keep the LacZ sensor at the deletion breakpoint (see Materials and Methods). With the TAMERE strategy, we also obtained a large duplication, reciprocal to del3 (S2 Fig.). All three deletions led to a complete loss of LacZ expression in the embryonic heart and forebrain (Fig. 2B) suggesting that the enhancers detected by SB-A1 and SB-B(3end) lie in the region encompassed by del1. Dup3-lacZ embryos showed LacZ expression in the heart similar to SB-B(3end), corroborating the presence of the heart enhancer(s) at the 3′ side of Bmp7 (S2 Fig.). These deletions also provided information on the locations of additional enhancers associated with other expression domains (S2 Fig.).


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)

Deletion alleles localise enhancers.(A) A schematic representation of the deletions generated. The loxP sites used for CRE-mediated recombination are depicted as filled red triangles for the one carried with the transposon. Open triangles indicate both the position of the static loxP at the 3′end of Bmp7 and the position of the LacZ reporter gene in deletions produced by TAMERE. (B) LacZ staining patterns of the three deletion lines in forebrain (top) and heart (bottom) in E11.5 embryos, in comparison to SB-A1 and SB-B3(3end), respectively. The deletions led to complete loss of the both lateral and medial expression in the forebrain (blue arrows in the SB-A1 embryo). (C) Relative expression levels of the Tfap2c and Bmp7 mRNAs in the heart, lateral and medial forebrains from E11.5 embryos measured by RT-qPCR in del1 homozygous, heterozygous, and control (wt) genotypes. For each gene, expression levels were normalized with Gapdh. Expression of the wild type allele in the lateral forebrain for Tfap2c and in the medial forebrain for Bmp7, respectively, was set as 1. The error bars represent s.d. from three biological replicates. Statistical significance was assessed by a two-sided t-test. *p<0.05; **p<0.01; ***p<0.001. (D) In situ hybridization of the wild type and del1 embryos at E10.5 with the anti-sense RNA probes for Tfap2c and Bmp7. (E) Enhancer activity of FB1 on the LacZ reporter gene. 32 out of 52 transgenic embryos showed broad forebrain expression. (F) The mm75 element drives specific expression in the mouse embryos at E11.5 (from VISTA enhancer browser: http://enhancer.lbl.gov). (G). Regulatory domains along the Tfap2c-Bmp7 interval. The forebrain enhancer (FB1) and the heart enhancer (mm75) are depicted with blue and pink ovals. A light blue (resp. pink) rectangle represents the region encompassing the H3K27ac peaks present in the segment deleted in del1, in the forebrain (resp. heart) (S3 Fig.).
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pgen-1004897-g002: Deletion alleles localise enhancers.(A) A schematic representation of the deletions generated. The loxP sites used for CRE-mediated recombination are depicted as filled red triangles for the one carried with the transposon. Open triangles indicate both the position of the static loxP at the 3′end of Bmp7 and the position of the LacZ reporter gene in deletions produced by TAMERE. (B) LacZ staining patterns of the three deletion lines in forebrain (top) and heart (bottom) in E11.5 embryos, in comparison to SB-A1 and SB-B3(3end), respectively. The deletions led to complete loss of the both lateral and medial expression in the forebrain (blue arrows in the SB-A1 embryo). (C) Relative expression levels of the Tfap2c and Bmp7 mRNAs in the heart, lateral and medial forebrains from E11.5 embryos measured by RT-qPCR in del1 homozygous, heterozygous, and control (wt) genotypes. For each gene, expression levels were normalized with Gapdh. Expression of the wild type allele in the lateral forebrain for Tfap2c and in the medial forebrain for Bmp7, respectively, was set as 1. The error bars represent s.d. from three biological replicates. Statistical significance was assessed by a two-sided t-test. *p<0.05; **p<0.01; ***p<0.001. (D) In situ hybridization of the wild type and del1 embryos at E10.5 with the anti-sense RNA probes for Tfap2c and Bmp7. (E) Enhancer activity of FB1 on the LacZ reporter gene. 32 out of 52 transgenic embryos showed broad forebrain expression. (F) The mm75 element drives specific expression in the mouse embryos at E11.5 (from VISTA enhancer browser: http://enhancer.lbl.gov). (G). Regulatory domains along the Tfap2c-Bmp7 interval. The forebrain enhancer (FB1) and the heart enhancer (mm75) are depicted with blue and pink ovals. A light blue (resp. pink) rectangle represents the region encompassing the H3K27ac peaks present in the segment deleted in del1, in the forebrain (resp. heart) (S3 Fig.).
Mentions: To further characterize the functional relevance of these two domains and associated enhancers, we used in vivo Cre-mediated recombination to engineer chromosomal deletions removing either the telomeric half or the whole of the intergenic region (Fig. 2). Each deletion was produced using a combination of loxP sites in cis and trans[54] in order to keep the LacZ sensor at the deletion breakpoint (see Materials and Methods). With the TAMERE strategy, we also obtained a large duplication, reciprocal to del3 (S2 Fig.). All three deletions led to a complete loss of LacZ expression in the embryonic heart and forebrain (Fig. 2B) suggesting that the enhancers detected by SB-A1 and SB-B(3end) lie in the region encompassed by del1. Dup3-lacZ embryos showed LacZ expression in the heart similar to SB-B(3end), corroborating the presence of the heart enhancer(s) at the 3′ side of Bmp7 (S2 Fig.). These deletions also provided information on the locations of additional enhancers associated with other expression domains (S2 Fig.).

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