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The Pdx1-Bound Swi/Snf Chromatin Remodeling Complex Regulates Pancreatic Progenitor Cell Proliferation and Mature Islet B-Cell Function

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The Pdx1-Bound Swi/Snf Chromatin Remodeling Complex Regulates Pancreatic Progenitor Cell Proliferation and Mature Islet B-Cell Function 1806 Diabetes Volume 68, September 2019 The Pdx1-Bound Swi/Snf Chromatin Remodeling Complex Regulates Pancreatic Progenitor Cell Proliferation and Mature Islet b-Cell Function Jason M. Spaeth,1 Jin-Hua Liu,1 Daniel Peters,2 Min Guo,1 Anna B. Osipovich,1 Fardin Mohammadi,2 Nilotpal Roy,3 Anil Bhushan,3 Mark A. Magnuson,1 Matthias Hebrok,3 Christopher V.E. Wright,2 and Roland Stein1 Diabetes 2019;68:1806–1818 | https://doi.org/10.2337/db19-0349 Transcription factors positively and/or negatively im- The mammalian pancreas consists of two functionally pact gene expression by recruiting coregulatory fac- distinct compartments: the exocrine pancreas containing tors, which interact through protein-protein binding. acinar and ductal cells essential for secreting diges- Here we demonstrate that mouse pancreas size and tive enzymes, and the endocrine pancreas containing islet b-cell function are controlled by the ATP- hormone-secreting a- (glucagon), b- (insulin), d- (somato- dependent Swi/Snf chromatin remodeling coregulatory statin), e- (ghrelin), and pancreatic polypeptide cells of complex that physically associates with Pdx1, a diabetes- the islets of Langerhans that are essential for regulating linked transcription factor essential to pancreatic glucose homeostasis. All of these pancreatic cells derive morphogenesis and adult islet cell function and mainte- from a common pool of progenitor cells at mouse embry- nance. Early embryonic deletion of just the Swi/Snf Brg1 onic day (e)8.5 that express the pancreas and duodenum ATPase subunit reduced multipotent pancreatic progeni- homeobox 1 (Pdx1) transcription factor, a critical regulator ISLET STUDIES b tor cell proliferation and resulted in pancreas hypoplasia. of pancreas development, later -cell formation, and adult b In contrast, removal of both Swi/Snf ATPase subunits, islet -cell function. In fact, pancreas agenesis occurs in Brg1 and Brm, was necessary to compromise adult islet mice and humans that lack PDX1 (1,2), whereas hetero- b-cell activity, which included whole-animal glucose zygous mutations cause type 2 diabetes (T2D) because of islet b-cell dysfunction (3). intolerance, hyperglycemia, and impaired insulin secre- + tion. Notably, lineage-tracing analysis revealed Swi/ Embryonic Pdx1 pancreatic progenitor cells rapidly divide and acquire the expression of other transcription Snf-deficient b-cells lost the ability to produce the mRNAs for Ins and other key metabolic genes with- factors essential to organ expansion and lineage diver- fi out effecting the expression of many essential islet- si cation, including Ptf1a (4) and Sox9 (5). These + + + enriched transcription factors. Swi/Snf was necessary Pdx1 Ptf1a Sox9 cells form the highly proliferative multi- forPdx1tobindtotheIns gene enhancer, demon- potent pancreatic progenitor cell (MPC) pool that differ- strating the importance of this association in mediating entiates into the distinct exocrine, ductal, and islet cell chromatin accessibility. These results illustrate how types (6). Notably, pancreas mass is restricted in mice by fundamental the Pdx1:Swi/Snf coregulator complex is in the MPC pool size (7), which is affected by early embryonic the pancreas, and we discuss how disrupting their genetic removal of Pdx1, Ptf1a,orSox9 (2,4,5). Because association could influence type 1 and type 2 diabetes of considerable variations in pancreas mass (and there- susceptibility. fore variable b-cell number) between humans (8), 1Department of Molecular Physiology and Biophysics, Vanderbilt University, This article contains Supplementary Data online at http://diabetes Nashville, TN .diabetesjournals.org/lookup/suppl/doi:10.2337/db19-0349/-/DC1. 2 Department of Cell and Developmental Biology, Vanderbilt University, Nashville, J.M.S. is currently affiliated with the Department of Pediatrics, Indiana University TN School of Medicine, Indianapolis, IN. 3Diabetes Center, Department of Medicine, University of California, San Francisco, © 2019 by the American Diabetes Association. Readers may use this article as San Francisco, CA long as the work is properly cited, the use is educational and not for profit, and the Corresponding author: Roland Stein, [email protected] work is not altered. More information is available at http://www.diabetesjournals Received 4 April 2019 and accepted 6 June 2019 .org/content/license. diabetes.diabetesjournals.org Spaeth and Associates 1807 understanding how the transcriptional activities of these RESEARCH DESIGN AND METHODS essential MPC regulators control growth rate and final Animals fi ERT organ size is of signi cant importance. It has been pro- Ptf1a-Cre (15) and MIP-Cre (16) mice were used to fl posed that dissimilarities in pancreas mass in uence di- remove the Loxp sites surrounding exons 17 and 18 of abetes susceptibility, a proposal supported by the reduced the Brg1 locus (Brg1f/f [17]) and the Stop cassette in pancreas size of autoantibody-positive individuals with the Rosa26-Loxp-Stop-Loxp-tdTomato lineage reporter 2 2 type 1 diabetes (9). (R26LSL-tdTomato [18]). Brm / mice were generated using ho- In the postnatal pancreas, Pdx1 is produced at much mologous recombination to insert the neomycin gene into b higher levels in islet -cells than in other pancreas cell Brm exon 4 (19). The following genotypes were used for the types (10). Conditional removal of Pdx1 from these cells developmental studies: control, Ptf1a-Cre;Brg1f/+ or Ptf1a- b f/+ +/2 Dpanc in mice leads to a profound loss of -cell function and Cre;Brg1 ;Brm ; experimental, Brg1 (Ptf1a- identity, because these cells rapidly transdifferentiate to f/f 2/2 2/2 Dpanc 2 Cre;Brg1 ), Brm (Ptf1a-Cre;Brm ), and DKO + a f/f 2/2 glucagon and insulin -like cells (11). This remarkable (Ptf1a-Cre;Brg1 ;Brm ). Noon of the day of the vaginal control derives not only from the positive actions of Pdx1 plug discovery was designated day e0.5. For BrdU injec- on target gene transcription but also from its repression tions, 100 mg of BrdU (B5002; Sigma-Aldrich) per kilo- a b of key -cell functional genes in -cells, such as MafB and gram of pregnant dam body weight was injected 30 min Gcg. As a result of these novel and fundamental proper- before embryo harvest. ties, Pdx1 is viewed as one of the most critical pancreas- The adult studies consisted of these genotypes: con- 2 enriched transcription factors (6). trol, MIP-CreERT;Brg1f/+;Brm+/ ;R26LSL-tdTomato/+;and Db 2 2 Transcription factors like Pdx1 predominantly control experimental, Brg1 ;Brm+/ (MIP-CreERT;Brg1f/f;Brm+/ ; Db 2 2 gene expression through the recruitment of coregulators, R26LSLtdTomato/+), Brg1 /+;Brm / (MIP-CreERT; 2 2 often operating as large multiprotein complexes. These co- Brg1f/+; Brm / ; R26LSLtdTomato/+), and bDKO (MIP- 2 2 regulators affect transcriptional activity positively and/or CreERT; Brg1f/f; Brm / ; R26LSLtdTomato/+). CreERT- negatively, for example, by displacing nucleosomes, epige- mediated recombination of Brg1f/f and the R26LSLtdTomato netically modifying histones/DNA, and affecting recruit- was achieved by administration of 4 mg tamoxifen (T5648; ment of the RNA polymerase II transcriptional machinery. Sigma-Aldrich) by oral gavage three times over a 5-day period. Because of limited knowledge of the coregulators influencing Pdx1 activity, an unbiased chemical crosslinking, immuno- Intraperitoneal Glucose Tolerance Test and Serum precipitation, and mass spectrometry analysis was used to Insulin Measurements identify interacting proteins in rodent b-cell lines. Numer- Mice (n =5–12) were given intraperitoneal injection of ous proteins with an array of cellular functions were found D-glucose (2 mg/g body wt) after a 6-h fast. Blood glucose to associate with Pdx1, including the ATP-dependent was measured using a FreeStyle glucometer (Abbott Di- Swi/Snf chromatin remodeling complex (12). Significantly, abetes Care). Serum insulin was measured by radio immu- Pdx1 was the principal islet b-cell–enriched transcription noassay at the Vanderbilt Hormone Assay and Analytical factor binding to Swi/Snf in mouse b-cell lines (12). Services Core. The multisubunit Swi/Snf complex uses the energy of ATP hydrolysis, through the actions of the two mutually Glucose-Stimulated Insulin Secretion exclusive Brg1 (i.e., also referred to as Smarca4) and Brm Secreted insulin from isolated control and bDKO mice (i.e., Smarca2) ATPase subunits (13,14) (Fig. 1A), to disrupt (n =8–10) islets was performed as described previously DNA-nucleosome contacts and influence DNA accessibil- (20). The outcome was presented as the fold change ity. We previously discovered that in vitro knockdown of between the percentage of secreted insulin (relative to Brg1 in rodent b-cell lines negatively affected Pdx1 target insulin content) at 16.7 mmol/L glucose and the per- gene expression (e.g., Ins, MafA, and Glut2). Moreover, centage of secreted insulin (relative to insulin content) Pdx1:Swi/Snf interactions were not only acutely enhanced at 2.8 mmol/L glucose. Islet insulin content was calcu- in islet b-cells by increased glucose concentrations but lated as the concentration of insulin per islet in each were also reduced in human T2D b-cells (12). Here we reaction (ng/mL/islet). show that the Pdx1:Brg1/Swi/Snf complex is critical for mouse MPC proliferation, with embryonic conditional Tissue Preparation and Immunostaining deletion of only the Brg1 gene encoding ATPase resulting Whole embryos and adult pancreata were fixed in 4% (v/v) in a ;50% smaller pancreas. In contrast, removing both paraformaldehyde, embedded,
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