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Male-biased aganglionic megacolon in the TashT mouse line due to perturbation of silencer elements in a large gene desert of chromosome 10.

Bergeron KF, Cardinal T, Touré AM, Béland M, Raiwet DL, Silversides DW, Pilon N - PLoS Genet. (2015)

Bottom Line: The TashT insertional mutation is localized in a gene desert containing multiple highly conserved elements that exhibit repressive activity in reporter assays.RNAseq analyses and 3C assays revealed that the TashT insertion results, at least in part, in NCC-specific relief of repression of the uncharacterized gene Fam162b; an outcome independently confirmed via transient transgenesis.The transcriptional signature of enteric NCC from homozygous TashT embryos is also characterized by the deregulation of genes encoding members of the most important signaling pathways for ENS formation-Gdnf/Ret and Edn3/Ednrb-and, intriguingly, the downregulation of specific subsets of X-linked genes.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics of Development Laboratory, Department of Biological Sciences and BioMed Research Center, University of Quebec at Montreal (UQAM), Quebec, Canada.

ABSTRACT
Neural crest cells (NCC) are a transient migratory cell population that generates diverse cell types such as neurons and glia of the enteric nervous system (ENS). Via an insertional mutation screen for loci affecting NCC development in mice, we identified one line-named TashT-that displays a partially penetrant aganglionic megacolon phenotype in a strong male-biased manner. Interestingly, this phenotype is highly reminiscent of human Hirschsprung's disease, a neurocristopathy with a still unexplained male sex bias. In contrast to the megacolon phenotype, colonic aganglionosis is almost fully penetrant in homozygous TashT animals. The sex bias in megacolon expressivity can be explained by the fact that the male ENS ends, on average, around a "tipping point" of minimal colonic ganglionosis while the female ENS ends, on average, just beyond it. Detailed analysis of embryonic intestines revealed that aganglionosis in homozygous TashT animals is due to slower migration of enteric NCC. The TashT insertional mutation is localized in a gene desert containing multiple highly conserved elements that exhibit repressive activity in reporter assays. RNAseq analyses and 3C assays revealed that the TashT insertion results, at least in part, in NCC-specific relief of repression of the uncharacterized gene Fam162b; an outcome independently confirmed via transient transgenesis. The transcriptional signature of enteric NCC from homozygous TashT embryos is also characterized by the deregulation of genes encoding members of the most important signaling pathways for ENS formation-Gdnf/Ret and Edn3/Ednrb-and, intriguingly, the downregulation of specific subsets of X-linked genes. In conclusion, this study not only allowed the identification of Fam162b coding and regulatory sequences as novel candidate loci for Hirschsprung's disease but also provides important new insights into its male sex bias.

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The TashT transgene insertion site is enriched in highly conserved regions possessing silencer activity.(a) Mapping of TashTTg/Tg genome sequencing reads on mouse chromosome 10 B2. Note the approximately twofold increase in reads over a 26 kb region and, flanking it, translocation-like events from chromosome 10 to chromosomes 7 (Tyrosinase gene locus) and Y (Sry gene locus), in light blue and light green respectively. A schematic representation of the TashT transgene insertion site as deduced from the mapping results is shown at the bottom. Transgenic sequences are estimated to be ~700 kb in length and surrounded by a 26 kb duplication. The position of genotyping PCR primers used in S7b Fig is indicated. (b) Map of the chromosome 10 B2–B3 region showing insertion of the TashT transgenes within a 3.3 Mb gene desert. The relative position of protein-coding genes around the locus is indicated by red rectangles. As indicated at the bottom with the GERP (Genomic Evolutionary Rate Profiling) conservation scores for 35 eutherian mammals (taken from www.ensembl.org using the NCBIm37 assembly), this region is enriched in constrained elements. Blue boxes delineate the ~1 kb sub-regions containing the constrained elements that were assessed for transcriptional activity (CE1 to CE7). (c) Evaluation of transcriptional activity for the 7 cloned regions in murine neuroblastoma (Neuro-2a) and embryocarcinoma (P19) cell lines. Luciferase assays were performed with reporter constructs driven by the cloned regions (CE1 to CE7) upstream of a minimal TK promoter. Luciferase activity is reported in fold induction relative to the empty vector (V) which is only driven by the TK minimal promoter. +/- symbols indicate sense and antisense orientation of the cloned fragments in relation to the reporter gene.
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pgen.1005093.g003: The TashT transgene insertion site is enriched in highly conserved regions possessing silencer activity.(a) Mapping of TashTTg/Tg genome sequencing reads on mouse chromosome 10 B2. Note the approximately twofold increase in reads over a 26 kb region and, flanking it, translocation-like events from chromosome 10 to chromosomes 7 (Tyrosinase gene locus) and Y (Sry gene locus), in light blue and light green respectively. A schematic representation of the TashT transgene insertion site as deduced from the mapping results is shown at the bottom. Transgenic sequences are estimated to be ~700 kb in length and surrounded by a 26 kb duplication. The position of genotyping PCR primers used in S7b Fig is indicated. (b) Map of the chromosome 10 B2–B3 region showing insertion of the TashT transgenes within a 3.3 Mb gene desert. The relative position of protein-coding genes around the locus is indicated by red rectangles. As indicated at the bottom with the GERP (Genomic Evolutionary Rate Profiling) conservation scores for 35 eutherian mammals (taken from www.ensembl.org using the NCBIm37 assembly), this region is enriched in constrained elements. Blue boxes delineate the ~1 kb sub-regions containing the constrained elements that were assessed for transcriptional activity (CE1 to CE7). (c) Evaluation of transcriptional activity for the 7 cloned regions in murine neuroblastoma (Neuro-2a) and embryocarcinoma (P19) cell lines. Luciferase assays were performed with reporter constructs driven by the cloned regions (CE1 to CE7) upstream of a minimal TK promoter. Luciferase activity is reported in fold induction relative to the empty vector (V) which is only driven by the TK minimal promoter. +/- symbols indicate sense and antisense orientation of the cloned fragments in relation to the reporter gene.

Mentions: Breeding of the TashT line revealed systematic co-segregation of pigmentation with YFP fluorescence, meaning co-integration of both transgenes into a single autosomal locus which is frequent when an equimolar mixture of each transgene is micro-injected [15]. FISH analysis first allowed a rough estimate of the localization of the transgene insertion site on chromosome 10 at bands B2–B3 (S7a Fig). To obtain a more precise localization, we sequenced the whole genome of a TashTTg/Tg mouse. Mapping of high-throughput paired sequencing reads allowed us to localize the transgenic insertion around the middle of a 3.3Mb gene desert between Hace1 and Grik2 (Fig. 3a,b). Twice as many reads were observed in a 26kb non-coding region of chromosome 10B2, indicating a duplication. Flanking this duplicated region were paired reads with one end mapping to chromosome 10 and the other end mapping to sequences corresponding to either one or the other transgene (Fig. 3a). A schematic representation of the inferred organization of the TashT transgene insertion site is shown at the bottom of Fig. 3a. The number of transgene copies was estimated from the mapping data and the total size of the insertion calculated to be about 700kb.


Male-biased aganglionic megacolon in the TashT mouse line due to perturbation of silencer elements in a large gene desert of chromosome 10.

Bergeron KF, Cardinal T, Touré AM, Béland M, Raiwet DL, Silversides DW, Pilon N - PLoS Genet. (2015)

The TashT transgene insertion site is enriched in highly conserved regions possessing silencer activity.(a) Mapping of TashTTg/Tg genome sequencing reads on mouse chromosome 10 B2. Note the approximately twofold increase in reads over a 26 kb region and, flanking it, translocation-like events from chromosome 10 to chromosomes 7 (Tyrosinase gene locus) and Y (Sry gene locus), in light blue and light green respectively. A schematic representation of the TashT transgene insertion site as deduced from the mapping results is shown at the bottom. Transgenic sequences are estimated to be ~700 kb in length and surrounded by a 26 kb duplication. The position of genotyping PCR primers used in S7b Fig is indicated. (b) Map of the chromosome 10 B2–B3 region showing insertion of the TashT transgenes within a 3.3 Mb gene desert. The relative position of protein-coding genes around the locus is indicated by red rectangles. As indicated at the bottom with the GERP (Genomic Evolutionary Rate Profiling) conservation scores for 35 eutherian mammals (taken from www.ensembl.org using the NCBIm37 assembly), this region is enriched in constrained elements. Blue boxes delineate the ~1 kb sub-regions containing the constrained elements that were assessed for transcriptional activity (CE1 to CE7). (c) Evaluation of transcriptional activity for the 7 cloned regions in murine neuroblastoma (Neuro-2a) and embryocarcinoma (P19) cell lines. Luciferase assays were performed with reporter constructs driven by the cloned regions (CE1 to CE7) upstream of a minimal TK promoter. Luciferase activity is reported in fold induction relative to the empty vector (V) which is only driven by the TK minimal promoter. +/- symbols indicate sense and antisense orientation of the cloned fragments in relation to the reporter gene.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4364714&req=5

pgen.1005093.g003: The TashT transgene insertion site is enriched in highly conserved regions possessing silencer activity.(a) Mapping of TashTTg/Tg genome sequencing reads on mouse chromosome 10 B2. Note the approximately twofold increase in reads over a 26 kb region and, flanking it, translocation-like events from chromosome 10 to chromosomes 7 (Tyrosinase gene locus) and Y (Sry gene locus), in light blue and light green respectively. A schematic representation of the TashT transgene insertion site as deduced from the mapping results is shown at the bottom. Transgenic sequences are estimated to be ~700 kb in length and surrounded by a 26 kb duplication. The position of genotyping PCR primers used in S7b Fig is indicated. (b) Map of the chromosome 10 B2–B3 region showing insertion of the TashT transgenes within a 3.3 Mb gene desert. The relative position of protein-coding genes around the locus is indicated by red rectangles. As indicated at the bottom with the GERP (Genomic Evolutionary Rate Profiling) conservation scores for 35 eutherian mammals (taken from www.ensembl.org using the NCBIm37 assembly), this region is enriched in constrained elements. Blue boxes delineate the ~1 kb sub-regions containing the constrained elements that were assessed for transcriptional activity (CE1 to CE7). (c) Evaluation of transcriptional activity for the 7 cloned regions in murine neuroblastoma (Neuro-2a) and embryocarcinoma (P19) cell lines. Luciferase assays were performed with reporter constructs driven by the cloned regions (CE1 to CE7) upstream of a minimal TK promoter. Luciferase activity is reported in fold induction relative to the empty vector (V) which is only driven by the TK minimal promoter. +/- symbols indicate sense and antisense orientation of the cloned fragments in relation to the reporter gene.
Mentions: Breeding of the TashT line revealed systematic co-segregation of pigmentation with YFP fluorescence, meaning co-integration of both transgenes into a single autosomal locus which is frequent when an equimolar mixture of each transgene is micro-injected [15]. FISH analysis first allowed a rough estimate of the localization of the transgene insertion site on chromosome 10 at bands B2–B3 (S7a Fig). To obtain a more precise localization, we sequenced the whole genome of a TashTTg/Tg mouse. Mapping of high-throughput paired sequencing reads allowed us to localize the transgenic insertion around the middle of a 3.3Mb gene desert between Hace1 and Grik2 (Fig. 3a,b). Twice as many reads were observed in a 26kb non-coding region of chromosome 10B2, indicating a duplication. Flanking this duplicated region were paired reads with one end mapping to chromosome 10 and the other end mapping to sequences corresponding to either one or the other transgene (Fig. 3a). A schematic representation of the inferred organization of the TashT transgene insertion site is shown at the bottom of Fig. 3a. The number of transgene copies was estimated from the mapping data and the total size of the insertion calculated to be about 700kb.

Bottom Line: The TashT insertional mutation is localized in a gene desert containing multiple highly conserved elements that exhibit repressive activity in reporter assays.RNAseq analyses and 3C assays revealed that the TashT insertion results, at least in part, in NCC-specific relief of repression of the uncharacterized gene Fam162b; an outcome independently confirmed via transient transgenesis.The transcriptional signature of enteric NCC from homozygous TashT embryos is also characterized by the deregulation of genes encoding members of the most important signaling pathways for ENS formation-Gdnf/Ret and Edn3/Ednrb-and, intriguingly, the downregulation of specific subsets of X-linked genes.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics of Development Laboratory, Department of Biological Sciences and BioMed Research Center, University of Quebec at Montreal (UQAM), Quebec, Canada.

ABSTRACT
Neural crest cells (NCC) are a transient migratory cell population that generates diverse cell types such as neurons and glia of the enteric nervous system (ENS). Via an insertional mutation screen for loci affecting NCC development in mice, we identified one line-named TashT-that displays a partially penetrant aganglionic megacolon phenotype in a strong male-biased manner. Interestingly, this phenotype is highly reminiscent of human Hirschsprung's disease, a neurocristopathy with a still unexplained male sex bias. In contrast to the megacolon phenotype, colonic aganglionosis is almost fully penetrant in homozygous TashT animals. The sex bias in megacolon expressivity can be explained by the fact that the male ENS ends, on average, around a "tipping point" of minimal colonic ganglionosis while the female ENS ends, on average, just beyond it. Detailed analysis of embryonic intestines revealed that aganglionosis in homozygous TashT animals is due to slower migration of enteric NCC. The TashT insertional mutation is localized in a gene desert containing multiple highly conserved elements that exhibit repressive activity in reporter assays. RNAseq analyses and 3C assays revealed that the TashT insertion results, at least in part, in NCC-specific relief of repression of the uncharacterized gene Fam162b; an outcome independently confirmed via transient transgenesis. The transcriptional signature of enteric NCC from homozygous TashT embryos is also characterized by the deregulation of genes encoding members of the most important signaling pathways for ENS formation-Gdnf/Ret and Edn3/Ednrb-and, intriguingly, the downregulation of specific subsets of X-linked genes. In conclusion, this study not only allowed the identification of Fam162b coding and regulatory sequences as novel candidate loci for Hirschsprung's disease but also provides important new insights into its male sex bias.

Show MeSH
Related in: MedlinePlus