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A novel TGFβ modulator that uncouples R-Smad/I-Smad-mediated negative feedback from R-Smad/ligand-driven positive feedback.

Gu W, Monteiro R, Zuo J, Simões FC, Martella A, Andrieu-Soler C, Grosveld F, Sauka-Spengler T, Patient R - PLoS Biol. (2015)

Bottom Line: Expression of ldb2a is itself activated by TGFβ signals, suggesting potential feed-forward loops that might delay the negative input of Ldb2a to the positive feedback, as well as the positive input of Ldb2a to the negative feedback.In Ldb2a-deficient zebrafish embryos, homeostasis of TGFβ signalling is perturbed and signalling is stably enhanced, giving rise to excess mesoderm and endoderm, an effect that can be rescued by reducing signalling by the TGFβ family members, Nodal and BMP.Thus, Ldb2a is critical to the homeostatic control of TGFβ signalling and thereby embryonic patterning.

View Article: PubMed Central - PubMed

Affiliation: Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.

ABSTRACT
As some of the most widely utilised intercellular signalling molecules, transforming growth factor β (TGFβ) superfamily members play critical roles in normal development and become disrupted in human disease. Establishing appropriate levels of TGFβ signalling involves positive and negative feedback, which are coupled and driven by the same signal transduction components (R-Smad transcription factor complexes), but whether and how the regulation of the two can be distinguished are unknown. Genome-wide comparison of published ChIP-seq datasets suggests that LIM domain binding proteins (Ldbs) co-localise with R-Smads at a substantial subset of R-Smad target genes including the locus of inhibitory Smad7 (I-Smad7), which mediates negative feedback for TGFβ signalling. We present evidence suggesting that zebrafish Ldb2a binds and directly activates the I-Smad7 gene, whereas it binds and represses the ligand gene, Squint (Sqt), which drives positive feedback. Thus, the fine tuning of TGFβ signalling derives from positive and negative control by Ldb2a. Expression of ldb2a is itself activated by TGFβ signals, suggesting potential feed-forward loops that might delay the negative input of Ldb2a to the positive feedback, as well as the positive input of Ldb2a to the negative feedback. In this way, precise gene expression control by Ldb2a enables an initial build-up of signalling via a fully active positive feedback in the absence of buffering by the negative feedback. In Ldb2a-deficient zebrafish embryos, homeostasis of TGFβ signalling is perturbed and signalling is stably enhanced, giving rise to excess mesoderm and endoderm, an effect that can be rescued by reducing signalling by the TGFβ family members, Nodal and BMP. Thus, Ldb2a is critical to the homeostatic control of TGFβ signalling and thereby embryonic patterning.

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Ldb1, R-Smad1, and R-Smad3 share a substantial subset of target genes across the genome.Genome-wide comparison of different ChIP-seq datasets shows that Ldb1, R-Smad1, and R-Smad3 co-localise at a substantial subset of R-Smad binding sites. (A) For each R-Smad1 binding site (y-axis), the relative locations of sites bound by R-Smad1 itself, as the positive control (blue), and Ldb1 (green) are displayed within a 5-kb window centred on the R-Smad1 binding site (position 0). High intensity at position 0 indicates co-occupancy. (B) For each R-Smad3 binding site (y-axis), the relative locations of sites bound by R-Smad3 itself (red) and Ldb1 (green) are displayed. (C) Ldb1 and R-Smad1 co-localise at the I-Smad6 locus. (D) Ldb1 and R-Smad3 co-occupy the I-Smad7 locus. ChIP-seq datasets analysed in (A–D) were obtained in different cell types, with Ldb1 in murine Flk1+ cells, and R-Smad1 in murine G1ER cells. R-Smad3 ChIP-seq was performed in murine pro-B cells. (E) Ldb1 binding was enriched at the Smad6 BS2 region (see C) and the Smad7 promoter (pr-BS3, see D) regions in murine day 4 EB-derived Flk1+ cells. In day 2.5 EBs, Ldb1 was enriched at Smad7 and at Smad6 in day 4 EBs. Shown as the negative control, Ldb1 binding was not enriched at Smad6 pr-BS1, Smad7 pr-BS1, Smad7 I-BRE, and Smad7 BS4 in Flk1+ cells, neither at Smad6 BS2 in day 2.5 EBs, nor at Smad7 BS4 in day 4 EBs. Error bars (standard deviation [SD]) are based on three biological replicates, each with three technical replicates.
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pbio.1002051.g001: Ldb1, R-Smad1, and R-Smad3 share a substantial subset of target genes across the genome.Genome-wide comparison of different ChIP-seq datasets shows that Ldb1, R-Smad1, and R-Smad3 co-localise at a substantial subset of R-Smad binding sites. (A) For each R-Smad1 binding site (y-axis), the relative locations of sites bound by R-Smad1 itself, as the positive control (blue), and Ldb1 (green) are displayed within a 5-kb window centred on the R-Smad1 binding site (position 0). High intensity at position 0 indicates co-occupancy. (B) For each R-Smad3 binding site (y-axis), the relative locations of sites bound by R-Smad3 itself (red) and Ldb1 (green) are displayed. (C) Ldb1 and R-Smad1 co-localise at the I-Smad6 locus. (D) Ldb1 and R-Smad3 co-occupy the I-Smad7 locus. ChIP-seq datasets analysed in (A–D) were obtained in different cell types, with Ldb1 in murine Flk1+ cells, and R-Smad1 in murine G1ER cells. R-Smad3 ChIP-seq was performed in murine pro-B cells. (E) Ldb1 binding was enriched at the Smad6 BS2 region (see C) and the Smad7 promoter (pr-BS3, see D) regions in murine day 4 EB-derived Flk1+ cells. In day 2.5 EBs, Ldb1 was enriched at Smad7 and at Smad6 in day 4 EBs. Shown as the negative control, Ldb1 binding was not enriched at Smad6 pr-BS1, Smad7 pr-BS1, Smad7 I-BRE, and Smad7 BS4 in Flk1+ cells, neither at Smad6 BS2 in day 2.5 EBs, nor at Smad7 BS4 in day 4 EBs. Error bars (standard deviation [SD]) are based on three biological replicates, each with three technical replicates.

Mentions: We compared published ChIP-seq datasets of Ldb1, the BMP effector, R-Smad1, and the Nodal/Activin/TGFβ effector, R-Smad3 [8,9,21,29]. We found that the binding of Ldb1 overlaps R-Smad1 or R-Smad3 binding at a substantial subset of R-Smad targets across the genome (Fig. 1A and 1B), including at the known TGFβ target genes, I-Smad6 and I-Smad7 (Fig. 1C and 1D). Ldb1 binding at these loci was validated in murine cells by ChIP-quantitative PCR (qPCR) (Fig. 1E). The ChIP-seq of Ldb1 had been performed in murine bone marrow cells or day 4 embryoid body (EB)-derived Flk1+ haemato-endothelial precursor cells [21,29], whereas the ChIP-seq of R-Smad1 and R-Smad3 had been carried out in murine G1ER erythroid progenitor cells and murine pro-B cells, respectively [8,9]. Nevertheless, the widespread co-localisation of Ldb1 and R-Smads, albeit in different cell types, suggests the potential for functional cooperation between these factors.


A novel TGFβ modulator that uncouples R-Smad/I-Smad-mediated negative feedback from R-Smad/ligand-driven positive feedback.

Gu W, Monteiro R, Zuo J, Simões FC, Martella A, Andrieu-Soler C, Grosveld F, Sauka-Spengler T, Patient R - PLoS Biol. (2015)

Ldb1, R-Smad1, and R-Smad3 share a substantial subset of target genes across the genome.Genome-wide comparison of different ChIP-seq datasets shows that Ldb1, R-Smad1, and R-Smad3 co-localise at a substantial subset of R-Smad binding sites. (A) For each R-Smad1 binding site (y-axis), the relative locations of sites bound by R-Smad1 itself, as the positive control (blue), and Ldb1 (green) are displayed within a 5-kb window centred on the R-Smad1 binding site (position 0). High intensity at position 0 indicates co-occupancy. (B) For each R-Smad3 binding site (y-axis), the relative locations of sites bound by R-Smad3 itself (red) and Ldb1 (green) are displayed. (C) Ldb1 and R-Smad1 co-localise at the I-Smad6 locus. (D) Ldb1 and R-Smad3 co-occupy the I-Smad7 locus. ChIP-seq datasets analysed in (A–D) were obtained in different cell types, with Ldb1 in murine Flk1+ cells, and R-Smad1 in murine G1ER cells. R-Smad3 ChIP-seq was performed in murine pro-B cells. (E) Ldb1 binding was enriched at the Smad6 BS2 region (see C) and the Smad7 promoter (pr-BS3, see D) regions in murine day 4 EB-derived Flk1+ cells. In day 2.5 EBs, Ldb1 was enriched at Smad7 and at Smad6 in day 4 EBs. Shown as the negative control, Ldb1 binding was not enriched at Smad6 pr-BS1, Smad7 pr-BS1, Smad7 I-BRE, and Smad7 BS4 in Flk1+ cells, neither at Smad6 BS2 in day 2.5 EBs, nor at Smad7 BS4 in day 4 EBs. Error bars (standard deviation [SD]) are based on three biological replicates, each with three technical replicates.
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pbio.1002051.g001: Ldb1, R-Smad1, and R-Smad3 share a substantial subset of target genes across the genome.Genome-wide comparison of different ChIP-seq datasets shows that Ldb1, R-Smad1, and R-Smad3 co-localise at a substantial subset of R-Smad binding sites. (A) For each R-Smad1 binding site (y-axis), the relative locations of sites bound by R-Smad1 itself, as the positive control (blue), and Ldb1 (green) are displayed within a 5-kb window centred on the R-Smad1 binding site (position 0). High intensity at position 0 indicates co-occupancy. (B) For each R-Smad3 binding site (y-axis), the relative locations of sites bound by R-Smad3 itself (red) and Ldb1 (green) are displayed. (C) Ldb1 and R-Smad1 co-localise at the I-Smad6 locus. (D) Ldb1 and R-Smad3 co-occupy the I-Smad7 locus. ChIP-seq datasets analysed in (A–D) were obtained in different cell types, with Ldb1 in murine Flk1+ cells, and R-Smad1 in murine G1ER cells. R-Smad3 ChIP-seq was performed in murine pro-B cells. (E) Ldb1 binding was enriched at the Smad6 BS2 region (see C) and the Smad7 promoter (pr-BS3, see D) regions in murine day 4 EB-derived Flk1+ cells. In day 2.5 EBs, Ldb1 was enriched at Smad7 and at Smad6 in day 4 EBs. Shown as the negative control, Ldb1 binding was not enriched at Smad6 pr-BS1, Smad7 pr-BS1, Smad7 I-BRE, and Smad7 BS4 in Flk1+ cells, neither at Smad6 BS2 in day 2.5 EBs, nor at Smad7 BS4 in day 4 EBs. Error bars (standard deviation [SD]) are based on three biological replicates, each with three technical replicates.
Mentions: We compared published ChIP-seq datasets of Ldb1, the BMP effector, R-Smad1, and the Nodal/Activin/TGFβ effector, R-Smad3 [8,9,21,29]. We found that the binding of Ldb1 overlaps R-Smad1 or R-Smad3 binding at a substantial subset of R-Smad targets across the genome (Fig. 1A and 1B), including at the known TGFβ target genes, I-Smad6 and I-Smad7 (Fig. 1C and 1D). Ldb1 binding at these loci was validated in murine cells by ChIP-quantitative PCR (qPCR) (Fig. 1E). The ChIP-seq of Ldb1 had been performed in murine bone marrow cells or day 4 embryoid body (EB)-derived Flk1+ haemato-endothelial precursor cells [21,29], whereas the ChIP-seq of R-Smad1 and R-Smad3 had been carried out in murine G1ER erythroid progenitor cells and murine pro-B cells, respectively [8,9]. Nevertheless, the widespread co-localisation of Ldb1 and R-Smads, albeit in different cell types, suggests the potential for functional cooperation between these factors.

Bottom Line: Expression of ldb2a is itself activated by TGFβ signals, suggesting potential feed-forward loops that might delay the negative input of Ldb2a to the positive feedback, as well as the positive input of Ldb2a to the negative feedback.In Ldb2a-deficient zebrafish embryos, homeostasis of TGFβ signalling is perturbed and signalling is stably enhanced, giving rise to excess mesoderm and endoderm, an effect that can be rescued by reducing signalling by the TGFβ family members, Nodal and BMP.Thus, Ldb2a is critical to the homeostatic control of TGFβ signalling and thereby embryonic patterning.

View Article: PubMed Central - PubMed

Affiliation: Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.

ABSTRACT
As some of the most widely utilised intercellular signalling molecules, transforming growth factor β (TGFβ) superfamily members play critical roles in normal development and become disrupted in human disease. Establishing appropriate levels of TGFβ signalling involves positive and negative feedback, which are coupled and driven by the same signal transduction components (R-Smad transcription factor complexes), but whether and how the regulation of the two can be distinguished are unknown. Genome-wide comparison of published ChIP-seq datasets suggests that LIM domain binding proteins (Ldbs) co-localise with R-Smads at a substantial subset of R-Smad target genes including the locus of inhibitory Smad7 (I-Smad7), which mediates negative feedback for TGFβ signalling. We present evidence suggesting that zebrafish Ldb2a binds and directly activates the I-Smad7 gene, whereas it binds and represses the ligand gene, Squint (Sqt), which drives positive feedback. Thus, the fine tuning of TGFβ signalling derives from positive and negative control by Ldb2a. Expression of ldb2a is itself activated by TGFβ signals, suggesting potential feed-forward loops that might delay the negative input of Ldb2a to the positive feedback, as well as the positive input of Ldb2a to the negative feedback. In this way, precise gene expression control by Ldb2a enables an initial build-up of signalling via a fully active positive feedback in the absence of buffering by the negative feedback. In Ldb2a-deficient zebrafish embryos, homeostasis of TGFβ signalling is perturbed and signalling is stably enhanced, giving rise to excess mesoderm and endoderm, an effect that can be rescued by reducing signalling by the TGFβ family members, Nodal and BMP. Thus, Ldb2a is critical to the homeostatic control of TGFβ signalling and thereby embryonic patterning.

Show MeSH
Related in: MedlinePlus