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Insm1 cooperates with Neurod1 and Foxa2 to maintain mature pancreatic β-cell function.

Jia S, Ivanov A, Blasevic D, Müller T, Purfürst B, Sun W, Chen W, Poy MN, Rajewsky N, Birchmeier C - EMBO J. (2015)

Bottom Line: We defined Insm1, Neurod1 and Foxa2 binding sites associated with genes deregulated in Insm1 mutant β-cells.Human genomic sequences corresponding to the murine sites occupied by Insm1/Neurod1/Foxa2 were enriched in single nucleotide polymorphisms associated with glycolytic traits.Thus, our data explain part of the mechanisms by which β-cells maintain maturity: Combinatorial Insm1/Neurod1/Foxa2 binding identifies regulatory sequences that maintain the mature gene expression program in β-cells, and disruption of this network results in functional failure.

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany cbirch@mdc-berlin.de jshiqi@mdc-berlin.de.

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Direct interaction of Insm1, Neurod1 and Foxa2reChIP-PCR analysis of selected binding sites in chromatin from SJ β-cells using antibodies against Insm1, Neurod1 and control IgG. Displayed is the percentage of the re-precipitated chromatin divided by chromatin input.reChIP-PCR analysis of chromatin from isolated islets using antibodies against Insm1, Neurod1 and control IgG. Displayed is the percentage of the re-precipitated chromatin divided by chromatin input.reChIP-PCR analysis of chromatin from SJ β-cell using antibodies against Insm1, Foxa2 and control IgG.reChIP-PCR analysis of chromatin from isolated islets using antibodies against Insm1, Foxa2 and control IgG.Co-immunoprecipitation of Insm1 and Neurod1, Insm1 and Foxa2 (but not Insm1 and MafA) in the presence or absence of ethidium bromide (EtBr). IP: antibody used for immunoprecipitation. The antibody used for Western blotting is also indicated. Representative results from one of three experiments are shownLuciferase reporter assay for cis-regulatory activity of DNA fragments associated with deregulated genes and Insm1 binding sites; luciferase reporter assays were performed in SJ β-cells, HepG2 and HEK293 cells. y-axis: luciferase activity relative to that of a plasmid with minimal promoter (pGL4-minP). x-axis: DNA regions co-occupied by Insm1/Neurod1/Foxa2 (category 3), Insm1/Neurod1 or Insm1/Foxa2 sites (category 2), or ‘Insm1 only’ (category 1) (n = 4).Synergistic transcriptional activation of fragments from the Glp1r and Pcx genes by Neurod1 and Insm1 in HepG2 cells (n = 4); *P < 0.05.Data information: (A–D) Representative results from experiments done in triplicate (A, C) and duplicate (B, D) are shown. Data in (F, G) are presented as means ± SEM. Source data are available online for this figure.
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fig07: Direct interaction of Insm1, Neurod1 and Foxa2reChIP-PCR analysis of selected binding sites in chromatin from SJ β-cells using antibodies against Insm1, Neurod1 and control IgG. Displayed is the percentage of the re-precipitated chromatin divided by chromatin input.reChIP-PCR analysis of chromatin from isolated islets using antibodies against Insm1, Neurod1 and control IgG. Displayed is the percentage of the re-precipitated chromatin divided by chromatin input.reChIP-PCR analysis of chromatin from SJ β-cell using antibodies against Insm1, Foxa2 and control IgG.reChIP-PCR analysis of chromatin from isolated islets using antibodies against Insm1, Foxa2 and control IgG.Co-immunoprecipitation of Insm1 and Neurod1, Insm1 and Foxa2 (but not Insm1 and MafA) in the presence or absence of ethidium bromide (EtBr). IP: antibody used for immunoprecipitation. The antibody used for Western blotting is also indicated. Representative results from one of three experiments are shownLuciferase reporter assay for cis-regulatory activity of DNA fragments associated with deregulated genes and Insm1 binding sites; luciferase reporter assays were performed in SJ β-cells, HepG2 and HEK293 cells. y-axis: luciferase activity relative to that of a plasmid with minimal promoter (pGL4-minP). x-axis: DNA regions co-occupied by Insm1/Neurod1/Foxa2 (category 3), Insm1/Neurod1 or Insm1/Foxa2 sites (category 2), or ‘Insm1 only’ (category 1) (n = 4).Synergistic transcriptional activation of fragments from the Glp1r and Pcx genes by Neurod1 and Insm1 in HepG2 cells (n = 4); *P < 0.05.Data information: (A–D) Representative results from experiments done in triplicate (A, C) and duplicate (B, D) are shown. Data in (F, G) are presented as means ± SEM. Source data are available online for this figure.

Mentions: We next tested whether Insm1 and Neurod1 and/or Foxa2 bind simultaneously or competitively by sequential chromatin immunoprecipitation (ChIP-reChIP) (Truax & Greer, 2012). After precipitation of chromatin from SJ β-cells and murine islets by anti-Insm1 antibodies, we observed substantial re-precipitation with anti-Neurod1 or anti-Foxa2 antibodies (Fig 7A–D). Thus, Insm1/Neurod1 and Insm1/Foxa2 can bind simultaneously. We also tested whether Insm1 directly interacts with Neurod1 or Foxa2 using immunoprecipitation and Western blotting. Anti-Insm1 antibodies co-precipitated endogenous Neurod1, and to a lesser extend Foxa2 in SJ β-cells (Fig 7E). Co-precipitation was also observed in the presence of ethidium bromide (Fig 7E), indicating that Insm1/Neurod1 and Insm1/Foxa2 interactions are independent of DNA binding (Lai & Herr, 1992). Conversely, the endogenous Insm1 was immunoprecipitated by anti-Neurod1 and anti-Foxa2 antibodies in the presence and absence of ethidium bromide, but not by control or anti-MafA antibodies (Fig 7E). Thus, Insm1 physically interacts strongly with Neurod1, and to a lesser extent with Foxa2, indicating that Insm1 can be recruited also indirectly to chromatin.


Insm1 cooperates with Neurod1 and Foxa2 to maintain mature pancreatic β-cell function.

Jia S, Ivanov A, Blasevic D, Müller T, Purfürst B, Sun W, Chen W, Poy MN, Rajewsky N, Birchmeier C - EMBO J. (2015)

Direct interaction of Insm1, Neurod1 and Foxa2reChIP-PCR analysis of selected binding sites in chromatin from SJ β-cells using antibodies against Insm1, Neurod1 and control IgG. Displayed is the percentage of the re-precipitated chromatin divided by chromatin input.reChIP-PCR analysis of chromatin from isolated islets using antibodies against Insm1, Neurod1 and control IgG. Displayed is the percentage of the re-precipitated chromatin divided by chromatin input.reChIP-PCR analysis of chromatin from SJ β-cell using antibodies against Insm1, Foxa2 and control IgG.reChIP-PCR analysis of chromatin from isolated islets using antibodies against Insm1, Foxa2 and control IgG.Co-immunoprecipitation of Insm1 and Neurod1, Insm1 and Foxa2 (but not Insm1 and MafA) in the presence or absence of ethidium bromide (EtBr). IP: antibody used for immunoprecipitation. The antibody used for Western blotting is also indicated. Representative results from one of three experiments are shownLuciferase reporter assay for cis-regulatory activity of DNA fragments associated with deregulated genes and Insm1 binding sites; luciferase reporter assays were performed in SJ β-cells, HepG2 and HEK293 cells. y-axis: luciferase activity relative to that of a plasmid with minimal promoter (pGL4-minP). x-axis: DNA regions co-occupied by Insm1/Neurod1/Foxa2 (category 3), Insm1/Neurod1 or Insm1/Foxa2 sites (category 2), or ‘Insm1 only’ (category 1) (n = 4).Synergistic transcriptional activation of fragments from the Glp1r and Pcx genes by Neurod1 and Insm1 in HepG2 cells (n = 4); *P < 0.05.Data information: (A–D) Representative results from experiments done in triplicate (A, C) and duplicate (B, D) are shown. Data in (F, G) are presented as means ± SEM. Source data are available online for this figure.
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fig07: Direct interaction of Insm1, Neurod1 and Foxa2reChIP-PCR analysis of selected binding sites in chromatin from SJ β-cells using antibodies against Insm1, Neurod1 and control IgG. Displayed is the percentage of the re-precipitated chromatin divided by chromatin input.reChIP-PCR analysis of chromatin from isolated islets using antibodies against Insm1, Neurod1 and control IgG. Displayed is the percentage of the re-precipitated chromatin divided by chromatin input.reChIP-PCR analysis of chromatin from SJ β-cell using antibodies against Insm1, Foxa2 and control IgG.reChIP-PCR analysis of chromatin from isolated islets using antibodies against Insm1, Foxa2 and control IgG.Co-immunoprecipitation of Insm1 and Neurod1, Insm1 and Foxa2 (but not Insm1 and MafA) in the presence or absence of ethidium bromide (EtBr). IP: antibody used for immunoprecipitation. The antibody used for Western blotting is also indicated. Representative results from one of three experiments are shownLuciferase reporter assay for cis-regulatory activity of DNA fragments associated with deregulated genes and Insm1 binding sites; luciferase reporter assays were performed in SJ β-cells, HepG2 and HEK293 cells. y-axis: luciferase activity relative to that of a plasmid with minimal promoter (pGL4-minP). x-axis: DNA regions co-occupied by Insm1/Neurod1/Foxa2 (category 3), Insm1/Neurod1 or Insm1/Foxa2 sites (category 2), or ‘Insm1 only’ (category 1) (n = 4).Synergistic transcriptional activation of fragments from the Glp1r and Pcx genes by Neurod1 and Insm1 in HepG2 cells (n = 4); *P < 0.05.Data information: (A–D) Representative results from experiments done in triplicate (A, C) and duplicate (B, D) are shown. Data in (F, G) are presented as means ± SEM. Source data are available online for this figure.
Mentions: We next tested whether Insm1 and Neurod1 and/or Foxa2 bind simultaneously or competitively by sequential chromatin immunoprecipitation (ChIP-reChIP) (Truax & Greer, 2012). After precipitation of chromatin from SJ β-cells and murine islets by anti-Insm1 antibodies, we observed substantial re-precipitation with anti-Neurod1 or anti-Foxa2 antibodies (Fig 7A–D). Thus, Insm1/Neurod1 and Insm1/Foxa2 can bind simultaneously. We also tested whether Insm1 directly interacts with Neurod1 or Foxa2 using immunoprecipitation and Western blotting. Anti-Insm1 antibodies co-precipitated endogenous Neurod1, and to a lesser extend Foxa2 in SJ β-cells (Fig 7E). Co-precipitation was also observed in the presence of ethidium bromide (Fig 7E), indicating that Insm1/Neurod1 and Insm1/Foxa2 interactions are independent of DNA binding (Lai & Herr, 1992). Conversely, the endogenous Insm1 was immunoprecipitated by anti-Neurod1 and anti-Foxa2 antibodies in the presence and absence of ethidium bromide, but not by control or anti-MafA antibodies (Fig 7E). Thus, Insm1 physically interacts strongly with Neurod1, and to a lesser extent with Foxa2, indicating that Insm1 can be recruited also indirectly to chromatin.

Bottom Line: We defined Insm1, Neurod1 and Foxa2 binding sites associated with genes deregulated in Insm1 mutant β-cells.Human genomic sequences corresponding to the murine sites occupied by Insm1/Neurod1/Foxa2 were enriched in single nucleotide polymorphisms associated with glycolytic traits.Thus, our data explain part of the mechanisms by which β-cells maintain maturity: Combinatorial Insm1/Neurod1/Foxa2 binding identifies regulatory sequences that maintain the mature gene expression program in β-cells, and disruption of this network results in functional failure.

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany cbirch@mdc-berlin.de jshiqi@mdc-berlin.de.

Show MeSH
Related in: MedlinePlus