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, 20 (1), 73-84

BRD7 Regulates XBP1s' Activity and Glucose Homeostasis Through Its Interaction With the Regulatory Subunits of PI3K

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BRD7 Regulates XBP1s' Activity and Glucose Homeostasis Through Its Interaction With the Regulatory Subunits of PI3K

Sang Won Park et al. Cell Metab.

Erratum in

  • Cell Metab. 2014 Dec 2;20(6):1088

Abstract

Bromodomain-containing protein 7 (BRD7) is a member of the bromodomain-containing protein family that is known to play a role as tumor suppressors. Here, we show that BRD7 is a component of the unfolded protein response (UPR) signaling through its ability to regulate X-box binding protein 1 (XBP1) nuclear translocation. BRD7 interacts with the regulatory subunits of phosphatidylinositol 3-kinase (PI3K) and increases the nuclear translocation of both p85α and p85β and the spliced form of XBP1 (XBP1s). Deficiency of BRD7 blocks the nuclear translocation of XBP1s. Furthermore, our in vivo studies have shown that BRD7 protein levels are reduced in the liver of obese mice, and reinstating BRD7 levels in the liver restores XBP1s nuclear translocation, improves glucose homeostasis, and ultimately reduces the blood glucose levels in the obese and diabetic mouse models.

Figures

Figure 1
Figure 1. BRD7 binds to p85α and increases its nuclear translocation
(A) Immunoblotting for BRD7 and flag-tagged p85α proteins after immunoprecipitation of p85α from 293HEK cells that were infected with Ad-BRD7 alone; or Ad-p85α-flag alone; or Ad-BRD7 and Ad-p85α-flag. Total lysates were immunoblotted for BRD7 and tubulin. (B) Immunoblots of flag-p85α and BRD7 after BRD7 immunoprecipitation from 293HEK cells that were infected with the indicated adenoviruses. (C) Nuclear protein levels of p85α in 293HEK cells infected with increasing doses of Ad-BRD7 and a constant dose of Ad-p85α-flag. LaminA/C was used as a control for nuclear protein level. (D) Nuclear protein levels of XBP-1s in 293HEK cells infected with increasing doses of Ad-BRD7 and a constant dose of XBP1s. (E) Immunoblotting for BRD7 and XBP1s following immunoprecipitation with XBP1-specific antibody. Total protein levels of BRD7 and tubulin are shown below. (F) BRD7 immunoblotting after XBP1 immunoprecipitation from DKD (p85α and p85β double knock down) and PLKO (control) cells that were infected with Ad-BRD7 and Ad-XBP1s. Total protein amounts of p85s, BRD7, XBP1s, and tubulin are shown below. (G) Immunoblotting for BRD7 protein after XBP1 immunoprecipitation from DKO (p85α and p85β double knock out) and its control cells that were infected with Ad-BRD7 and Ad-XBP1s. Total protein amounts of p85s, BRD7, XBP1 and tubulin are shown below. (H) Nuclear protein amounts of XBP1s in PLKO and DKD cells infected with increasing doses of Ad-BRD7 and a constant dose of XBP1s. NUP98 was used as a control for nuclear protein level. (I) Western blot for XBP1s in PLKO and DKO cells infected with Ad-BRD7 and Ad-XBP1s. LaminA/C was used as a control. Each experiments was independently repeated three times.
Figure 2
Figure 2. BRD7 increases the nuclear translocation of XBP1s
(A) Flag-p85α and flag-XBP1s immunoblots after XBP1 immunoprecipitation from cytoplasmic and nuclear protein fractions of 293HEK cells that were infected with Ad-p85α-flag and Ad-XBP1s-flag; or together with Ad-BRD7. Total lysates were immunoblotted for BRD7 and p85α. NUP98 and tubulin were used as a control for nuclear and cytoplasmic protein levels, respectively. (B) BRD7 and flag-p85α immunoblots after flag immunoprecipitation from cytoplasmic and nuclear protein fractions of 293HEK cells that were infected with Ad-p85α-flag and Ad-BRD7, followed by insulin treatment at 500 nM for indicated times. Each cytoplasmic and nuclear protein lysates was immunoblotted for BRD7, flag, and tubulin/or NUP98. (C) BRD7 immunoblotting in XBP1 immunoprecipitates after insulin (500 nM) stimulation. Protein lysates were immunoblotted for indicated antibodies. (D) Nuclear protein amounts of p85α and p85β in 293HEK cells infected with a constant dose of Ad-85α-flag and Ad-p85β-myc and increasing doses of Ad-BRD7. (E) Expression levels of XBP1 target genes, Erdj4, Herp, and Ero1α in MEFs infected with Ad-LacZ or Ad-BRD7. (F) Western blot analysis for nuclear XBP1s proteins in 293HEK cells that were infected with the indicated adenoviruses. (G-I) Eight-week-old male wild-type mice were injected with Ad-LacZshRNA or Ad-BRD7shRNA (1.5×108 pfu/g, n=6 for each group) through the tail vein. (G) Nuclear XBP1s protein amounts at 24 hours of fasting and during one hour of refeeding on day seven post-injection (left). NUP98 was used as a control. Quantification of the western blot showing the ratio of XBP1s to NUP98 (right). (H) XBP1 mRNA splicing assay in Ad-LacZshRNA or Ad-BRD7shRNA injected mice at 24 hours of fasting and one hour after refeeding. (I) Relative mRNA levels of Erdj4, Herp, and CHOP in the liver of Ad-LacZshRNA or Ad-BRD7shRNA injected mice after six hours of fasting. Error bars are represented as mean ± SEM., P values were determined by Student's t-test. *P<0.05, **P <0.01, ***P <0.001. Each experiments was independently repeated three times.
Figure 3
Figure 3. Restoration of BRD7 in the liver of the ob/ob mice improves glucose tolerance and establishes euglycemia
(A) Total BRD7 protein amounts in the liver of lean wild-type and ob/ob mice at six hours of fasting (top). Quantification of the western blot showing the ratio of BRD7 to tubulin (bottom). (B) BRD7 mRNA levels in the wild-type and ob/ob mice's livers during 24 hours of fasting and one and three hours of refeeding states. (C-G) Eight-week-old male ob/ob mice were injected with Ad-BRD7 or Ad-LacZ (1×108 pfu/g, n=6 for each group) as a control through the tail vein. (C) Blood glucose levels (mg/dl) after six hours fasting on day three of the injections. (D) Glucose tolerance test (GTT) on day five post-injection (left). Area under the curve of GTT (right). (E) Insulin tolerance test (ITT) on day seven post-injection (left). Area under the curve of ITT (right). (F) In vivo insulin receptor signaling in the liver of Ad-LacZ or Ad-BRD7 injected ob/ob mice on day eight after injection (left). Quantifications of the western blots (right). (G) Immunoblots of IRS1, IRS2, and p85 after p85 immunoprecipitation of the liver tissues that were infused with insulin in Ad-LacZ and Ad-BRD7 injected ob/ob mice (left). Quantifications of the western blots (right). Error bars are represented as mean ± SEM., P values in (B) and (C) were determined by Student's t-test. Significance in (D) and (E) was determined by two-way ANOVA with Bonferroni multiple-comparison analysis. *P<0.05, **P <0.01, ***P <0.001. Experiments were repeated in three independent cohorts.
Figure 4
Figure 4. Restoration of BRD7 in the liver of the ob/ob mice releases ER stress and displays improved phenotypes
Eight-week-old male ob/ob mice were injected with Ad-BRD7 or Ad-LacZ (1×108 pfu/g, n=6 for each group) as a control through the tail vein. (A) Western blots for nuclear XBP1s protein on day eight post-injection (top). Quantification of the western blot showing the ratio of BRD7 to tubulin (bottom). (B-C) mRNA and protein levels of XBP1s target genes in the liver of Ad-LacZ and Ad-BRD7 injected mice after six hours of fasting were analyzed by qPCR (B) and western blot (C, left). Quantifications of the western blots (C, right). (D) PERK phosphorylation on Thr980, total PERK, eIF2α phosphorylation on Ser51, and total eIF2α protein levels in the liver of Ad-LacZ and Ad-BRD7 injected ob/ob mice were analyzed by western blot. (E) Total and nuclear amounts of FoxO1 protein in the liver of Ad-LacZ- or Ad-BRD7-injected ob/ob mice. (F) Relative mRNA levels of G6p, Fbp, and Pepck. (G) Macroscopic view of the liver of Ad-LacZ injected (top) and Ad-BRD7 injected (bottom) ob/ob mice. (H) H&E staining of liver sections in 4x (left) and 10x (right) magnifications. (I) Triglyceride contents (mg/g) in the liver. (J) Relative mRNA levels of Acc1, FasN, and Dgat2. Error bars are represented as mean ± SEM., P values were determined by Student's t-test. (*p<0.05, **p<0.01, ***p<0.001). Experiments were repeated in three independent cohorts.
Figure 5
Figure 5. Restoration of BRD7 in the liver of the diet-induced obese (DIO) mice improves glucose tolerance and increases XBP1s nuclear translocation
(A) Total BRD7 protein amounts in the liver of lean wild-type that were fed either on normal chow diet (NCD) or high fat diet (HFD) at six hours of fasting (left). Quantification of the western blot showing the ratio of BRD7 to tubulin (right). (B) Western blot for BRD7 protein levels from the total lysates of wild-type (NCD), ob/ob, and HFD mice (left). Tubulin was used as a control. Quantification of the western blot showing the ratio of BRD7 to tubulin (right). (C-H) Mice that were fed either on NCD or HFD were injected with Ad-BRD7 or Ad-LacZ (5×107 pfu/g, n=6 for each group) through the tail vein. (C) The graph shows the blood glucose levels (mg/dl) after six hour fasting on day three of the injections. (D) Glucose tolerance test (GTT) on post-injection day five post-injection (left). Area under the curve of GTT (right). (E) XBP1s nuclear protein amounts in the liver lysates (top). Quantification of the western blot showing the ratio of XBP1s to tubulin (bottom). (F-H) Mice were fasted for 24 hours and refed for 0 and 1 hour. Relative mRNA levels of Erdj4, Herp, and Ero1α were determined by qPCR. Error bars are represented as mean ± SEM., P values in (A-C and E-H) were determined by Student's t-test. Significance in (D) was determined by two-way ANOVA with Bonferroni multiple-comparison analysis. *P<0.05, **P <0.01, ***P <0.001. Experiments were repeated in three independent cohorts.

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