Regulation of Early Adipose Commitment by Zfp521

While there has been significant progress in determining the transcriptional cascade involved in terminal adipocyte differentiation, less is known about early events leading to lineage commitment and cell fate choice. It has been recently discovered that zinc finger protein 423 (Zfp423) is an early actor in adipose determination. Here, we show that a close paralog of Zfp423, Zfp521, acts as a key regulator of adipose commitment and differentiation in vitro and in vivo. Zfp521 exerts its actions by binding to early B cell factor 1 (Ebf1), a transcription factor required for the generation of adipocyte progenitors, and inhibiting the expression of Zfp423. Overexpression of Zfp521 in cells greatly inhibits adipogenic potential, whereas RNAi-mediated knock-down or genetic ablation of Zfp521 enhances differentiation. In addition, $Zfp521^{−/−}$ embryos exhibit increased mass of interscapular brown adipose tissue and subcutaneous white adipocytes, a cell autonomous effect. Finally, Ebf1 participates in a negative feedback loop to repress Zfp521 as differentiation proceeds. Because Zfp521 is known to promote bone development, our results suggest that it acts as a critical switch in the commitment decision between the adipogenic and osteogenic lineages

A critical role for Ebf1 and Ebf2 in the adipogenic transcriptional cascade.

The Ebf (O/E) family of helix-loop-helix transcription factors plays a significant role in B lymphocyte and neuronal development. The three primary members of this family, Ebf1, 2, and 3, are all expressed in adipocytes, and Ebf1 promotes adipogenesis when overexpressed in NIH 3T3 fibroblasts. Here we report that these three proteins have adipogenic potential in multiple cellular models and that peroxisome proliferator-activated receptor gamma (PPAR gamma) is required for this effect, at least in part due to direct activation of the PPAR gamma 1 promoter by Ebf1. Ebf1 also directly binds to and activates the C/EBP alpha promoter, which exerts positive feedback on C/EBP delta expression. Despite this, C/EBP alpha is dispensable for the adipogenic action of Ebf proteins. Ebf1 itself is induced by C/EBP beta and delta, which bind and activate its promoter. Reduction of Ebf1 and Ebf2 proteins by specific short hairpin RNA blocks differentiation of 3T3-L1 cells, suggesting a critical role for these factors and the absence of functional redundancy between members of this family. Altogether, these data place Ebf1 within the known transcriptional cascade of adipogenesis and suggest critical roles for Ebf1 and Ebf2

Joining chain-expressing and -nonexpressing B cell populations in the mouse

The diphtheria toxin A chain (DTA) was gene targeted into the Joining chain (J chain) locus to create a mouse strain selecting against J chain-expressing cells, JDTA mice. Serum immunoglobulin (Ig)M and serum IgG were reduced six to eightfold, while serum IgA was elevated 14-fold in these mice. JDTA mice were immune competent although the serum Ig response compared with wild-type mice was reduced sixfold at day 14 but only fourfold at day 45 after immunization. Exchanging the DTA gene with a cDNA for c-myc resulted in mice with a distinct phenotype with increased Ig production and enhanced humoral immune responses. Analysis of single B cells stimulated by lipopolysaccharide in vitro using reverse transcription-polymerase chain reaction showed that J chain-nonexpressing B cells could be detected that had a secretory phenotype as determined by an abundance of transcript for secretory IgM. Finally, limiting dilution analysis of peripheral B cells showed that J chain expression was a clonal property already established in naive, peripheral B lymphocytes

Serum disposition of sertraline, N-desmethylsertraline and paroxetine: A pharmacokinetic evaluation of repeated drug concentration measurements during 6 months of treatment for major depression

Sertraline and paroxetine are frequently prescribed SSRIs for long-term treatment of major depression. Nevertheless, continuous follow-ups of drug concentrations prevailing in patients during the whole treatment period are not available. Hence, in a large phase IV clinical trial, a total of 353 patients with major depression were enrolled for a 6-month comparison of sertraline (50-150 mg daily) and paroxetine (20-60 mg daily). The present study reports the pharmacokinetic results of up to eight serum samples per patient. 1. A profound variability was found in the interindividual steady state and trough serum levels of sertraline, desmethylsertraline and paroxetine: the coefficient of variation (CV) was 59% for sertraline, 51% for desmethylsertraline, 27% for the ratio desmethylsertraline/sertraline (50 mg/day), and 71% for paroxetine (20 mg/day). The intraindividual CV for the ratio desmethylsertraline/sertraline was only 19%, indicating intraindividual metabolizing stability over time. Both sertraline and paroxetine displayed sex differences in the dose-concentration correlation. 2. It was possible to predict sertraline, but not paroxetine, steady state levels. 3. The terminal elimination t1/2 for both drugs after 6 months of treatments was similar to data previously reported from short-term withdrawal studies. 4. No correlation between serum drug concentrations and clinical effect was detected for either sertraline or paroxetine. For the future, continuous efforts are warranted to perform PK investigations in the natural clinical setting in which the drugs are usually prescribed

The CXCL12, periostin and CCL9 genes are direct targets for early B-cell factor (EBF) in OP-9 stroma cells.

The development of blood cells from hematopoietic stem cells in the bone marrow is dependent on communication with bone marrow stroma cells, making these cells central for the appropriate regulation of hematopoiesis. To identify transcription factors that may play a role in gene regulation in stroma cells, we performed comparative gene expression analysis of fibroblastic NIH3T3 cells, unable to support hematopoiesis in vitro, and OP-9 stroma cells, highly efficient in this regard. These experiments revealed that transcription factors of the early B cell factor (EBF) family were highly expressed in OP-9 cells as compared with the NIH3T3 cells. To identify potential targets genes for EBF proteins in stroma cells, we overexpressed EBF in fibroblasts and analyzed the pattern of induced genes by microarray analysis. This revealed that EBF was able to up-regulate expression of among others the Cxcl12, Ccl9, and Periostin genes. The identification of relevant promoters revealed that they all contained functional EBF binding sites able to interact with EBF in OP-9 cells. Furthermore, ectopic expression of a dominant negative EBF protein or antisense EBF-1 RNA in OP-9 stroma cells resulted in reduced expression of these target genes. These data suggest that EBF proteins might have dual roles in hematopoiesis acting both as intrinsic regulators of B-lymphopoiesis and as regulators of genes in bone marrow stroma cells

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 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

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