Limits...
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.

Show MeSH
Insm1 binds chromatin sites that are co-occupied by Neurod1 and Foxa2Insm1, Neurod1 and Foxa2 ChIP-seq binding tracks identified in chromatin from SJ β-cells for Pdx1 and Glut2 (Slc2a2).De novo motif analysis of Insm1 peaks identified consensus binding sequences of basic helix-loop-helix and forkhead factors (i and ii); in addition, an Insm1 motif (iii) identified in ‘Insm1 only’ sites that resembles the known Insm1 binding sequence is shown.Overlap between Insm1, Neurod1 and Foxa2 binding sites.Distance between the summits of Insm1 and Neurod1 (violet), Insm1 and Foxa2 (green) as well as Foxa2 and Neurod1 (light blue) peaks; displayed are the fraction of peaks versus the distance between binding sites in base pairs.Heat map showing read tracks for Insm1, Neurod1 and Foxa2 at sites co-occupied by Insm1/Neurod1/Foxa2 (I+F+N; top), Insm1/Neurod1 and Insm1/Foxa2 (I+N and I+F; middle), or at sites bound by Insm1 only (I; bottom). The color code indicates read density ±2 kb around binding sites as log2 of the number of reads.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4492000&req=5

fig05: Insm1 binds chromatin sites that are co-occupied by Neurod1 and Foxa2Insm1, Neurod1 and Foxa2 ChIP-seq binding tracks identified in chromatin from SJ β-cells for Pdx1 and Glut2 (Slc2a2).De novo motif analysis of Insm1 peaks identified consensus binding sequences of basic helix-loop-helix and forkhead factors (i and ii); in addition, an Insm1 motif (iii) identified in ‘Insm1 only’ sites that resembles the known Insm1 binding sequence is shown.Overlap between Insm1, Neurod1 and Foxa2 binding sites.Distance between the summits of Insm1 and Neurod1 (violet), Insm1 and Foxa2 (green) as well as Foxa2 and Neurod1 (light blue) peaks; displayed are the fraction of peaks versus the distance between binding sites in base pairs.Heat map showing read tracks for Insm1, Neurod1 and Foxa2 at sites co-occupied by Insm1/Neurod1/Foxa2 (I+F+N; top), Insm1/Neurod1 and Insm1/Foxa2 (I+N and I+F; middle), or at sites bound by Insm1 only (I; bottom). The color code indicates read density ±2 kb around binding sites as log2 of the number of reads.

Mentions: To understand the molecular mechanism of Insm1 function in β-cells, ChIP-seq experiments were performed. As a chromatin source, we used early passages of an immortalized pancreatic β-cell line (referred to as SJ β-cells) that we established (Radvanyi et al, 1993). Insulin secretion from SJ β-cells was stimulated eightfold to tenfold in response to glucose, which is comparable to the response observed in dissociated cells from adult islets, but lower than the one in intact islets, and these cells respond to exendin-4 and GIP (Supplementary Fig S4A; compare with Blum et al, 2012; Halban et al, 1982). Two independent ChIP-seq experiments (GSE54046) identified 17,453 high confidence Insm1 binding sites in chromatin of SJ β-cells that overlapped between experiments (see Supplementary Fig S4B–D for replicate comparisons, antibody specificity and binding site distribution). Example traces are shown in Fig 5A and Supplementary Fig S4E (upper traces). Examination of these binding sites by de novo motif analyses revealed an enrichment of two sequences within ±25 bp of the summits of binding sites (Fig 5Bi and ii; E-value = 8 × 10−92 and 8 × 10−41 for the two motifs shown in i and ii, respectively). The first corresponds to an E-box (present in 22% of all Insm1 binding sites) and the second to the consensus sequence of forkhead factors (present in 21% of all Insm1 binding sites; Newburger & Bulyk, 2009). The previously described consensus Insm1 binding sequence (Breslin et al, 2002) was not identified by de novo motif analysis, but a related sequence (Fig 5B iii) was observed in 19% of all Insm1 binding sites (E-value = 5.5 × 10−55; see also below).


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)

Insm1 binds chromatin sites that are co-occupied by Neurod1 and Foxa2Insm1, Neurod1 and Foxa2 ChIP-seq binding tracks identified in chromatin from SJ β-cells for Pdx1 and Glut2 (Slc2a2).De novo motif analysis of Insm1 peaks identified consensus binding sequences of basic helix-loop-helix and forkhead factors (i and ii); in addition, an Insm1 motif (iii) identified in ‘Insm1 only’ sites that resembles the known Insm1 binding sequence is shown.Overlap between Insm1, Neurod1 and Foxa2 binding sites.Distance between the summits of Insm1 and Neurod1 (violet), Insm1 and Foxa2 (green) as well as Foxa2 and Neurod1 (light blue) peaks; displayed are the fraction of peaks versus the distance between binding sites in base pairs.Heat map showing read tracks for Insm1, Neurod1 and Foxa2 at sites co-occupied by Insm1/Neurod1/Foxa2 (I+F+N; top), Insm1/Neurod1 and Insm1/Foxa2 (I+N and I+F; middle), or at sites bound by Insm1 only (I; bottom). The color code indicates read density ±2 kb around binding sites as log2 of the number of reads.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492000&req=5

fig05: Insm1 binds chromatin sites that are co-occupied by Neurod1 and Foxa2Insm1, Neurod1 and Foxa2 ChIP-seq binding tracks identified in chromatin from SJ β-cells for Pdx1 and Glut2 (Slc2a2).De novo motif analysis of Insm1 peaks identified consensus binding sequences of basic helix-loop-helix and forkhead factors (i and ii); in addition, an Insm1 motif (iii) identified in ‘Insm1 only’ sites that resembles the known Insm1 binding sequence is shown.Overlap between Insm1, Neurod1 and Foxa2 binding sites.Distance between the summits of Insm1 and Neurod1 (violet), Insm1 and Foxa2 (green) as well as Foxa2 and Neurod1 (light blue) peaks; displayed are the fraction of peaks versus the distance between binding sites in base pairs.Heat map showing read tracks for Insm1, Neurod1 and Foxa2 at sites co-occupied by Insm1/Neurod1/Foxa2 (I+F+N; top), Insm1/Neurod1 and Insm1/Foxa2 (I+N and I+F; middle), or at sites bound by Insm1 only (I; bottom). The color code indicates read density ±2 kb around binding sites as log2 of the number of reads.
Mentions: To understand the molecular mechanism of Insm1 function in β-cells, ChIP-seq experiments were performed. As a chromatin source, we used early passages of an immortalized pancreatic β-cell line (referred to as SJ β-cells) that we established (Radvanyi et al, 1993). Insulin secretion from SJ β-cells was stimulated eightfold to tenfold in response to glucose, which is comparable to the response observed in dissociated cells from adult islets, but lower than the one in intact islets, and these cells respond to exendin-4 and GIP (Supplementary Fig S4A; compare with Blum et al, 2012; Halban et al, 1982). Two independent ChIP-seq experiments (GSE54046) identified 17,453 high confidence Insm1 binding sites in chromatin of SJ β-cells that overlapped between experiments (see Supplementary Fig S4B–D for replicate comparisons, antibody specificity and binding site distribution). Example traces are shown in Fig 5A and Supplementary Fig S4E (upper traces). Examination of these binding sites by de novo motif analyses revealed an enrichment of two sequences within ±25 bp of the summits of binding sites (Fig 5Bi and ii; E-value = 8 × 10−92 and 8 × 10−41 for the two motifs shown in i and ii, respectively). The first corresponds to an E-box (present in 22% of all Insm1 binding sites) and the second to the consensus sequence of forkhead factors (present in 21% of all Insm1 binding sites; Newburger & Bulyk, 2009). The previously described consensus Insm1 binding sequence (Breslin et al, 2002) was not identified by de novo motif analysis, but a related sequence (Fig 5B iii) was observed in 19% of all Insm1 binding sites (E-value = 5.5 × 10−55; see also below).

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