Zfp423 Expression Research Articles

Thyroid hormones regulate Zfp423 expression in regionally distinct adipose depots through direct and cell-autonomous action.

The hypothalamic pituitary thyroid axis is a major regulator of many differentiation processes, including adipose tissue. However, it remains unclear whether and how thyroid hormone (TH) signaling contributes to preadipocyte commitment and differentiation into mature adipocytes. Here, we show a cell-autonomous effect of TH on the transcriptional regulation of zinc finger protein 423 (Zfp423), an early adipogenic determination factor, in murine adipose depots. Mechanistically, binding of the unliganded TH receptor to a negative TH responsive element within the Zfp423 promoter activates transcriptional activity that is reversed upon TH binding. Zfp423 upregulation is associated with increased GFP+ preadipocyte recruitment in stromal vascular fraction isolated from white fat of hypothyroid Zfp423GFP reporter mice. RNA sequencing identified Zfp423-driven gene programs that are modulated in response to TH during adipogenic differentiation. Collectively, we identified Zfp423 as a key molecule that integrates TH signaling into the regulation of adipose tissue plasticity.

Increased prostaglandin-D2 in male STAT3-deficient hearts shifts cardiac progenitor cells from endothelial to white adipocyte differentiation.

Cardiac levels of the signal transducer and activator of transcription factor-3 (STAT3) decline with age, and male but not female mice with a cardiomyocyte-specific STAT3 deficiency conditional knockout (CKO) display premature age-related heart failure associated with reduced cardiac capillary density. In the present study, isolated male and female CKO-cardiomyocytes exhibit increased prostaglandin (PG)-generating cyclooxygenase-2 (COX-2) expression. The PG-degrading hydroxyprostaglandin-dehydrogenase-15 (HPGD) expression is only reduced in male cardiomyocytes, which is associated with increased prostaglandin D2 (PGD2) secretion from isolated male but not female CKO-cardiomyocytes. Reduced HPGD expression in male cardiomyocytes derive from impaired androgen receptor (AR)-signaling due to loss of its cofactor STAT3. Elevated PGD2 secretion in males is associated with increased white adipocyte accumulation in aged male but not female hearts. Adipocyte differentiation is enhanced in isolated stem cell antigen-1 (SCA-1)+ cardiac progenitor cells (CPC) from young male CKO-mice compared with the adipocyte differentiation of male wild-type (WT)-CPC and CPC isolated from female mice. Epigenetic analysis in freshly isolated male CKO-CPC display hypermethylation in pro-angiogenic genes (Fgfr2, Epas1) and hypomethylation in the white adipocyte differentiation gene Zfp423 associated with up-regulated ZFP423 expression and a shift from endothelial to white adipocyte differentiation compared with WT-CPC. The expression of the histone-methyltransferase EZH2 is reduced in male CKO-CPC compared with male WT-CPC, whereas no differences in the EZH2 expression in female CPC were observed. Clonally expanded CPC can differentiate into endothelial cells or into adipocytes depending on the differentiation conditions. ZFP423 overexpression is sufficient to induce white adipocyte differentiation of clonal CPC. In isolated WT-CPC, PGD2 stimulation reduces the expression of EZH2, thereby up-regulating ZFP423 expression and promoting white adipocyte differentiation. The treatment of young male CKO mice with the COX inhibitor Ibuprofen or the PGD2 receptor (DP)2 receptor antagonist BAY-u 3405 in vivo increased EZH2 expression and reduced ZFP423 expression and adipocyte differentiation in CKO-CPC. Thus, cardiomyocyte STAT3 deficiency leads to age-related and sex-specific cardiac remodeling and failure in part due to sex-specific alterations in PGD2 secretion and subsequent epigenetic impairment of the differentiation potential of CPC. Causally involved is the impaired AR signaling in absence of STAT3, which reduces the expression of the PG-degrading enzyme HPGD.

Epigenetic modifications of the Zfp/ZNF423 gene control murine adipogenic commitment and are dysregulated in human hypertrophic obesity

Aims/hypothesisSubcutaneous adipocyte hypertrophy is associated with insulin resistance and increased risk of type 2 diabetes, and predicts its future development independent of obesity. In humans, subcutaneous adipose tissue hypertrophy is a consequence of impaired adipocyte precursor cell recruitment into the adipogenic pathway rather than a lack of precursor cells. The zinc finger transcription factor known as zinc finger protein (ZFP) 423 has been identified as a major determinant of pre-adipocyte commitment and maintained white adipose cell function. Although its levels do not change during adipogenesis, ectopic expression of Zfp423 in non-adipogenic murine cells is sufficient to activate expression of the gene encoding peroxisome proliferator-activated receptor γ (Pparγ; also known as Pparg) and increase the adipogenic potential of these cells. We investigated whether the Zfp423 gene is under epigenetic regulation and whether this plays a role in the restricted adipogenesis associated with hypertrophic obesity.MethodsMurine 3T3-L1 and NIH-3T3 cells were used as fibroblasts committed and uncommitted to the adipocyte lineage, respectively. Human pre-adipocytes were isolated from the stromal vascular fraction of subcutaneous adipose tissue of 20 lean non-diabetic individuals with a wide adipose cell size range. mRNA levels were measured by quantitative real-time PCR, while methylation levels were analysed by bisulphite sequencing. Chromatin structure was analysed by micrococcal nuclease protection assay, and DNA-methyltransferases were chemically inhibited by 5-azacytidine. Adipocyte differentiation rate was evaluated by Oil Red O staining.ResultsComparison of uncommitted (NIH-3T3) and committed (3T3-L1) adipose precursor cells revealed that Zfp423 expression increased (p < 0.01) in parallel with the ability of the cells to differentiate into mature adipocytes owing to both decreased promoter DNA methylation (p < 0.001) and nucleosome occupancy (nucleosome [NUC] 1 p < 0.01; NUC2 p < 0.001) in the 3T3-L1 compared with NIH-3T3 cells. Interestingly, non-adipogenic epigenetic profiles can be reverted in NIH-3T3 cells as 5-azacytidine treatment increased Zfp423 mRNA levels (p < 0.01), reduced DNA methylation at a specific CpG site (p < 0.01), decreased nucleosome occupancy (NUC1, NUC2: p < 0.001) and induced adipocyte differentiation (p < 0.05). These epigenetic modifications can also be initiated in response to changes in the pre-adipose cell microenvironment, in which bone morphogenetic protein 4 (BMP4) plays a key role. We finally showed that, in human adipocyte precursor cells, impaired epigenetic regulation of zinc nuclear factor (ZNF)423 (the human orthologue of murine Zfp423) was associated with inappropriate subcutaneous adipose cell hypertrophy. As in NIH-3T3 cells, the normal ZNF423 epigenetic profile was rescued by 5-azacytidine exposure.Conclusions/interpretationOur results show that epigenetic events regulate the ability of precursor cells to commit and differentiate into mature adipocytes by modulating ZNF423, and indicate that dysregulation of these mechanisms accompanies subcutaneous adipose tissue hypertrophy in humans.

Retinoic acid inhibits white adipogenesis by disrupting GADD45A-mediated Zfp423 DNA demethylation.

Retinoic acid (RA), a bioactive metabolite of vitamin A, is a critical mediator of cell differentiation. RA blocks adipogenesis, but mechanisms remain to be established. ZFP423 is a key transcription factor maintaining white adipose identity. We found that RA inhibits Zfp423 expression and adipogenesis via blocking DNA demethylation in the promoter of Zfp423, a process mediated by growth arrest and DNA-damage-inducible protein alpha (GADD45A). RA induces the partnering between retinoic acid receptor (RAR) and tumor suppressor inhibitor of growth protein 1 (ING1), which prevents the formation of GADD45A and ING1 complex necessary for locus-specific Zfp423 DNA demethylation. In vivo, vitamin A supplementation prevents obesity, downregulates Gadd45a expression, and reduces GADD45A binding and DNA demethylation in the Zfp423 promoter. Inhibition of Zfp423 expression due to RA contributes to the enhanced brown adipogenesis. In summary, RA inhibits white adipogenesis by inducing RAR and ING1 interaction and inhibiting Gadd45a expression, which prevents GADD45A-mediated DNA demethylation.

Zfp423 Maintains White Adipocyte Identity through Suppression of the Beige Cell Thermogenic Gene Program

The transcriptional regulators Ebf2 and Prdm16 establish and maintain the brown and/or beige fat cell identity. However, the mechanisms operating in white adipocytes to suppress the thermogenic gene program and maintain an energy-storing phenotype are less understood. Here, we report that the transcriptional regulator Zfp423 is critical for maintaining white adipocyte identity through suppression of the thermogenic gene program. Zfp423 expression is enriched in white versus brown adipocytes and suppressed upon cold exposure. Doxycycline-inducible inactivation of Zfp423 in mature adipocytes, combined with β-adrenergic stimulation, triggers a conversion of differentiated adiponectin-expressing inguinal and gonadal adipocytes into beige-like adipocytes; this reprogramming event is sufficient to prevent and reverse diet-induced obesity and insulin resistance. Mechanistically, Zfp423 acts in adipocytes to inhibit the activity of Ebf2 and suppress Prdm16 activation. These data identify Zfp423 as a molecular brake on adipocyte thermogenesis and suggest a therapeutic strategy to unlock the thermogenic potential of white adipocytes in obesity.

All‐trans‐Retinoic Acid Inhibits Adipogenesis by Interrupting Gadd45α Induced Zfp423

Retinoic acid (RA) is known to maintain the preadipocyte phenotype and inhibit adipocyte differentiation and maturation. Although RA has been showed to induce epigenetic changes during embryonic stem cell differentiation, the effect of RA on epigenetic modifications of adipogenic genes are unknown. C3H10T1/2 mesenchymal progenitor cells were induced adipogenesis with/without 1mM all‐trans retinoic acid (ATRA), and epigenetic changes in the zfp423 promoter, a critical transcription factor initiating adipogenesis, were analyzed. We found that Growth arrest and DNA‐damage‐inducible protein GADD45 alpha (gadd45α) is involved in adipogenesis of C3H10T1/2 by inducing DNA demethylation in the zfp423 promoter. During adipogenesis, both gadd45α and zfp423 expression was upregulated. Gadd45α overexpression upregulated zfp423 expression and enhanced adipogenesis whereas gadd45α knock down had opposite effects. RA strongly inhibited the expression of zfp423 and down‐stream adipogenic genes in both control and gadd45α overexpressing cells and completely blocked lipid accumulation, which was associated with a reduction in gadd45α expression and binding to the zfp423 promoter. Consistently, in vivo, we found that 30 IU/mL vitamin A supplementation (through water) prevented high fat diet (60 kcal% fat) induced obesity, which was correlated with attenuated zfp423 and pparg expression in the adipose tissue of mice. After one month of high fat diet challenge, vitamin A supplemented mice had lower body weight gain, less white fat deposition, smaller adipocytes size and higher insulin sensitivity compared with control mice. These data demonstrate that GADD45α mediated zfp423 promoter demethylation is required for adipogenesis. RA inhibits adipogenesis by preventing DNA demethylation of the zfp423 promoter and lipid accumulation. Thus, RA is beneficial for the prevention of high fat diet induced obesity.Support or Funding InformationSupported by NIH R01HD067449 and USDA‐NIFA 2015‐67015‐23219

MiR-195a inhibits adipocyte differentiation by targeting the preadipogenic determinator Zfp423.

MicroRNAs (miRNAs) play essential roles in various cellular processes including proliferation and differentiation. In this study, we identified miRNA-195a (miR-195a) as a regulator of adipocyte differentiation. Differential expression of miR-195a in preadipocytes and adipocytes suggests its role in lipid accumulation and adipocyte differentiation. Forced expression of miR-195a mimics suppressed lipid accumulation and inhibited expression of adipocyte markers such as PPARγ and aP2 in 3T3-L1 and C3H10T1/2 cells. Conversely, downregulation of miR-195a by anti-miR-195a increased lipid accumulation and expression of adipocyte markers. Target prediction analysis suggested zinc finger protein 423 (Zfp423), a preadipogenic determinator, as a potential gene recognized by miR-195a. In line with this, mimicked expression of miR-195a reduced the expression of Zfp423, whereas anti-miR-195a increased its expression. Predicted targeting sequences in Zfp423 3'UTR, but not mutated sequences fused to luciferase, were regulated by miR-195a. Ectopic Zfp423 expression in 3T3-L1 cells increased lipid accumulation and expression of adipocyte markers, consistent with the observation that miR-195a targets Zfp423, resulting in suppressed adipocyte differentiation. In addition, miR-195a and Zfp423 were inversely correlated in obese fat tissues, raising the possibility of miRNA's role in obesity. Together, our data show that miR-195a is an anti-adipogenic regulator, which acts by targeting Zfp423, and further suggest the roles of miR-195a in obesity and metabolic diseases.

Effect of lipopolysaccharides on adipogenic potential and premature senescence of adipocyte progenitors.

The elevation of circulating LPS has been associated with obesity and aging. However, whether and how LPS contributes to adipose tissue dysfunction is unclear. In this study, we investigated the effect of LPS on the adipogenic capacity and cellular senescence of adipocyte progenitors. Stromal-vascular cells were isolated from inguinal adipose tissue of C57BL/6 mice and treated with LPS during the different time periods of adipocyte differentiation. We found that LPS treatment for 24 h prior to the induction of differentiation led to the most profound effect on the inhibition of adipogenesis, as evidenced by the morphological changes and the decreased mRNA expression of adipocyte marker genes. In addition, LPS induced features of premature senescence of SV cells, including the activation of p53, the elevation of SA-β-gal activity, and increased hydrogen peroxide production, but not telomere length. Upon LPS treatment, SV cells also developed senescence-associated secretory phenotype (SASP), as demonstrated by the increased expression of TNFα, IL-1β, IL-6, MCP-1, and VEGFα. Blocking LPS-induced NF-κB activation and cytokine production by Bay 11-7082 failed to rescue the impaired adipogenesis and the reduction in PPARγ and Zfp423 expression. On the contrary, rosiglitazone had little effect on cytokine production but corrected the defective adipogenic potential. In conclusion, we demonstrate that LPS inhibits adipogenesis by disrupting the differentiation of adipocyte progenitors in a NF-κB-independent manner; LPS also induces premature senescence of adipocyte progenitors. Our data suggest that LPS could be a potential contributor to the defective adipogenesis and the development of cellular senescence in adipose tissue during obesity and aging.

Maternal Obesity Induces Epigenetic Modifications to Facilitate Zfp423 Expression and Enhance Adipogenic Differentiation in Fetal Mice

Maternal obesity (MO) predisposes offspring to obesity and type 2 diabetes despite poorly defined mechanisms. Zfp423 is the key transcription factor committing cells to the adipogenic lineage, with exceptionally dense CpG sites in its promoter. We hypothesized that MO enhances adipogenic differentiation during fetal development through inducing epigenetic changes in the Zfp423 promoter and elevating its expression. Female mice were subjected to a control (Con) or obesogenic (OB) diet for 2 months, mated, and maintained on their diets during pregnancy. Fetal tissue was harvested at embryonic day 14.5 (E14.5), when the early adipogenic commitment is initiated. The Zfp423 expression was 3.6-fold higher and DNA methylation in the Zfp423 promoter was lower in OB compared with Con. Correspondingly, repressive histone methylation (H3K27me3) was lower in the Zfp423 promoter of OB fetal tissue, accompanied by reduced binding of enhancer of zeste 2 (EZH2). Gain- and loss-of-function analysis showed that Zfp423 regulates early adipogenic differentiation in fetal progenitor cells. In summary, MO enhanced Zfp423 expression and adipogenic differentiation during fetal development, at least partially through reducing DNA methylation in the Zfp423 promoter, which is expected to durably elevate adipogenic differentiation of progenitor cells in adult tissue, programming adiposity and metabolic dysfunction later in life.

Zfp423 Binds Autoregulatory Sites in P19 Cell Culture Model

Zfp423 is a 30 zinc finger transcription factor that forms regulatory complexes with EBF family members and factors targeted by canonical signaling pathways. Zfp423 mutations produce a range of developmental abnormalities in mice and humans related to the ciliopathies. Surprisingly, computational analysis of clustered Zfp423 and partner motifs in conserved genomic sequences predicts enrichment in Zfp423 and Ebf genes. In cell culture models selected for Zfp423 and EBF expression, we identify strong and reproducible occupancy of two Zfp423 intronic sites using chromatin immunoprecipitation with multiple independent antibodies. Both sites are significantly enriched in either quantitative PCR or massively parallel sequencing assays. A site in intron 5 acts as a classical enhancer in transient assays, but does not require the consensus motif for activity, suggesting a redundant or modulatory role for Zfp423 binding in this context. We speculate that Zfp423 may repress this enhancer as part of a developmental ratchet.

Maternal obesity enhances ZFP423 expression and adipogenic differentiation of E14.5 fetal muscle of mice

Obesity including maternal obesity, is increasing rapidly, which impairs fetal skeletal muscle development. ZFP423 is the earliest marker of adipogenic commitment currently identified and the 5′‐UTR region of ZFP423 has extremely enriched CG islands. We hypothesized that maternal obesity enhanced the expression of ZFP423 and thus adipogenesis through altering DNA methylation in the ZFP423 promoter. Female mice (one month old) were fed either a control (Con) or obesogenic (OB) diet to induce obesity before pregnancy, and maintained in their respective diets during pregnancy. Muscle samples were harvested from fetal mice at E14.5, a very early stage for adipogenic differentiation. The methylation status of ZFP423 5′‐UTR region was analyzed using bisulfite sequencing. The mRNA expression of ZFP423 was higher in OB fetal muscle, which was associated with attenuated methylation in the ZFP423 promoter. The primary cultured cells prepared from OB fetal muscle demonstrated enhanced adipogenic potential. Our data show that maternal obesity enhances adipogenic differentiation of multipotent cells in fetal muscle, at least partially through altering the DNA methylation of ZFP423, a critical initiator of adipogenesis. This research is funded by NIH grant R01HD067449.

Zfp423 Expression Identifies Committed Preadipocytes and Localizes to Adipose Endothelial and Perivascular Cells

Progress has been made in elucidating the cell-surface phenotype of primary adipose progenitors; however, specific functional markers and distinct molecular signatures of fat depot-specific preadipocytes have remained elusive. In this study, we label committed murine adipose progenitors through expression of GFP from the genetic locus for Zfp423, a gene controlling preadipocyte determination. Selection of GFP-expressing fibroblasts from either subcutaneous or visceral adipose-derived stromal vascular cultures isolates stably committed preadipocytes that undergo robust adipogenesis. Immunohistochemistry for Zfp423-driven GFP expression invivo confirms a perivascular origin of preadipocytes within both white and brown adipose tissues. Interestingly, a small subset of capillary endothelial cells within white and brown fat also express this marker, suggesting a contribution of specialized endothelial cells to the adipose lineage. Zfp423(GFP) mice represent a simple tool for the specific localization and isolation of molecularly defined preadipocytes from distinct adipose tissue depots.

ZFP423 Coordinates Notch and Bone Morphogenetic Protein Signaling, Selectively Up-regulating Hes5 Gene Expression

Zinc finger protein 423 encodes a 30 Zn-finger transcription factor involved in cerebellar and olfactory development. ZFP423 is a known interactor of SMAD1-SMAD4 and of Collier/Olf-1/EBF proteins, and acts as a modifier of retinoic acid-induced differentiation. In the present article, we show that ZFP423 interacts with the Notch1 intracellular domain in mammalian cell lines and in Xenopus neurula embryos, to activate the expression of the Notch1 target Hes5/ESR1. This effect is antagonized by EBF transcription factors, both in cultured cells and in Xenopus embryos, and amplified in vitro by BMP4, suggesting that ZFP423 acts to integrate BMP and Notch signaling, selectively promoting their convergence onto the Hes5 gene promoter.

Transcriptional control of preadipocyte determination by Zfp423

The worldwide epidemic of obesity has increased the urgency of developing a deeper understanding of physiological systems related to energy balance and energy storage, including the mechanisms controlling the development of fat cells (adipocytes). The differentiation of committed preadipocytes to adipocytes is controlled by PPARγ and several other transcription factors 1, but the molecular basis for preadipocyte determination is not understood. Using a novel method for the quantitative analysis of transcriptional components, we identified the zinc-finger protein Zfp423 as a factor enriched in preadipose versus non-preadipose fibroblasts. Ectopic expression of Zfp423 in non-adipogenic NIH 3T3 fibroblasts robustly activates expression of PPARγ in undifferentiated cells and permits cells to undergo adipocyte differentiation under permissive conditions. ShRNA-mediated reduction of Zfp423 expression in 3T3-L1 cells blunts preadipocyte PPARγ expression and diminishes the ability of these cells to differentiate. Furthermore, both brown and white adipocyte differentiation is strikingly impaired in Zfp423-deficient mouse embryos. Zfp423 regulates PPARγ expression, in part, through amplification of the BMP signaling pathway, an effect dependent on the SMAD binding capacity of Zfp423. This study identifies Zfp423 as a transcriptional regulator of preadipocyte determination.

Enhanced expression of p210BCR/ABL and aberrant expression of Zfp423/ZNF423 induce blast crisis of chronic myelogenous leukemia

AbstractChronic myelogenous leukemia (CML) is a hematopoietic disorder originating from p210BCR/ABL-transformed stem cells, which begins as indolent chronic phase (CP) but progresses into fatal blast crisis (BC). To investigate molecular mechanism(s) underlying disease evolution, CML-exhibiting p210BCR/ABL transgenic mice were crossed with BXH2 mice that transmit a replication-competent retrovirus. Whereas nontransgenic mice in the BXH2 background exclusively developed acute myeloid leukemia, p210BCR/ABL transgenic littermates developed nonmyeloid leukemias, in which inverse polymerase chain reaction detected 2 common viral integration sites (CISs). Interestingly, one CIS was transgene’s own promoter, which up-regulated p210BCR/ABL expression. The other was the 5′ noncoding region of a transcription factor, Zfp423, which induced aberrant Zfp423 expression. The cooperative activities of Zfp423 and p210BCR/ABL were demonstrated as follows: (1) introduction of Zfp423 in p210BCR/ABL transgenic bone marrow (BM) cells increased colony-forming ability, (2) suppression of ZNF423 (human homologue of Zfp423) in ZNF423-expressing, p210BCR/ABL-positive hematopoietic cells retarded cell growth, (3) mice that received a transplant of BM cells transduced with Zfp423 and p210BCR/ABL developed acute leukemia, and (4) expression of ZNF423 was found in human BCR/ABL-positive cell lines and CML BC samples. These results demonstrate that enhanced expression of p210BCR/ABL and deregulated expression of Zfp423/ZNF423 contribute to CML BC.