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Dynamic zebrafish interactome reveals transcriptional mechanisms of dioxin toxicity.

Alexeyenko A, Wassenberg DM, Lobenhofer EK, Yen J, Linney E, Sonnhammer EL, Meyer JN - PLoS ONE (2010)

Bottom Line: The most notable processes altered at later developmental stages were calcium and iron metabolism, embryonic morphogenesis including neuronal and retinal development, a variety of mitochondria-related functions, and generalized stress response (not including induction of antioxidant genes).Within the interactome, many of these responses were connected to cytochrome P4501A (cyp1a) as well as other genes that were dioxin-regulated one day after exposure.This suggests that cyp1a may play a key role initiating the toxic dysregulation of those processes, rather than serving simply as a passive marker of dioxin exposure, as suggested by earlier research.

View Article: PubMed Central - PubMed

Affiliation: Stockholm Bioinformatics Centre, Stockholm University, Stockholm, Sweden.

ABSTRACT

Background: In order to generate hypotheses regarding the mechanisms by which 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) causes toxicity, we analyzed global gene expression changes in developing zebrafish embryos exposed to this potent toxicant in the context of a dynamic gene network. For this purpose, we also computationally inferred a zebrafish (Danio rerio) interactome based on orthologs and interaction data from other eukaryotes.

Methodology/principal findings: Using novel computational tools to analyze this interactome, we distinguished between dioxin-dependent and dioxin-independent interactions between proteins, and tracked the temporal propagation of dioxin-dependent transcriptional changes from a few genes that were altered initially, to large groups of biologically coherent genes at later times. The most notable processes altered at later developmental stages were calcium and iron metabolism, embryonic morphogenesis including neuronal and retinal development, a variety of mitochondria-related functions, and generalized stress response (not including induction of antioxidant genes). Within the interactome, many of these responses were connected to cytochrome P4501A (cyp1a) as well as other genes that were dioxin-regulated one day after exposure. This suggests that cyp1a may play a key role initiating the toxic dysregulation of those processes, rather than serving simply as a passive marker of dioxin exposure, as suggested by earlier research.

Conclusions/significance: Thus, a powerful microarray experiment coupled with a flexible interactome and multi-pronged interactome tools (which are now made publicly available for microarray analysis and related work) suggest the hypothesis that dioxin, best known in fish as a potent cardioteratogen, has many other targets. Many of these types of toxicity have been observed in mammalian species and are potentially caused by alterations to cyp1a.

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FunCoup sub-networks with genes and links affected by dioxin.A. Network region around the originally (on day 1) altered CYP1A includes links to gene groups altered later (as discovered with jActiveModules and temporal GO relation analyses): calmodulins (triangles), members of heme biosynthesis (crosses), and neuronal development (stars) pathways. B. Network region with three adjoining clusters enriched in differentially expressed links. cnn2 (calponin 2) is co-expressed with many surrounding genes only after dioxin exposure (cluster P-916), while ung (uracil DNA glycosylase; cluster N-609), on the contrary, is normally co-expressed with its network neighbors but not after dioxin exposure. Meanwhile, several “border” genes lost functional coupling to genes from clusters N-609 and N-63, while gaining coupling to cluster P-916 genes, after dioxin exposure. The clusters are named P-* when containing positive (dioxin-enabled) links, N-* for negative (dioxin-sensitive), or PN-* for both positive and negative links. C. Members of cluster N-632 include DNA/RNA processing, cell division, and embryonic forebrain patterning genes. Interactions: Grey, FunCoup links at confidence FBS>9 (Fig. B) and FBS>3 (elsewhere) (every blue and brown link must overlap with a FunCoup link at FBS>3). For every sub-network, only a sub-set of links was retrieved, filtering for confidence and relevance to query genes with algorithm (1) (see description at http://funcoup.sbc.su.se/algo.html#noislets). Brown, clustered (co-expression + FunCoup) links caused by dioxin treatment; Blue, clustered (co-expression + FunCoup) links disabled by dioxin treatment; Green, developmental-stage specific co-expression. Color lines at A indicate data types evident of functional coupling and are explained in right panel of the screenshot. Nodes: Diamonds: members of respective clusters (i.e., genes participating in a DEL). Squares: other genes with measured expression in the course of the experiment; Circles: other genes having evidence from orthologs in FunCoup but for which expression data was lacking in our experiment. Shades of red and green, up- and down-regulation (main factor “DIOXIN TREATMENT”) with dioxin, respectively; genes missing microarray data colored grey. All modules can be found and manipulated at http://funcoup.sbc.su.se/zfish_supplementary.html, and GO enrichment analysis of modules significantly enriched in at least one GO biological process are presented in Data File S5. Both clusters and individual genes can be accessed at http://funcoup.sbc.su.se/zfish.html. See Methods S1 for details of link inference and other analysis.
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pone-0010465-g003: FunCoup sub-networks with genes and links affected by dioxin.A. Network region around the originally (on day 1) altered CYP1A includes links to gene groups altered later (as discovered with jActiveModules and temporal GO relation analyses): calmodulins (triangles), members of heme biosynthesis (crosses), and neuronal development (stars) pathways. B. Network region with three adjoining clusters enriched in differentially expressed links. cnn2 (calponin 2) is co-expressed with many surrounding genes only after dioxin exposure (cluster P-916), while ung (uracil DNA glycosylase; cluster N-609), on the contrary, is normally co-expressed with its network neighbors but not after dioxin exposure. Meanwhile, several “border” genes lost functional coupling to genes from clusters N-609 and N-63, while gaining coupling to cluster P-916 genes, after dioxin exposure. The clusters are named P-* when containing positive (dioxin-enabled) links, N-* for negative (dioxin-sensitive), or PN-* for both positive and negative links. C. Members of cluster N-632 include DNA/RNA processing, cell division, and embryonic forebrain patterning genes. Interactions: Grey, FunCoup links at confidence FBS>9 (Fig. B) and FBS>3 (elsewhere) (every blue and brown link must overlap with a FunCoup link at FBS>3). For every sub-network, only a sub-set of links was retrieved, filtering for confidence and relevance to query genes with algorithm (1) (see description at http://funcoup.sbc.su.se/algo.html#noislets). Brown, clustered (co-expression + FunCoup) links caused by dioxin treatment; Blue, clustered (co-expression + FunCoup) links disabled by dioxin treatment; Green, developmental-stage specific co-expression. Color lines at A indicate data types evident of functional coupling and are explained in right panel of the screenshot. Nodes: Diamonds: members of respective clusters (i.e., genes participating in a DEL). Squares: other genes with measured expression in the course of the experiment; Circles: other genes having evidence from orthologs in FunCoup but for which expression data was lacking in our experiment. Shades of red and green, up- and down-regulation (main factor “DIOXIN TREATMENT”) with dioxin, respectively; genes missing microarray data colored grey. All modules can be found and manipulated at http://funcoup.sbc.su.se/zfish_supplementary.html, and GO enrichment analysis of modules significantly enriched in at least one GO biological process are presented in Data File S5. Both clusters and individual genes can be accessed at http://funcoup.sbc.su.se/zfish.html. See Methods S1 for details of link inference and other analysis.

Mentions: We next identified network modules significantly enriched in DELs using an in-house clustering program (CohTop; see Methods S1). Thus, dioxin-enabled modules are enriched in links that appeared only in the treated condition (e.g., Fig. 2E), and dioxin-sensitive modules were enriched in links observed only in the normal condition (i.e., were vulnerable to disruption by dioxin). We defined modules enriched in dioxin-enabled, -sensitive, or mixture of both types. We identified 151 dioxin-enabled modules, 142 dioxin-sensitive modules, and 186 modules enriched in both dioxin-enabled and dioxin-sensitive links. These modules ranged in size from only a few nodes to dozens (maximally 30 for dioxin-sensitive modules, 86 for dioxin-enabled modules, and 122 for mixed dioxin-sensitive and-enabled). For a complete list of all DEL-enriched modules presented as interactive clusters, go to http://funcoup.sbc.su.se/zfish_supplementary.html. The modules on the website identify core nodes (those participating in a DEL) as diamonds, but also show their immediate network neighbors. The website contains rich functionality for graphical and tabular analysis and allows components of interaction evidence to be aligned and studied both within and across species. Data File S5 contains GO enrichment analysis of modules that were significantly enriched in at least one GO biological process. While not a focus of this manuscript, we also defined developmental links: edges connecting genes co-expressed synchronously in the course of development (exemplified at Fig. 3A; see Methods S1 for details of the calculation).


Dynamic zebrafish interactome reveals transcriptional mechanisms of dioxin toxicity.

Alexeyenko A, Wassenberg DM, Lobenhofer EK, Yen J, Linney E, Sonnhammer EL, Meyer JN - PLoS ONE (2010)

FunCoup sub-networks with genes and links affected by dioxin.A. Network region around the originally (on day 1) altered CYP1A includes links to gene groups altered later (as discovered with jActiveModules and temporal GO relation analyses): calmodulins (triangles), members of heme biosynthesis (crosses), and neuronal development (stars) pathways. B. Network region with three adjoining clusters enriched in differentially expressed links. cnn2 (calponin 2) is co-expressed with many surrounding genes only after dioxin exposure (cluster P-916), while ung (uracil DNA glycosylase; cluster N-609), on the contrary, is normally co-expressed with its network neighbors but not after dioxin exposure. Meanwhile, several “border” genes lost functional coupling to genes from clusters N-609 and N-63, while gaining coupling to cluster P-916 genes, after dioxin exposure. The clusters are named P-* when containing positive (dioxin-enabled) links, N-* for negative (dioxin-sensitive), or PN-* for both positive and negative links. C. Members of cluster N-632 include DNA/RNA processing, cell division, and embryonic forebrain patterning genes. Interactions: Grey, FunCoup links at confidence FBS>9 (Fig. B) and FBS>3 (elsewhere) (every blue and brown link must overlap with a FunCoup link at FBS>3). For every sub-network, only a sub-set of links was retrieved, filtering for confidence and relevance to query genes with algorithm (1) (see description at http://funcoup.sbc.su.se/algo.html#noislets). Brown, clustered (co-expression + FunCoup) links caused by dioxin treatment; Blue, clustered (co-expression + FunCoup) links disabled by dioxin treatment; Green, developmental-stage specific co-expression. Color lines at A indicate data types evident of functional coupling and are explained in right panel of the screenshot. Nodes: Diamonds: members of respective clusters (i.e., genes participating in a DEL). Squares: other genes with measured expression in the course of the experiment; Circles: other genes having evidence from orthologs in FunCoup but for which expression data was lacking in our experiment. Shades of red and green, up- and down-regulation (main factor “DIOXIN TREATMENT”) with dioxin, respectively; genes missing microarray data colored grey. All modules can be found and manipulated at http://funcoup.sbc.su.se/zfish_supplementary.html, and GO enrichment analysis of modules significantly enriched in at least one GO biological process are presented in Data File S5. Both clusters and individual genes can be accessed at http://funcoup.sbc.su.se/zfish.html. See Methods S1 for details of link inference and other analysis.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2864754&req=5

pone-0010465-g003: FunCoup sub-networks with genes and links affected by dioxin.A. Network region around the originally (on day 1) altered CYP1A includes links to gene groups altered later (as discovered with jActiveModules and temporal GO relation analyses): calmodulins (triangles), members of heme biosynthesis (crosses), and neuronal development (stars) pathways. B. Network region with three adjoining clusters enriched in differentially expressed links. cnn2 (calponin 2) is co-expressed with many surrounding genes only after dioxin exposure (cluster P-916), while ung (uracil DNA glycosylase; cluster N-609), on the contrary, is normally co-expressed with its network neighbors but not after dioxin exposure. Meanwhile, several “border” genes lost functional coupling to genes from clusters N-609 and N-63, while gaining coupling to cluster P-916 genes, after dioxin exposure. The clusters are named P-* when containing positive (dioxin-enabled) links, N-* for negative (dioxin-sensitive), or PN-* for both positive and negative links. C. Members of cluster N-632 include DNA/RNA processing, cell division, and embryonic forebrain patterning genes. Interactions: Grey, FunCoup links at confidence FBS>9 (Fig. B) and FBS>3 (elsewhere) (every blue and brown link must overlap with a FunCoup link at FBS>3). For every sub-network, only a sub-set of links was retrieved, filtering for confidence and relevance to query genes with algorithm (1) (see description at http://funcoup.sbc.su.se/algo.html#noislets). Brown, clustered (co-expression + FunCoup) links caused by dioxin treatment; Blue, clustered (co-expression + FunCoup) links disabled by dioxin treatment; Green, developmental-stage specific co-expression. Color lines at A indicate data types evident of functional coupling and are explained in right panel of the screenshot. Nodes: Diamonds: members of respective clusters (i.e., genes participating in a DEL). Squares: other genes with measured expression in the course of the experiment; Circles: other genes having evidence from orthologs in FunCoup but for which expression data was lacking in our experiment. Shades of red and green, up- and down-regulation (main factor “DIOXIN TREATMENT”) with dioxin, respectively; genes missing microarray data colored grey. All modules can be found and manipulated at http://funcoup.sbc.su.se/zfish_supplementary.html, and GO enrichment analysis of modules significantly enriched in at least one GO biological process are presented in Data File S5. Both clusters and individual genes can be accessed at http://funcoup.sbc.su.se/zfish.html. See Methods S1 for details of link inference and other analysis.
Mentions: We next identified network modules significantly enriched in DELs using an in-house clustering program (CohTop; see Methods S1). Thus, dioxin-enabled modules are enriched in links that appeared only in the treated condition (e.g., Fig. 2E), and dioxin-sensitive modules were enriched in links observed only in the normal condition (i.e., were vulnerable to disruption by dioxin). We defined modules enriched in dioxin-enabled, -sensitive, or mixture of both types. We identified 151 dioxin-enabled modules, 142 dioxin-sensitive modules, and 186 modules enriched in both dioxin-enabled and dioxin-sensitive links. These modules ranged in size from only a few nodes to dozens (maximally 30 for dioxin-sensitive modules, 86 for dioxin-enabled modules, and 122 for mixed dioxin-sensitive and-enabled). For a complete list of all DEL-enriched modules presented as interactive clusters, go to http://funcoup.sbc.su.se/zfish_supplementary.html. The modules on the website identify core nodes (those participating in a DEL) as diamonds, but also show their immediate network neighbors. The website contains rich functionality for graphical and tabular analysis and allows components of interaction evidence to be aligned and studied both within and across species. Data File S5 contains GO enrichment analysis of modules that were significantly enriched in at least one GO biological process. While not a focus of this manuscript, we also defined developmental links: edges connecting genes co-expressed synchronously in the course of development (exemplified at Fig. 3A; see Methods S1 for details of the calculation).

Bottom Line: The most notable processes altered at later developmental stages were calcium and iron metabolism, embryonic morphogenesis including neuronal and retinal development, a variety of mitochondria-related functions, and generalized stress response (not including induction of antioxidant genes).Within the interactome, many of these responses were connected to cytochrome P4501A (cyp1a) as well as other genes that were dioxin-regulated one day after exposure.This suggests that cyp1a may play a key role initiating the toxic dysregulation of those processes, rather than serving simply as a passive marker of dioxin exposure, as suggested by earlier research.

View Article: PubMed Central - PubMed

Affiliation: Stockholm Bioinformatics Centre, Stockholm University, Stockholm, Sweden.

ABSTRACT

Background: In order to generate hypotheses regarding the mechanisms by which 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) causes toxicity, we analyzed global gene expression changes in developing zebrafish embryos exposed to this potent toxicant in the context of a dynamic gene network. For this purpose, we also computationally inferred a zebrafish (Danio rerio) interactome based on orthologs and interaction data from other eukaryotes.

Methodology/principal findings: Using novel computational tools to analyze this interactome, we distinguished between dioxin-dependent and dioxin-independent interactions between proteins, and tracked the temporal propagation of dioxin-dependent transcriptional changes from a few genes that were altered initially, to large groups of biologically coherent genes at later times. The most notable processes altered at later developmental stages were calcium and iron metabolism, embryonic morphogenesis including neuronal and retinal development, a variety of mitochondria-related functions, and generalized stress response (not including induction of antioxidant genes). Within the interactome, many of these responses were connected to cytochrome P4501A (cyp1a) as well as other genes that were dioxin-regulated one day after exposure. This suggests that cyp1a may play a key role initiating the toxic dysregulation of those processes, rather than serving simply as a passive marker of dioxin exposure, as suggested by earlier research.

Conclusions/significance: Thus, a powerful microarray experiment coupled with a flexible interactome and multi-pronged interactome tools (which are now made publicly available for microarray analysis and related work) suggest the hypothesis that dioxin, best known in fish as a potent cardioteratogen, has many other targets. Many of these types of toxicity have been observed in mammalian species and are potentially caused by alterations to cyp1a.

Show MeSH
Related in: MedlinePlus