Type 2 diabetes (T2D) is associated with loss of transcription factors (TFs) from a subset of failing β-cells. Among these TFs is Pdx1, which controls the expression of numerous genes involved in maintaining β-cell function and identity. Pdx1 activity is modulated by transcriptional coregulators and has recently been shown, through an unbiased screen, to interact with the Chd4 ATPase subunit of the Nucleosome Remodeling and Deacetylase complex. Chd4 contributes to the maintenance of cellular identity and functional status of numerous different cell types. Here, we demonstrate Pdx1 dynamically interacts with Chd4 under physiological and stimulatory conditions within islet β-cells. We establish a fundamental role for Chd4 in regulating insulin secretion and modulating numerous Pdx1 bound genes in vitro, including the MafA TF, where we discovered Chd4 is bound at the MafA Region 3 enhancer. Furthermore, we found that Pdx1:Chd4 interactions are significantly compromised in islet β-cells under metabolically-induced stress in vivo and in human donor tissues with T2D. Our findings establish a fundamental role for Chd4 in regulating insulin secretion and modulating Pdx1-bound genes in vitro, and disruption of Pdx1:Chd4 interactions coincides with β-cell dysfunction associated with T2D.
Signal Transducer and Activator of Transcription 3 (STAT3) has recently been shown to be involved in bone development and has been implicated in bone diseases, such as Job's Syndrome. Bone growth and changes have been known for many years to differ between sexes with male bones tending to have higher bone mass than female bones and older females tending to lose bone mass at faster rates than older males. Previous studies using conditional knock mice with Stat3 specifically deleted from the osteoblasts showed both sexes exhibited decreased bone mineral density (BMD) and strength. Using the Cre-Lox system with Cathepsin K promotor driving Cre to target the deletion of the Stat3 gene in mature osteoclasts (STAT3-cKO mice), we observed that 8-week old STAT3-cKO female femurs exhibited significantly lower BMD and bone mineral content (BMC) compared to littermate control (CN) females. There were no differences in BMD and BMC observed between male knockout and male CN femurs. However, micro-computed tomography (μCT) analysis showed that both male and female STAT3-cKO mice had significant decreases in bone volume/tissue volume (BV/TV). Bone histomorphometry analysis of the distal femur, further revealed a decrease in bone formation rate and mineralizing surface/ bone surface (MS/BS) with a significant decrease in osteoclast surface in female, but not male, STAT3-cKO mice. Profiling gene expression in an osteoclastic cell line with a knockdown of STAT3 showed an upregulation of a number of genes that are directly regulated by estrogen receptors. These data collectively suggest that regulation of STAT3 differs in male and female osteoclasts and that inactivation of STAT3 in osteoclasts affects bone turnover more in females than males, demonstrating the complicated nature of STAT3 signaling pathways in osteoclastogenesis. Drugs targeting the STAT3 pathway may be used for treatment of diseases such as Job's Syndrome and osteoporosis.
Islet β-cell dysfunction that leads to impaired insulin secretion is a principal source of pathology of diabetes. In type 2 diabetes, this breakdown in β-cell health is associated with compromised islet-enriched transcription factor (TF) activity that disrupts gene expression programs essential for cell function and identity. TF activity is modulated by recruited coregulators that govern activation and/or repression of target gene expression, thereby providing a supporting layer of control. To date, over 350 coregulators have been discovered that coordinate nucleosome rearrangements, modify histones, and physically bridge general transcriptional machinery to recruited TFs; however, relatively few have been attributed to β-cell function. Here, we will describe recent findings on those coregulators with direct roles in maintaining islet β-cell health and identity and discuss how disruption of coregulator activity is associated with diabetes pathogenesis.
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 β-cells in vivo, we generated an inducible-β-cell-specific Chd4 knockout mouse model. Removal of Chd4 from mature islet β-cells rendered mutant animals glucose intolerant, in part due to defects in insulin secretion. We observed an increased ratio of immature:mature insulin granules in Chd4-deficient β-cells that correlated with elevated levels of proinsulin both within isolated islets and from plasma following glucose stimulation in vivo. RNA- and ATACSequencing showed that lineage-labeled Chd4-deficient β-cells have alterations in chromatin accessibility and altered expression of genes critical for β-cell function, including MafA, Slc2a2, Chga, and Chgb. Knockdown of CHD4 from a human β-cell line revealed similar defects in insulin secretion and alterations in several β-cell-enriched gene targets. These results illustrate how critical Chd4 activities are in controlling genes essential for maintaining β-cell function. Article Highlights Pdx1:Chd4 interactions were previously shown to be compromised in β-cells from human donors with type 2 diabetes. β-cell-specific removal of Chd4 impairs insulin secretion and leads to glucose intolerance in mice. Expression of key β-cell functional genes and chromatin accessibility are compromised in Chd4-deficient β-cells. Chromatin remodeling activities enacted by Chd4 are essential for β-cell function under normal physiological conditions.
<p> </p> <p>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 β-cells <em>in vivo</em>, we generated an inducible-β-cell-specific Chd4 knockout mouse model. Removal of <em>Chd4</em> from mature islet β-cells rendered mutant animals glucose intolerant, in part due to defects in insulin secretion. We observed an increased ratio of immature:mature insulin granules in <em>Chd4</em>-deficient β-cells that correlated with elevated levels of proinsulin both within isolated islets and from plasma following glucose stimulation <em>in vivo</em>. RNA- and ATAC-Sequencing showed that lineage-labeled <em>Chd4</em>-deficient β-cells have alterations in chromatin accessibility and altered expression of genes critical for β-cell function, including <em>MafA</em>,<em> Slc2a2</em>,<em> Chga</em>, and <em>Chgb</em>. Knockdown of <em>CHD4 </em>from a human β-cell line revealed similar defects in insulin secretion and alterations in several β-cell-enriched gene targets. These results illustrate how critical Chd4 activities are in controlling genes essential for maintaining β-cell function.</p>
A key feature of diabetes is insufficient functional β-cell mass. Strategies to augment functional β-cell mass include directed differentiation of stem cells towards a β-cell fate, which requires extensive knowledge of transcriptional programs governing differentiation in vivo. Pdx1, an essential transcription factor involved in β-cell differentiation, dynamically recruits coregulators to drive different gene expression programs. A recent study identified the ATP-dependent Swi/Snf chromatin remodeling complex as an important Pdx1-interacting partner. In the developing pancreas, conditional removal of the Brg Swi/Snf ATPase subunit compromised final pancreas mass, and loss of both ATPase subunits, Brg1 and Brm, led to glucose dyshomeostasis. While Swi/Snf is essential during early pancreas development and mature β-cell function, the role of Swi/Snf during endocrine cell development has yet to be explored. Here, we generated a conditional knockout of either Brg1 (Brg1ΔendoBrm+/-), Brm (Brg1Δendo/+Brm-/-), or both subunits (DKOΔendo) during endocrine development using Neurogenin 3 Cre. Four-week-old Brg1ΔendoBrm+/- mice are glucose intolerant, hyperglycemic, and hypoinsulinemic, with no phenotype observed in Brg1Δendo/+Brm-/-mice. Impaired glucose homeostasis of Brg1ΔendoBrm+/- mice was due in part to reduced islet number, impaired insulin secretion, and altered gene expression programs. DKOΔendo mice have not been found at weaning; however, postnatal day 6 DKOΔendo mice are severely hyperglycemic with reduced plasma insulin levels. Single-cell RNA-Seq of embryonic day 15.5 lineage-labeled cells revealed reduced insulin and glucagon transcript in the β-cell and α-cell clusters, respectively, in both Brg1ΔendoBrm+/- and DKOΔendo mice and reduced Neurogenin 3 transcript in DKOΔendo endocrine progenitor clusters. These data suggest Swi/Snf plays a critical role in governing gene-expression programs essential for endocrine cell development. Disclosure R.K.Davidson: Employee; Eli Lilly and Company. S.Kanojia: None. M.E.Osmulski: None. J.Spaeth: None. Funding National Institutes of Health (R01DK129287, R03DK127129)
Evaluating the role of Chd3 helicase subunit on β-cell function Pancreatic β-cells play a crucial role in maintaining whole-body glucose homeostasis by secreting insulin upon increased blood glucose levels. To develop effective therapies to combat hyperglycemia, it is of vital importance to understand how β-cells function normally and the mechanisms driving their dysfunction in settings of diabetes. Pdx1, an essential transcription factor involved in β-cell development and function, dynamically recruits coregulators to drive gene expression programs. Recently, Pdx1 was shown to interact with Chd4, a helicase of Nucleosome Remodeling and Deacetylase (NuRD) complex within pancreatic β-cells. Chd4 was found to regulate insulin secretion, modulate expression of β-cell functional genes by modifying chromatin accessibility. We discovered that Chd4 removal from mature β-cells (Chd4Δβ) increased Chd3 levels, an alternate Chd subunit of the NuRD complex. This led us to evaluate whether Chd3 alone plays a role in β-cell function and/or does it compensate in absence of Chd4. To investigate this possibility, we generated tamoxifen inducible, β-cell-specific Chd3 (Chd3Δβ) and Chd3/Chd4 double knockout (Chd3/4Δβ) mouse models. Whereas 4-weeks following Chd3 removal, Chd3Δβ mice did not display glucose intolerance, the Chd3/4Δβ mutants were profoundly glucose intolerant with elevated ad libitum fed blood glucose levels and near complete loss of insulin secretion in response to glucose, a phenotype much more severe than Chd4Δβ mutants previously characterized. This data suggests Chd3 plays a pertinent role in the absence of Chd4 within the β-cell. Current efforts are focused on determining the insulin secretion capacity of Chd3/4Δβ islets and evaluating gene expression and chromatin accessibility of Chd3/Chd4Δβ β-cells. Disclosure S.Kanojia: None. M.E.Osmulski: None. R.K.Davidson: Employee; Eli Lilly and Company. J.Spaeth: None. Funding National Institutes of Health (DK129287)
<p> </p> <p>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 β-cells <em>in vivo</em>, we generated an inducible-β-cell-specific Chd4 knockout mouse model. Removal of <em>Chd4</em> from mature islet β-cells rendered mutant animals glucose intolerant, in part due to defects in insulin secretion. We observed an increased ratio of immature:mature insulin granules in <em>Chd4</em>-deficient β-cells that correlated with elevated levels of proinsulin both within isolated islets and from plasma following glucose stimulation <em>in vivo</em>. RNA- and ATAC-Sequencing showed that lineage-labeled <em>Chd4</em>-deficient β-cells have alterations in chromatin accessibility and altered expression of genes critical for β-cell function, including <em>MafA</em>,<em> Slc2a2</em>,<em> Chga</em>, and <em>Chgb</em>. Knockdown of <em>CHD4 </em>from a human β-cell line revealed similar defects in insulin secretion and alterations in several β-cell-enriched gene targets. These results illustrate how critical Chd4 activities are in controlling genes essential for maintaining β-cell function.</p>
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