Brain-specific homeobox factor as a target selector for glucocorticoid receptor in energy balance

Abstract

The molecular basis underlying the physiologically well-defined orexigenic function of glucocorticoid (Gc) is unclear. Brain-specific homeobox factor (Bsx) is a positive regulator of the orexigenic neuropeptide, agouti-related peptide (AgRP), in AgRP neurons of the hypothalamic arcuate nucleus. Here, we show that in response to fasting-elevated Gc levels, Gc receptor (GR) and Bsx synergize to direct activation of AgRP transcription. This synergy is dictated by unique sequence features in a novel Gc response element in AgRP (AgRP-GRE). In contrast to AgRP-GRE, Bsx suppresses transactivation directed by many conventional GREs, functioning as a gene context-dependent modulator of GR actions or a target selector for GR. Consistent with this finding, AgRP-GRE drives fasting-dependent activation of a target gene specifically in GR(+) Bsx(+) AgRP neurons. These results define AgRP as a common orexigenic target gene of GR and Bsx and provide an opportunity to identify their additional common targets, facilitating our understanding of the molecular basis underlying the orexigenic activity of Gc and Bsx.

Fig 1

In vivo evidence for the involvement of GR in regulating expression of AgRP. (A) The coronal sections of the ARC region of mice fasted for 24 h were immunostained with antibodies against GR and Bsx. DAPI staining is included as a control for all of the nuclei in the section. The yellow scale bar indicates 100 μm. (B) In situ hybridizations for AgRP and NPY were carried out with serial coronal 12-μm sections of the ARC region of mice (n = 3 in each group) intraperitoneally injected with either vehicle or 50 mg/kg of RU486. Representative images are shown. (C) For all the images shown in panel B, the in situ signal intensity of AgRP was quantified using ImageJ and plotted against the intensity of samples injected with vehicle alone. (D) The in situ signal intensity of AgRP and NPY of serial coronal 12-μm sections of the ARC region of control (n = 3) and GR^f/f:AgRP-Cre (GR-KO) mice (n = 5) was quantified using ImageJ and plotted against the intensity of fed control samples. P values of less than 0.001 and 0.0001 are denoted ** and ***, respectively (C and D).

Fig 2

Identification of AgRP-GRE. (A) Schematic representation of the AgRP promoter. AgRP-GRE is highly conserved in mammals, as indicated. The MMTV-GRE and the consensus GRE sequences are shown for comparison. (B and C) Luciferase reporter assays with AgRP-1kb:LUC reporter (B) and its derivative with a mutation in the first half site of the AgRP-GRE (m3) (C) in HEK293 cells. (D) Luciferase reporter assays with the (AgRP-GRE)₂:LUC reporter in HEK293 cells. The 27-mer sequences, as well as a schematic representation of the reporter, are shown. (E and F) ChIP for GR binding in the hypothalamus lysates of mice intraperitoneally injected with either vehicle or 10 mg/kg of Dex (E) or in the hypothalamus lysates of mice either fed or fasted for 24 h (F).

Fig 3

Synergy of GR and Bsx in activating expression of AgRP. (A to C) Luciferase reporter assays with AgRP-1kb:LUC reporter (A and C) and (AgRP-GRE)₂:LUC reporter (B) in HEK293 cells transfected with expression vectors as indicated. Bsx-N160A is a DNA binding-defective mutant form of Bsx (C). (D) Anti-HA antibody-coimmunoprecipitated HA-tagged Bsx and Flag-tagged GR from HEK293 cells transfected with expression vectors for HA-Bsx and Flag-GR. Immunoprecipitation with IgG was carried out as a negative control. IP, immunoprecipitation; WB, Western blotting. (E) Coimmunoprecipitation of endogenous GR and Bsx in the hypothalamus lysates of mice either fed or fasted for 24 h. Association of GR and Bsx observed in fed mice was further enhanced in fasted mice by approximately 1.7-fold. (F) ChIP for Bsx binding in the hypothalamus lysates of mice either fed or fasted for 24 h (top) or of mice intraperitoneally injected with either vehicle or 10 mg/kg of Dex (lower).

Fig 4

AgRP-GRE as a prototypic tool to find additional targets of GR and Bsx. (A to E and G) Luciferase reporter assays with MMTV:LUC (A), (AgRP-GRE)₃:LUC (B), (AgRP-GRE-m6)₃:LUC (C), (AgRP-GRE-m9)₃:LUC (D), and (AgRP-GRE-m10)₄:LUC (E) in HEK293 cells and Per1:LUC and Asb4:LUC (G) reporters in P19 cells transfected with the indicated expression vectors. Sequences of each hybrid GRE are shown (C to E). (F) Sequences of AgRP-GRE, Per1-GRE, and Asb4-GRE as well as of MMTV-GRE and other known GREs that are negatively regulated by Bsx are shown. The unique sequences in AgRP-GRE and AgRP-GRE-like motifs are indicated with an asterisk.

Fig 5

Bsx as an in vivo target selector of GR- and Gc-triggered assembly of active chromatin on the AgRP promoter. (A) Dex-dependent expression of AgRP and Sgk1, which are positively and negatively regulated by Bsx, respectively, were compared between N42 cells expressing either control siRNA or si-Bsx using RT-PCR. (B and C) Dex-dependent expression of AgRP (B) and formation of active chromatin on the AgRP promoter (C) were compared between P19 cells expressing either GR alone or both GR and Bsx using RT-PCR and ChIP, respectively. H3K4me3 and H3Ac indicate two open chromatin marks, trimethylated histone H3 lysine 4 and acetylated H3, respectively (C).

Fig 6

AgRP neuronal expression of GFP directed by 1-kb AgRP promoter in transgenic mice. (A) Schematic representation of an EGFP reporter flanked by two insulators and driven by a minimal CMV promoter fused to the 1-kb AgRP promoter. (B to D) The coronal sections of the ARC region of transgenic mice with the 1-kb AgRP promoter linked to EGFP, either fed (B) or fasted for 24 h (B to D), were immunostained with antibodies against GFP, Bsx, and GR. Yellow scale bars, 100 μm.

Fig 7

Roles of Bsx in AgRP neurons. (A) Dual function of Bsx segregates GREs into two groups, positive targets, such as AgRP-GRE, and negative targets. The positive targets can be associated with critical orexigenic genes in AgRP neurons. (B) The working model for the synergistic transactivation of the AgRP gene by GR and Bsx. Fasting increases nuclear GR due to increased Gc levels. Both GR and Bsx likely are required to form a transcriptionally active enhanceosome at the AgRP promoter. Protein-protein interactions between GR and Bsx, as well as binding sites for both GR and Bsx, likely play critical roles in the synergy.