The SWI/SNF chromatin remodelling complex regulates pancreatic endocrine cell expansion and differentiation in mice in vivo.

AIMS/HYPOTHESIS: Strategies to augment functional beta cell mass include directed differentiation of stem cells towards a beta cell fate, which requires extensive knowledge of transcriptional programs governing endocrine progenitor cell differentiation in vivo. We aimed to study the contributions of the Brahma-related gene-1 (BRG1) and Brahma (BRM) ATPase subunits of the SWI/SNF chromatin remodelling complex to endocrine cell development. METHODS: We generated mice with endocrine progenitor-specific Neurog3-Cre BRG1 removal in the presence of heterozygous (Brg1Δendo;Brm+/-) or homozygous (double knockout: DKOΔendo) BRM deficiency. Whole-body metabolic phenotyping, islet function characterisation, islet quantitative PCR and histological characterisation were performed on animals and tissues postnatally. To test the mechanistic actions of SWI/SNF in controlling gene expression during endocrine cell development, single-cell RNA-seq was performed on flow-sorted endocrine-committed cells from embryonic day 15.5 control and mutant embryos. RESULTS: Brg1Δendo;Brm+/- mice exhibit severe glucose intolerance, hyperglycaemia and hypoinsulinaemia, resulting, in part, from reduced islet number; diminished alpha, beta and delta cell mass; compromised islet insulin secretion; and altered islet gene expression programs, including reductions in MAFA and urocortin 3 (UCN3). DKOΔendo mice were not recovered at weaning; however, postnatal day 6 DKOΔendo mice were severely hyperglycaemic with reduced serum insulin levels and beta cell area. Single-cell RNA-seq of embryonic day 15.5 lineage-labelled cells revealed endocrine progenitor, alpha and beta cell populations from SWI/SNF mutants have reduced expression of Mafa, Gcg, Ins1 and Ins2, suggesting limited differentiation capacity. Reduced Neurog3 transcripts were discovered in DKOΔendo endocrine progenitor clusters, and the proliferative capacity of neurogenin 3 (NEUROG3)+ cells was reduced in Brg1Δendo;Brm+/- and DKOΔendo mutants. CONCLUSIONS/INTERPRETATION: Loss of BRG1 from developing endocrine progenitor cells has a severe postnatal impact on glucose homeostasis, and loss of both subunits impedes animal survival, with both groups exhibiting alterations in hormone transcripts embryonically. Taken together, these data highlight the critical role SWI/SNF plays in governing gene expression programs essential for endocrine cell development and expansion. DATA AVAILABILITY: Raw and processed data for scRNA-seq have been deposited into the NCBI Gene Expression Omnibus (GEO) database under the accession number GSE248369.

Dynamic regulation of pancreatic ß cell function and gene expression by the SND1 coregulator in vitro.

The pancreatic ß cell synthesizes, packages, and secretes insulin in response to glucose-stimulation to maintain blood glucose homeostasis. Under diabetic conditions, a subset of ß cells fail and lose expression of key transcription factors (TFs) required for insulin secretion. Among these TFs is Pancreatic and duodenal homeobox 1 (PDX1), which recruits a unique subset of transcriptional coregulators to modulate its activity. Here we describe a novel interacting partner of PDX1, the Staphylococcal Nuclease and Tudor domain-containing protein (SND1), which has been shown to facilitate protein-protein interactions and transcriptional control through diverse mechanisms in a variety of tissues. PDX1:SND1 interactions were confirmed in rodent ß cell lines, mouse islets, and human islets. Utilizing CRISPR-Cas9 gene editing technology, we deleted Snd1 from the mouse ß cell lines, which revealed numerous differentially expressed genes linked to insulin secretion and cell proliferation, including limited expression of Glp1r. We observed Snd1 deficient ß cell lines had reduced cell expansion rates, GLP1R protein levels, and limited cAMP accumulation under stimulatory conditions, and further show that acute ablation of Snd1 impaired insulin secretion in rodent and human ß cell lines. Lastly, we discovered that PDX1:SND1 interactions were profoundly reduced in human ß cells from donors with type 2 diabetes (T2D). These observations suggest the PDX1:SND1 complex formation is critical for controlling a subset of genes important for ß cell function and is targeted in diabetes pathogenesis.

The Chd4 Helicase Regulates Chromatin Accessibility and Gene Expression Critical for ß-Cell Function In Vivo.

The transcriptional activity of Pdx1 is modulated by a diverse array of coregulatory factors that govern chromatin accessibility, histone modifications, and nucleosome distribution. We previously identified the Chd4 subunit of the nucleosome remodeling and deacetylase complex as a Pdx1-interacting factor. To identify how loss of Chd4 impacts glucose homeostasis and gene expression programs in ß-cells in vivo, we generated an inducible ß-cell-specific Chd4 knockout mouse model. Removal of Chd4 from mature islet ß-cells rendered mutant animals glucose intolerant, in part due to defects in insulin secretion. We observed an increased ratio of immature-to-mature insulin granules in Chd4-deficient ß-cells that correlated with elevated levels of proinsulin both within isolated islets and from plasma following glucose stimulation in vivo. RNA sequencing and assay for transposase-accessible chromatin with sequencing showed that lineage-labeled Chd4-deficient ß-cells have alterations in chromatin accessibility and altered expression of genes critical for ß-cell function, including MafA, Slc2a2, Chga, and Chgb. Knockdown of CHD4 from a human ß-cell line revealed similar defects in insulin secretion and alterations in several ß-cell-enriched gene targets. These results illustrate how critical Chd4 activities are in controlling genes essential for maintaining ß-cell function. ARTICLE HIGHLIGHTS: Pdx1-Chd4 interactions were previously shown to be compromised in ß-cells from human donors with type 2 diabetes. ß-Cell-specific removal of Chd4 impairs insulin secretion and leads to glucose intolerance in mice. Expression of key ß-cell functional genes and chromatin accessibility are compromised in Chd4-deficient ß-cells. Chromatin remodeling activities enacted by Chd4 are essential for ß-cell function under normal physiological conditions.