MLL3/MLL4-Associated PAGR1 Regulates Adipogenesis by Controlling Induction of C/EBPβ and C/EBPδ

Transcription factors C/EBPβ and C/EBPδ are induced within hours after initiation of adipogenesis in culture. They directly promote the expression of master adipogenic transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and C/EBPα and are required for adipogenesis in vivo. However, the mechanism that controls the induction of C/EBPβ and C/EBPδ remains elusive. We previously showed that histone methyltransferases MLL3/MLL4 and associated PTIP are required for the induction of PPARγ and C/EBPα during adipogenesis.

ABSTRACT

Transcription factors C/EBPβ and C/EBPδ are induced within hours after initiation of adipogenesis in culture. They directly promote the expression of master adipogenic transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and C/EBPα and are required for adipogenesis in vivo. However, the mechanism that controls the induction of C/EBPβ and C/EBPδ remains elusive. We previously showed that histone methyltransferases MLL3/MLL4 and associated PTIP are required for the induction of PPARγ and C/EBPα during adipogenesis. Here, we show MLL3/MLL4/PTIP-associated protein PAGR1 (also known as PA1) cooperates with phosphorylated CREB and ligand-activated glucocorticoid receptor to directly control the induction of C/EBPβ and C/EBPδ in the early phase of adipogenesis. Deletion of Pagr1 in white and brown preadipocytes prevents the induction of C/EBPβ and C/EBPδ and leads to severe defects in adipogenesis. Adipogenesis defects in PAGR1-deficient cells can be rescued by the ectopic expression of C/EBPβ or PPARγ. Finally, the deletion of Pagr1 in Myf5⁺ precursor cells impairs brown adipose tissue and muscle development. Thus, by controlling the induction of C/EBPβ and C/EBPδ, PAGR1 plays a critical role in adipogenesis.

INTRODUCTION

The differentiation of preadipocytes toward adipocytes (adipogenesis) is regulated by a transcriptional network of sequentially expressed transcription factors (TFs) (1–3). Peroxisome proliferator-activated receptor γ (PPARγ) and C/EBPα are two master regulators of adipogenesis (3, 4). The ectopic expression of either PPARγ or C/EBPα in fibroblasts can induce adipogenic conversion (5, 6). During adipogenesis in culture, the expression of Pparg and Cebpa in preadipocytes is induced at relatively late phase, 1 to 2 days after exposure to a standard adipogenic cocktail of isobutylmethylxanthine (IBMX), dexamethasone (DEX), and insulin (collectively known as MDI), and are maintained at high levels throughout the differentiation (7, 8).

C/EBPβ and C/EBPδ are induced within hours of initiation of adipogenesis, preceding the induction of PPARγ and C/EBPα (7, 9). C/EBPβ and C/EBPδ promote PPARγ and C/EBPα expression and are required for adipogenesis (9, 10). The induction of these early adipogenic TFs in preadipocytes is largely dependent on stimulation by the adipogenic cocktail MDI. C/EBPβ and C/EBPδ are induced by IBMX and DEX, respectively, in the absence of protein synthesis (11). Although many studies have expanded our knowledge on the transcriptional regulation of adipogenesis, little is known about the factors that link hormone signaling to the coordinated induction of C/EBPβ and C/EBPδ (12).

Unlike many adipogenic TFs that are induced during adipogenesis, cyclic AMP (cAMP) response element-binding protein (CREB) is constitutively expressed prior to and throughout adipogenesis, suggesting that CREB is one of the initiating adipogenic TFs (13). CREB is phosphorylated at the Ser-133 residue upon stimulation by cAMP inducers IBMX and forskolin (14). Phospho-CREB (pCREB), the active form of CREB, binds to CRE-like elements in the Cebpb promoter, activates Cebpb expression, and promotes adipogenesis. Conversely, expression of a dominant-negative CREB, A-CREB, blocks Cebpb expression and adipogenesis, indicating that CREB acts upstream of C/EBPβ during adipogenesis (15). DEX, a synthetic ligand for glucocorticoid receptor (GR), induces Cebpd expression, suggesting that GR directly activates Cebpd expression in the early phase of adipogenesis (11). Studies have shown that GR, when bound and activated by DEX, associates with transiently open chromatin regions during the early phase of adipogenesis (16, 17). Although GR is dispensable for adipose development in vivo, it accelerates adipogenesis in cells (18).

Studies have identified essential roles of epigenomic regulators in promoting the expression of adipogenic TFs (12). We previously showed that histone H3K4 methyltransferases MLL3/MLL4 and associated PTIP are required for the induction of PPARγ and C/EBPα and adipogenesis (19, 20). Along with PTIP, a novel protein, PAGR1 (also known as PA1), is a unique component of the MLL3/MLL4 complex (21). PAGR1 and PTIP also form a separate lower molecular weight complex of ∼200 kDa, which is independent of the MLL3/MLL4 complex of ∼2 MDa (22, 23). PAGR1 interacts directly with PTIP but has no recognizable domains. PAGR1 has been implicated in DNA damage response (22). However, the biological functions of PAGR1 under normal physiological conditions remain almost completely unknown, except that PAGR1 is essential for mouse embryonic development (24). Therefore, we sought to determine whether PAGR1 plays a role in regulating adipogenesis.

In this study, we show that PAGR1 is dispensable for cell proliferation but is required for adipogenesis in cells and in mice. PAGR1 cooperates with phosphorylated CREB and ligand-activated GR to directly control the induction of early adipogenic TFs C/EBPβ and C/EBPδ within hours of the initiation of adipogenesis.

RESULTS

Reduced expression of C/EBPβ and C/EBPδ in PAGR1 knockout mouse embryonic fibroblasts (MEFs).

To understand the biological functions of the novel protein PAGR1, we generated conditional knockout (KO) Pagr1^lox/lox mice as well as whole-body KO Pagr1^−/− mice (Fig. 1A to D). The mouse Pagr1 gene contains 3 exons. Exons 1 and 2 encode the first 187 amino acids of the 253-amino-acid-long PAGR1 protein and were flanked by two loxP sites in the conditional KO (lox) allele. The targeted allele contains a loxP site in front of exon 1 and a neomycin (neo) selection cassette flanked by 2 loxP sites located in intron 2. Deletion of the neo selection cassette from the targeted allele by Cre recombinase generates the lox allele. Deletion of the entire loxed region, including exons 1 and 2 from the targeted allele, generates the null allele (Fig. 1A). Pagr1^−/− mice showed embryonic lethality and died at around embyronic day 8.5 (E8.5) to ∼E9.5 (24).

FIG 1.

PAGR1 is required for Cebpb and Cebpd expression in MEFs. (A) Schematic representation of mouse Pagr1 genomic locus (wild-type allele), targeted allele, lox allele, and knockout (KO) allele. Restriction endonuclease sites and Southern blot probe are indicated. The Pagr1 gene contains 3 exons, represented by the 3 black rectangles. (B) Genomic DNA prepared from ES cells was digested with NotI and EcoRV and hybridized with 5′ flanking probe in Southern blotting. Southern blotting detected an expected 12.8-kb band from the wild-type allele and an expected 8.0-kb band from the targeted allele. (C) Mating strategies for generating PAGR1 conditional KO and whole-body KO mice. (D) Confirmation of 3-week-old Pagr1^lox/lox mice by PCR genotyping. (E) Schematic of experimental approach to identify PAGR1-regulated genes in MEFs. Immortalized Pagr1^lox/lox MEFs were infected with retrovirus MSCVpuro expressing Cre or vector (Vec) alone. Total RNA was extracted for microarray analysis (Table S1). The resulting putative PAGR1-regulated genes were further confirmed by qRT-PCR and ChIP assays. (F) Western blot confirmation of Pagr1 deletion in MEFs. The asterisk indicates a nonspecific band. (G) Cell growth curves of Vec- and Cre-infected Pagr1^lox/lox MEFs. (H) Gene ontology analysis of genes that show over 2.5-fold decrease in PAGR1 null MEFs. Transcriptional domain coverage shows the most significant biological functions of annotated genes. (I) qRT-PCR confirmation of putative PAGR1-regulated genes in Pagr1^lox/lox MEFs. (J) ChIP assays of PAGR1 recruitment to target gene promoters in Vec- and Cre-infected Pagr1^lox/lox MEFs. All results are representative of two to four independent experiments. Quantitative PCR data in all figures are presented as means ± SD.

To identify PAGR1-regulated genes, we isolated and immortalized Pagr1^lox/lox mouse embryonic fibroblasts (MEFs). Cells were infected with retroviruses expressing Cre recombinase (Fig. 1E). Deletion of Pagr1 was confirmed by Western blotting and quantitative reverse transcriptase PCR (qRT-PCR) (Fig. 1F and I). KO of PAGR1 in MEFs did not affect cell growth (Fig. 1G). Microarray analysis using mouse whole-genome expression arrays revealed that deletion of Pagr1 led to a more than 2.5-fold decreased expression of 61 genes and increased expression of 3 genes (see Table S1 in the supplemental material). Gene ontology analysis of the downregulated genes identified Bcl3, Bmp4, Cebpb, Gap43, Igfbp5, Ntrk3, Sfrp1, and Col11a1 as the general cell differentiation genes and Cebpb, Cebpd, Klf4, and Krox20 as the fat cell differentiation genes (Fig. 1H). PAGR1-regulated genes identified from the microarray analysis were further confirmed by qRT-PCR. Specifically, deletion of Pagr1 in MEFs led to a more than 4-fold decreased expression of genes encoding the pioneering adipogenic TFs C/EBPβ and C/EBPδ (Fig. 1I). By chromatin immunoprecipitation (ChIP) assays in Vec- and Cre-infected Pagr1^lox/lox MEFs, we observed specific enrichment of endogenous PAGR1 protein on the promoters of Cebpb and Cebpd but not those of Pparg1 and Cebpa, which work in the later phase of adipogenesis (Fig. 1J). Taken together, these data indicate that PAGR1 directly controls the expression of early adipogenic TFs C/EBPβ and C/EBPδ in MEFs.

PAGR1 is required for adipogenesis in culture.

Since PAGR1 was required for expression of C/EBPβ and C/EBPδ in MEFs, we tested whether PAGR1 was required for adipogenesis. For this purpose, we isolated primary white and brown preadipocytes from Pagr1^lox/lox mice. Cells were infected with adenoviral Cre followed by adipogenesis assays. Cre-mediated Pagr1 deletion was confirmed by qRT-PCR (Fig. 2A and E). Deletion of Pagr1 had little effect on cell growth but led to severe adipogenesis defects in primary white and brown preadipocytes, as determined by oil red O staining (Fig. 2B, C, and F). Accordingly, induction of adipogenesis markers Pparg1, Pparg2, Cebpa, and Fabp4 was severely impaired in PAGR1 null white preadipocytes (Fig. 2D). Induction of adipogenesis markers and brown adipocyte markers was also inhibited by Pagr1 deletion in primary brown preadipocytes (Fig. 2G). Consistent with data obtained from primary preadipocytes, short hairpin RNA (shRNA)-mediated knockdown of Pagr1 inhibited adipogenesis in immortalized 3T3-L1 preadipocytes (Fig. 2H and I). Deletion of Pagr1 in immortalized brown preadipocytes also resulted in adipogenesis defects (Fig. 2J to L). These data demonstrate that PAGR1 is dispensable for cell growth but is required for adipogenesis in culture.

FIG 2.

PAGR1 is required for adipogenesis in culture. (A to G) Primary white (A to D) and brown (E to G) preadipocytes isolated from Pagr1^lox/lox mice were infected with adenoviruses expressing Cre or GFP, followed by adipogenesis assay. (A and E) qRT-PCR analysis of Cre-mediated Pagr1 gene deletion before adipogenesis. (B) Cell growth curves of GFP- and Cre-infected primary white preadipocytes. (C and F) Morphological differentiation with oil red O staining (top, stained dishes; lower, representative fields under phase-contrast microscope). (D) During differentiation of primary white preadipocytes, total RNA was isolated at indicated time points for qRT-PCR analysis of induction of adipocyte markers. (G) After differentiation of primary brown preadipocytes, total RNA was isolated for qRT-PCR analysis of expression of genes common to white and brown adipocytes (Pparg, Cebpa, and Fabp4), genes specific for brown adipocytes (Prdm16, Cidea, Eva1, and Ucp1), and mitochondrial components (Cox5b, Cox8b, and Cyt-c). All results are representative of two to four independent experiments. (H and I) 3T3-L1 preadipocytes were infected with lentiviral shRNA targeting PAGR1 (sh-Pagr1) or control virus alone (sh-Con), followed by adipogenesis assay. (H) qRT-PCR analysis of Pagr1 knockdown efficiency. (I) Oil red O staining of differentiated adipocytes. (J to L) SV40T-immortalized Pagr1^lox/lox brown preadipocytes were infected with retroviruses expressing Cre or Vec, followed by adipogenesis assay. (J) qRT-PCR analysis of Cre-mediated Pagr1 gene deletion before adipogenesis. (K) Cell growth curves of Vec- and Cre-infected immortalized brown preadipocytes. (L) Oil red O staining of differentiated adipocytes.

PAGR1 is required for induction of C/EBPβ and C/EBPδ during adipogenesis.

We examined expression patterns of adipogenic TFs during differentiation of primary white preadipocytes (Fig. 3A). Creb expression did not change during differentiation. Cebpb and Cebpd were immediately and transiently induced upon exposure to the adipogenic cocktail MDI, with the expression levels peaking at 4 h. Klf5 and Klf15, two downstream targets of C/EBPβ and C/EBPδ, were induced at 8 h. Pparg, Cebpa, and GR levels started to increase at day 1 and continued to increase throughout the differentiation. These results are highly consistent with what has been reported in the immortalized 3T3-L1 white preadipocyte cell line (2, 7, 13, 25).

FIG 3.

PAGR1 is required for induction of Cebpb and Cebpd during adipogenesis. (A) Expression of TFs implicated in adipogenesis during primary white preadipocyte differentiation. During adipogenesis of primary Pagr1^lox/lox white preadipocytes, total RNA was isolated at indicated time points and subjected to qRT-PCR analysis. The red lines indicate the time points at which each TF reaches its peak expression level. (B and C) PAGR1 controls the induction of Cebpb and Cebpd in the early phase of adipogenesis. Primary Pagr1^lox/lox white preadipocytes were infected with adenoviruses expressing Cre or GFP. Cells were replated and induced to undergo adipogenesis. Cells were collected at indicated time points for qRT-PCR (B) and ChIP (C) assays using PAGR1 antibody. (B) PAGR1 is required for induction of Cebpb and Cebpd but not Creb and Pparg in the early phase of adipogenesis (0 to 8 h). (C) Time course ChIP assays of endogenous PAGR1 enrichments on promoters of adipogenic TFs. (D) Pagr1 mRNA levels during primary white preadipocyte differentiation. (E) Western blot analysis of PAGR1 and PTIP in the early phase of adipogenesis of primary white preadipocytes. The nuclear protein RbBP5 served as a control. All results are representative of two to four independent experiments.

Consistent with the requirement of PAGR1 for Cebpb and Cebpd expression in MEFs, Pagr1 deletion by Cre in primary white preadipocytes prevented the robust induction of Cebpb and Cebpd but had no effect on Creb and Pparg levels in the early phase of differentiation (0 to 8 h) (Fig. 3B). Further, ChIP assays revealed that endogenous PAGR1 was recruited to the promoters of Cebpb and Cebpd in a manner that correlated with their expression patterns in the early phase of adipogenesis (Fig. 3C). In contrast, no enrichment of PAGR1 was detected on Creb and Pparg1 promoters. PAGR1 mRNA and protein levels remained unchanged in the early phase of adipogenesis (Fig. 3D and E), suggesting that PAGR1 controls its target gene expression through its binding to specific genomic regions. Together, these results indicate that PAGR1 directly controls the induction of Cebpb and Cebpd in the early phase of adipogenesis.

PAGR1 regulates CREB- and GR-dependent induction of C/EBPβ and C/EBPδ in culture.

Next, we asked which TFs recruit PAGR1 to the promoters of Cebpb and Cebpd in the early phase of adipogenesis, since PAGR1 itself does not contain any recognizable DNA-binding domain. Because induction of Cebpb depends on IBMX while induction of Cebpd requires DEX (8), we focused on CREB and GR, which are activated by IBMX and DEX, respectively, and are already expressed before the induction of adipogenesis.

Phospho-CREB (pCREB) directly binds to the Cebpb proximal promoter after induction of adipogenesis and activates endogenous Cebpb expression (15). By time course ChIP assays, we confirmed the enrichment of pCREB on the Cebpb promoter in the early phase of primary white preadipocyte differentiation. pCREB enrichment on the Cebpb promoter could be inhibited by adenovirus-mediated overexpression of a dominant-negative CREB (A-CREB) (Fig. 4A and B). A-CREB impaired the induction of Cebpb (Fig. 4C), indicating that pCREB directly controls Cebpb induction in the early phase of adipogenesis. Interestingly, A-CREB also blocked PAGR1 enrichment on the Cebpb promoter (Fig. 4D). Conversely and surprisingly, deletion of Pagr1 inhibited pCREB binding to the Cebpb promoter (Fig. 4E), suggesting an interdependent binding of pCREB and PAGR1 on the Cebpb promoter. Deletion of Pagr1 did not affect pCREB levels induced by the adipogenic cocktail MDI (Fig. 4F). To directly assess the role of PAGR1 in CREB-mediated Cebpb induction, primary white preadipocytes and simian virus 40T (SV40T)-immortalized brown preadipocytes were treated with forskolin, which induces phosphorylation and activation of endogenous CREB (14). Deletion of Pagr1 impaired the induction of Cebpb by activated CREB (Fig. 4G and H). Together, these results suggest that pCREB and PAGR1 cooperate to induce Cebpb expression during adipogenesis.

FIG 4.

pCREB and PAGR1 cooperate to induce Cebpb expression in culture. (A to C) Phospho-CREB (pCREB) directly controls the induction of Cebpb in the early phase of adipogenesis. Primary Pagr1^lox/lox white preadipocytes were infected with adenoviruses expressing the dominant-negative CREB (A-CREB) or GFP, followed by induction of adipogenesis. Cells were collected at the indicated time points for ChIP assays and qRT-PCR. (A) Expression of HA-tagged A-CREB before adipogenesis was confirmed by Western blotting with an HA antibody. (B) ChIP assays using a pCREB antibody. (C) qRT-PCR analysis of Cebpb and Cebpd. (D) pCREB recruits PAGR1 to Cebpb promoter in the early phase of adipogenesis. (E) PAGR1 is required for stable enrichment of pCREB on Cebpb promoter in the early phase of adipogenesis. Primary Pagr1^lox/lox white preadipocytes were infected with adenoviruses expressing Cre or GFP, followed by induction of adipogenesis. Samples were collected at indicated time points for ChIP assays using a pCREB antibody. (F) Deletion of Pagr1 does not affect overall pCREB levels induced by adipogenic cocktail MDI. (G and H) PAGR1 is required for Cebpb induction by activated CREB. (G) Primary Pagr1^lox/lox white preadipocytes infected with adenoviruses expressing Cre or GFP were treated with 10 μM forskolin (FSK) for 1 h and collected for qRT-PCR analysis. (H) Immortalized Pagr1^lox/lox; CreER brown preadipocytes were treated with 2 μM 4-OHT for 2 days to delete Pagr1. Cells were then treated with 10 μM forskolin (FSK) or MDI for 1 h and collected for qRT-PCR analysis.

By ChIP assays, we found that GR was enriched on Cebpb and Cebpd promoters at around 2 h after induction of adipogenesis in primary white preadipocytes. Deletion of Pagr1 impaired GR binding to the promoter of Cebpd but not that of Cebpb (Fig. 5A). In contrast, knockdown of GR in 3T3-L1 white preadipocytes did not affect PAGR1 recruitment (Fig. 5B). GR knockdown in 3T3-L1 cells impaired the induction of Cebpd but not Cebpb in the early phase of adipogenesis (Fig. 5C). Similarly, knockdown of GR inhibited DEX-induced Cebpd expression in 3T3-L1 (Fig. 5D). Deletion of Pagr1 in SV40T-immortalized brown preadipocytes blocked DEX-induced Cebpd expression, indicating that PAGR1 is required for GR-mediated induction of Cebpd (Fig. 5E). Taken together, these data suggest a model in which PAGR1 cooperates with IBMX-activated pCREB and DEX-activated GR to control the induction of Cebpb and Cebpd, respectively, in the early phase of adipogenesis (Fig. 5F).

FIG 5.

PAGR1 regulates GR-dependent induction of Cebpd in culture. (A) PAGR1 is required for GR enrichment on Cebpd promoter. Experiments were done as described for Fig. 4E. Samples were collected at the indicated time points for ChIP assays using a GR antibody. (B and C) Knockdown of GR inhibits the induction of Cebpd but has little effect on PAGR1 recruitment on Cebpd promoter in the early phase of adipogenesis. 3T3-L1 cells were infected with lentiviral shRNA targeting GR (sh-GR) or control virus alone (sh-Con), followed by induction of adipogenesis. Cells were collected at indicated time points for ChIP assays using a PAGR1 antibody (B) and qRT-PCR (C). All results are representative of two to four independent experiments. (D) Knockdown of GR inhibits DEX-induced Cebpd expression. 3T3-L1 cells infected with lentiviral sh-GR or sh-Con were treated with 1 μM DEX for 1 h and collected for qRT-PCR analysis. (E) PAGR1 is required for Cebpd induction by activated GR. Immortalized Pagr1^lox/lox; CreER brown preadipocytes were treated with 2 μM 4-OHT for 2 days to delete Pagr1. Cells were treated with 1 μM DEX for 1 h and collected for qRT-PCR analysis. (F) Model depicting the role of PAGR1 in transcriptional regulation of adipogenesis in culture. PAGR1 regulates adipogenesis by cooperating with IBMX-induced pCREB and DEX-activated GR to control the induction of C/EBPβ and C/EBPδ in the early phase of adipogenesis.

Ectopic C/EBPβ or PPARγ can rescue adipogenesis defect in PAGR1-deficient cells.

Since PAGR1 is required for Cebpb gene induction in the early stage of adipogenesis, we tested if the adipogenesis defects in PAGR1-deficient cells could be rescued by forced expression of C/EBPβ or PPARγ. We isolated and immortalized primary brown preadipocytes from inducible PAGR1 KO mice (Pagr1^lox/lox; CreER). Cells were treated with 4-hydroxytamoxifen (4-OHT) to delete Pagr1, followed by retroviral vector-mediated ectopic expression of C/EBPβ or PPARγ (Fig. 6A). Consistent with the data obtained from viral Cre-infected PAGR1 null cells, 4-OHT-induced deletion of Pagr1 resulted in adipogenesis defects. Such defects can be rescued by ectopic expression of C/EBPβ or PPARγ (Fig. 6B and C), suggesting that PAGR1 functions upstream of C/EBPβ during adipogenesis.

FIG 6.

Ectopic C/EBPβ or PPARγ can rescue adipogenesis defect in PAGR1-deficient cells. Immortalized Pagr1^lox/lox; CreER brown preadipocytes were treated with 2 μM 4-OHT for 2 days to delete Pagr1 and then infected with retroviruses expressing C/EBPβ or PPARγ, followed by adipogenesis assay. (A) Western blot analyses of PAGR1 deletion and ectopic expression of C/EBPβ and PPARγ before adipogenesis. (B) Oil red O staining. (C) qRT-PCR analysis of Pagr1, Pparg, Fabp4, and Ucp1.

PAGR1 is essential for brown adipose tissue and muscle development in mice.

To investigate PAGR1 function in adipogenesis in vivo, we crossed Pagr1^lox/lox mice with Myf5-Cre mice, in which Cre expression is under the control of the Myf5 promoter (26). Myf5-Cre is active in precursor cells that can develop into brown adipocytes and skeletal muscle cells in the back (27). The KO mice died within minutes after birth due to breathing defects caused by reduced muscles in the rib cage. Therefore, we removed E18.5 embryos by cesarean section. KO embryos exhibited abnormal cervical curvature and died instantly after dissection (Fig. 7A and B). Sagittal sections along the midline or ∼400 μm lateral to the midline of E18.5 embryos were used for hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) using antibodies specifically recognizing brown adipose tissue (BAT)-specific UCP1 and muscle-specific myosin (Fig. 7C and D). Compared with their littermate controls, KO mice showed a dramatic decrease of interscapular BAT and muscle. These data are consistent with the essential role for PAGR1 in adipogenesis in culture and indicate that PAGR1 is required for adipogenesis in vivo.

FIG 7.

PAGR1 is required for brown adipose tissue and muscle development. (A) Genotype of E18.5 embryos from crossing Pagr1^lox/+; Myf5-Cre with Pagr1^lox/lox mice. The expected ratios of the four resulting genotypes are 1:1:1:1. (B) Representative pictures of control (Con) and PAGR1 KO (Pagr1^lox/lox; Myf5-Cre) E18.5 embryos. KO embryos died instantly after cesarean section because of breathing defects. (C and D) Loss of interscapular brown adipose tissue (BAT) in E18.5 PAGR1 KO embryos. E18.5 embryos were sagitally sectioned along the midline (C) or ∼400 μm lateral to the midline (D). The sections were subjected to H&E staining (top) as well as IHC using antibodies recognizing BAT-specific UCP1 protein (green) and skeletal muscle-specific myosin (red) (bottom). SP indicates spine. B indicates BAT. Ventral is on the left side, dorsal is on the right side. Scale bar, 800 μm.

DISCUSSION

Early adipogenic TFs C/EBPβ and C/EBPδ are induced within hours after initiating adipogenesis and promote expression of the master adipogenic TFs PPARγ and C/EBPα (25). We previously showed that PTIP and MLL3/MLL4 control the induction of PPARγ and C/EBPα during adipogenesis (19, 20). In this paper, we report that the MLL3/MLL4/PTIP-associated PAGR1 protein controls the induction of C/EBPβ and C/EBPδ and, thus, is required for adipogenesis. Together, components of the MLL3/MLL4 complex regulate a key transcriptional cascade that governs adipogenesis.

Regulation of gene expression by PAGR1.

Deletion of Ptip in MEFs leads to a more than 2.5-fold decrease of 60 genes, including Pparg. PTIP directly controls Pparg and Cebpa expression in MEFs as well as during adipogenesis (19). On the other hand, Pagr1 deletion in MEFs led to a more than 2.5-fold decrease of 61 genes, including Cebpb, Cebpd, Klf4, and Krox20 (see Table S1 in the supplemental material), which were listed as fat cell differentiation genes in the gene ontology analysis. Because KLF4 and Krox20 have been shown to be dispensable for adipogenesis (28), we focused on PAGR1 regulation of C/EBPβ and C/EBPδ expression. Although Pparg showed an ∼2-fold decrease in PAGR1 null MEFs, PAGR1 is undetectable on the Pparg promoter in wild-type MEFs, suggesting the decreased Pparg expression in PAGR1 null MEFs is a secondary effect. Despite a direct physical interaction between them, PTIP and PAGR1 shared only 10 overlapping target genes in MEFs. These include Adamts2, Ahr, Ak5, Bmp4, Col11a1, Fbn2, Fmod3, Inhbb, Klf4, and Tnfrsf11b. Collectively, these results suggest that while PAGR1 and PTIP directly regulate several common target genes, they regulate adipogenesis by controlling the expression of distinct sets of adipogenic TFs. Despite the physical association of PAGR1 with the MLL3/MLL4-containing histone H3K4 methyltransferase complex (21), our data suggest that PAGR1 regulation of some of its target genes is independent of the MLL3/MLL4 complex. Indeed, induction of C/EBPβ is not altered in Mll3^−/−Mll4^−/− brown preadipocytes during adipogenesis (20).

PAGR1 cooperates with pCREB and GR to induce early adipogenic transcription factors.

Although PAGR1 protein levels show little change in the early phase of adipogenesis, PAGR1 is recruited to Cebpb and Cebpd promoters in a manner that highly correlates with the transient induction of these early adipogenic TFs by adipogenic cocktail. IBMX induces phosphorylation and activation of CREB while DEX binds and activates GR (14, 16). IBMX induces Cebpb expression while DEX induces Cebpd (8). Consistent with these previous reports, we show that PAGR1 cooperates with pCREB and GR to induce Cebpb and Cebpd in the early phase of adipogenesis.

CREB family members associate with the Cebpb promoter even before induction of adipogenesis, whereas pCREB is detected only after induction (15). Therefore, CREB may already sit on target gene promoters but IBMX-induced CREB phosphorylation triggers the recruitment of PAGR1 to the Cebpb promoter. PAGR1 recruitment may stabilize CREB complex assembly, because pCREB enrichment on the Cebpb promoter is severely impaired in PAGR1 null cells.

Our data suggest an updated model on transcriptional regulation of adipogenesis. After preadipocytes are treated with adipogenic cocktail MDI, IBMX induces phosphorylation and activation of CREB while DEX binds and activates GR. PAGR1 cooperates with pCREB and GR to directly induce Cebpb and Cebpd expression in the early phase of adipogenesis. The elevated levels of these early adipogenic TFs are critical for the induction of the master adipogenic TFs PPARγ and C/EBPα, which directly activate adipocyte gene expression and lead to phenotypic conversion of preadipocytes to adipocytes (Fig. 5F). Interestingly, physically interacting PAGR1 and PTIP control the induction of distinct sets of adipogenic TFs during adipogenesis. Together, PAGR1 and PTIP control the induction of the cascade of adipogenic TFs that governs adipogenesis.

Further studies are needed to understand the role of PAGR1 in undifferentiated preadipocytes. We cannot rule out the possibility that before induction of adipogenesis, PAGR1 interacts with preadipocyte TFs, such as ZFP423 (29), TCF7L1 (30), KAISO (31), and ATF4 (32), to regulate the expression of genes involved in maintaining an undifferentiated state or committing cells to the preadipogenic state. This possibility is supported by the fact that in MEFs, PAGR1 recruitment to its target genes occurs in the absence of an adipogenic cocktail and does not require activated CREB or GR.

Essential role of PAGR1 in adipose tissue development in vivo.

PAGR1 is required for IBMX- and DEX-induced expression of early adipogenic TFs and is essential for adipogenesis of both white and brown preadipocytes in culture. However, IBMX and DEX are synthetic chemicals that do not exist in vivo. To investigate the physiological importance of PAGR1 in adipose tissue development, we utilized Myf5-Cre mice. Myf5-expressing progenitor cells give rise to both skeletal muscle and BAT but not WAT (27). Myf5-Cre starts to be expressed at early embryonic stages (E10.5 or earlier), which is much earlier than the initial appearance of BAT (around E14.5 to E15.5) (33, 34). Therefore, Myf5-Cre mice enable us to study the roles of PAGR1 in the early stage of BAT development. We show that the deletion of Pagr1 leads to severe defects in BAT development. Myf5-Cre-mediated deletion of Ptip also leads to severe defects in BAT development, confirming the critical role of PTIP in adipogenesis in vivo (data not shown). Together, our animal data are highly consistent with the cell culture results and strongly suggest that, by controlling the induction of C/EBPβ and C/EBPδ, PAGR1 is essential for adipogenesis in vivo.

In MEFs, PAGR1 also controls the expression of BMP4, a cytokine that commits the multipotent mesenchymal cells to the adipocyte lineage (35). It will be interesting to determine the roles of PAGR1 in earlier developmental events, in particular the lineage commitment of mesenchymal stem cells. Future work will be needed to fully understand the molecular mechanism by which PAGR1 regulates induction of C/EBPβ and C/EBPδ as well as other target genes.

PAGR1 as a developmental regulator.

The facts that Pagr1^−/− mice are embryonic lethal and that Myf5-Cre-driven deletion of Pagr1 reduces BAT and muscle suggest that PAGR1 is important for both embryonic and tissue development. Pagr1^−/− mice die before embryonic day 10.5 due to the abnormal development of extraembryonic tissues, including amnion, chorion, and visceral yolk sac (24). At the molecular level, Pagr1^−/− embryos show reduced expression of BMP2, which is critical for extraembryonic development. While Cebpb^−/−Cebpd^−/− mice do not exhibit embryonic death, approximately 85% of them die at the early neonatal stage (10). The impaired expression of other PAGR1-regulated genes may also contribute to the embryonic lethality of Pagr1^−/− mice. For instance, whole-body KO of Bmp4, a PAGR1-regulated gene in MEFs, leads to early embryonic lethality due to defective mesoderm formation and patterning (36). Targeted deletion of many PAGR1-regulated genes, such as Klf4 (37) or Krox20 (38), also leads to defects in the postnatal development of various tissues and causes neonatal death. It remains to be investigated whether PAGR1 is required for development of other tissues.

MATERIALS AND METHODS

Generation of Pagr1^lox/lox mice.

PAGR1 conditional KO mice, Pagr1^lox/lox mice, were generated by following the published protocol (39). The mouse Pagr1 gene, also known as 2900092E17Rik, contains 3 exons. The targeting vector introduced a loxP site into the EcolCRI restriction enzyme site upstream of exon 1 and a neomycin cassette flanked by two loxP sites into the BglII site in intron 2 to generate the targeted allele (loxneo) in embryonic stem (ES) cells (Fig. 1A). Among the 160 ES cell clones that we screened using the Expand Long Template PCR system (Roche), clone 138 was identified as a Pagr1^loxneo/+ clone carrying a targeted allele and a wild-type allele. The correct targeting in clone 138 was confirmed by Southern blotting using a 5′ flanking probe generated by PCR amplification of the NotI-EcoRV genomic DNA fragment (Fig. 1B).

Pagr1^loxneo/+ ES cell clone 138 was injected into blastocysts to obtain male chimera mice, which were crossed with wild-type C57BL/6J females to screen for germ line transmission. Mice bearing germ line transmission (Pagr1^loxneo/+) were crossed with EIIa-Cre mice (no. 003724; The Jackson Laboratory) to generate both Pagr1^lox/+ and Pagr1^+/− mice by following a procedure described earlier (Fig. 1C) (40). Pagr1^lox/+ mice were crossed with C57BL/6J mice for 7 generations to obtain congenic mice. Pagr1^lox/lox mice were obtained by intercrossing between Pagr1^lox/+ mice. For genotyping the lox allele, allelic PCR was developed using forward primer 5′-TGGCCCAAACCTAAACATTAG-3′ and reverse primer 5′-TTATGGCGCTTCATGTCTGAG-3′. PCR amplified 200 bp from the wild-type allele and 250 bp from the lox allele (Fig. 1D).

Plasmids, antibodies, and chemicals.

The retroviral plasmids MSCVpuro-Cre, MSCVhygro-PPARγ2, and WZLhyg-C/EBPβ have been described already (20, 41). The lentiviral shRNA plasmid pLKO.1 targeting Pagr1 (clone ID TRCN0000177775) was purchased from Sigma. The following antibodies have been described: anti-PTIP#3, anti-PAGR1#2, anti-GAPDH, anti-p85α, anti-RbBP5, and all histone modification antibodies (19, 42). Anti-phospho-CREB (Ser133) (D1G6) was from Cell Signaling Technology. Anti-GR (sc-1004x) was from Santa Cruz Biotechnology. Rosiglitazone was from Cayman (71740). Other chemicals were from Sigma: IBMX (I-7018), DEX (D4902), T3 (T-2877), insulin (I-6634), indomethacin (I-7378), and forskolin (F3917).

Cell culture, adipogenesis, and virus infection.

Primary MEFs were isolated from two E13.5 Pagr1^lox/lox embryos. One was immortalized by transfecting cells with a SV40T-expressing plasmid, and the other was immortalized by following the 3T3 protocol (43). The isolation, culture, and adipogenesis of primary white and brown preadipocytes, the immortalization of brown preadipocytes, and the retroviral infection of the immortalized MEFs were done as described previously (19). Briefly, the adipogenesis of primary white preadipocytes was induced with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 0.5 mM IBMX, 1 μM DEX, 5 μg/ml insulin, 0.2 nM T3, and 0.5 μM rosiglitazone. For adipogenesis of primary and immortalized brown preadipocytes, cells were induced with DMEM supplemented with 10% FBS, 0.02 μM insulin, 1 nM T3, 0.5 mM IBMX, 2 μg/ml DEX, and 0.125 mM indomethacin. Adenovirus expressing the dominant-negative CREB (A-CREB) was described previously (44). Adenoviral infection of preadipocytes was done at a multiplicity of infection of 50 (19). 4-OHT treatment was done at 2 μM concentration for 2 days.

Microarray, gene ontology analysis, qRT-PCR, and ChIP.

Total RNA extraction, microarray analysis on the Mouse Genome 430 2.0 array (Affymetrix, Santa Clara, CA), and qRT-PCR were done as described previously (19). Gene ontology analysis of downregulated genes in PAGR1-deficient MEFs was done using the FunNet bioinformatics tool (www.funnet.info) (45). A total of 61 genes with a more than 2.5-fold decrease in PAGR1-deficient cells were subjected to gene ontology analysis. Of the 61 downregulated genes, the 51 annotated genes were analyzed with a 5% false discovery rate (FDR). qRT-PCR primer sequences are shown in Table S2 in the supplemental material. TaqMan probes for Pparg1 and Pparg2 have been described already (19).

ChIP was performed as described previously (46). The quantitation of enriched genomic DNA relative to inputs was performed in duplicate or triplicate using SYBR green master mix (Applied Biosystems). Primer sequences are listed in Table S3.

Generation of tissue-specific PAGR1 KO mice.

The Myf5-Cre mice with a mixed C57BL/6J and 129 Sv background (no. 007893) were obtained from The Jackson Laboratory. Pagr1^lox/lox mice with congenic C57BL/6J background were crossed with Myf5-Cre mice. The resulting heterozygous mice (Pagr1^lox/+; Cre) in F1 were then crossed with Pagr1^lox/lox mice to generate tissue-specific PAGR1 KO mice (Pagr1^lox/lox; Myf5-Cre). Pagr1^lox/lox mice were used as the control. Genotyping of Myf5-Cre was done by PCR using 3 primers: 5′-CGTAGACGCCTGAAGAAGGTCAACCA-3′, 5′-CACATTAGAAAACCTGCCAACACC-3′, and 5′-ACGAAGTTATTAGGTCCCTCGAC-3′. PCR amplified the wild-type (603 bp) and Myf5-Cre (400 bp) allele. All mouse work was approved by the Animal Care and Use Committee of NIDDK, NIH.

Histology and IHC.

E18.5 embryos were removed by cesarean section and fixed in 4% paraformaldehyde overnight at 4°C. Embryos were further dehydrated, embedded in paraffin, and sectioned at 7 to 10 μm with a microtome. Mouse inguinal adipose tissues were fixed in 10% formalin, embedded in paraffin, and sectioned at 5 μm with a microtome. H&E staining and immunohistochemistry on paraffin sections were done as described previously (47). The primary antibodies used for IHC were a 1:20 dilution of antimyosin (MF20; Developmental Studies Hybridoma Bank) and a 1:400 dilution of anti-UCP1 (ab10983; Abcam). Fluorescent secondary antibodies used were Alexa Fluor 488–goat anti-mouse IgG2b and Alexa Fluor 555–goat anti-rabbit IgG (Molecular Probes).

Data availability.

Microarray data have been deposited in the NCBI GEO database (accession number GSE20157).