Ebf1 suppresses <i>Zfp521</i> expression via an intronic binding site.

<p>(A) ChIP-Seq tracks corresponding to the Zfp521 locus are shown for three histone marks: H3K4me3 (green), H3K27Ac (black), and H3K36me3 (blue) at four time-points during 3T3-L1 adipogenesis. A cluster of Ebf1 peaks highlighted in the red box contains three putative Ebf1-responsive elements (EBF-RE). (B) ChIP-PCR analysis was performed with antibody against IgG or Ebf1 in 3T3-L1 preadipocytes. Immunoprecipitated DNA was amplified with Q-PCR using primers designed for the three putative EBF-REs shown in (A). (C) Zfp521 mRNA expression was measured in 3T3-L1 preadipocytes transduced with shScr or shEbf1. (D) Zfp521 mRNA expression was measured in confluent <i>Ebf1^+/+</i> and <i>Ebf1^−/−</i> MEFs. (E) Ebf1 and Zfp521 mRNA expression was measured in <i>Ebf1^+/+</i> and <i>Ebf1^−/−</i> MEFs transduced with Ebf1 or empty vector. Data presented as mean ± SD, <i>n</i> = 3, *<i>p</i><0.05. (F) A proposed model for the transcriptional cascade involving Zfp521, Ebf1, and Zfp423.</p

Zfp521 is a suppressor of adipogenesis in vitro.

<p>(A) C3H10T1/2 cells were differentiated and RNA isolated at the indicated time points. Gene expression of <i>Zfp521</i> and <i>Pparg</i> was measured by Q-PCR and normalized to cyclophilin. Data shown as mean of three biological replicates. (B) Protein lysates isolated during 3T3-L1 adipogenesis were subjected to western blotting with anti-Zfp521 antibody. (C, left) Zfp521 mRNA expression was measured in fractionated subcutaneous and epididymal fat tissue taken from wild-type mice (SV, stromal-vascular fraction; AD, adipocytes). (C, right) SV of epididymal fat tissue from Zfp423^GFP transgenic mice was sorted with GFP antibody and plated. After washing away floating cells, Zfp521 mRNA expression was measured in GFP− and GFP+ cells. (D–G) C3H10T1/2 cells were retrovirally transduced with Zfp521, empty vector, shRNA specific forZfp521 (sh521), or a scrambled hairpin (Scr). Overexpression and knock-down were confirmed by immunoblotting of Zfp521 prior to differentiation in the boxed insert. Cells were differentiated with DMI or DMI plus rosiglitazone (DMIR) and stained with oil red-O (D, F) and adipocyte markers were determined by Q-PCR (E, G) on day 8. Data presented as mean ± SD, <i>n</i> = 3, *<i>p</i><0.05.</p

Zfp521 suppresses Zfp423 expression.

<p>(A) 3T3-L1 preadipocytes were transduced with retrovirus expressing sh521, shScr, Zfp521, or empty vector. After puromycin selection, RNA was collected and submitted for analysis using Affymetrix arrays. The Venn diagram shows the number of genes up-regulated by sh521 (sh521/shScr>1.5-fold) and down-regulated by Zfp521 (Zfp521/pMSCV<0.7-fold). The heat map corresponds to genes in the intersecting set. (B) Overexpression of Zfp521 in C3H10T1/2 cells represses Zfp423 expression by Q-PCR. (C) Knockdown of Zfp521 in C3H10T1/2 cells enhances Zfp423 expression by Q-PCR. Data presented as mean ± SD, <i>n</i> = 3, *<i>p</i><0.05.</p

Zfp521 inhibits Ebf1 transcriptional activity through physical interaction.

<p>(A) 3T3-L1 preadipocytes were harvested and endogenous Ebf1 was immunoprecipitated using anti-Ebf1 beads and blotted with normal goat-IgG or anti-Zfp521. (B–D) 3T3-L1 preadipocytes were co-transfected with vectors expressing Flag-Zfp521, Myc-Ebf1, and various reporter plasmids containing the <i>mb1</i>-promoter (B), <i>Sncg</i>-promoter (C), or <i>Cebpa</i>-promoter (D). At 24 h after transfection, luciferase activity was normalized to β-galactosidase activity. Data presented as mean ± SD, <i>n</i> = 4, *<i>p</i><0.05. (E) 3T3-L1 preadipocytes were stably transduced with Flag-Ebf1 or Flag-Zfp521. ChIP assay was performed on 3T3-L1 cells that were treated with DMI for 1 h using anti-Flag antibody or an IgG control using PCR primers directed at regions of the Cebpa containing the putative Ebf sites. (F, G) Immortalized <i>Zfp521^+/+</i> and <i>Zfp521^−/−</i> MEFs were transduced with a retrovirus expressing Ebf1, Zfp521, or empty vector and differentiated prior to staining with oil red-O after 8 d (F) and adipocyte gene expression was measured by Q-PCR (G). (H, I) C3H10T1/2 cells were transduced with a retrovirus expressing Zfp521WT, Zfp521ΔZF27-30, Zfp521Δ13aa, or empty pMSCV vector. Cells were differentiated with DMIR and stained with oil red-O and gene expression was measured on day 6. (J) Zfp521 and Ebf1 were expressed in C3H10T1/2 cells alone or in combination, and expression of Zfp521, Ebf1, and Zfp423 was determined by Q-PCR. (K) Zfp521, Zfp521Δ27-30, or pMSCV was expressed in C3H10T1/2 cells and expression of Zfp521, Ebf1, and Zfp423 was determined by Q-PCR. Data presented as mean ± SD, <i>n</i> = 3, *<i>p</i><0.05.</p