Insm1 cooperates with Neurod1 and Foxa2 to maintain mature pancreatic β-cell function.
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.
Affiliation: Developmental Biology, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany email@example.com firstname.lastname@example.org.Show MeSH
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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.
Affiliation: Developmental Biology, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany email@example.com firstname.lastname@example.org.