Unraveling the complexity of thermogenic remodeling of white fat reveals potential antiobesity therapies.

Adipose tissue is a complex organ consisting of a mixture of mature adipocytes and stromal vascular cells. It displays a remarkable ability to adapt to environmental and dietary cues by changing its morphology and metabolic capacity. This plasticity is demonstrated by the emergence of interspersed thermogenic beige adipocytes within white depots in response to catecholamines secretion. Coordinated cellular interaction between different cell types within the tissue and a fine-tuned transcriptional program synergistically take place to promote beige remodeling. However, both cell-cell interactions and molecular mechanisms governing beige adipocyte appearance and maintenance are poorly understood. In this and the previous issue of Genes & Development, Shao and colleagues (pp. 1461-1474) and Shan and colleagues (pp. 1333-1338) advance our understanding of these issues and, in doing so, highlight potential therapeutic strategies to combat obesity-associated diseases.

ZFP423 controls EBF2 coactivator recruitment and PPARγ occupancy to determine the thermogenic plasticity of adipocytes.

Energy-storing white adipocytes maintain their identity by suppressing the energy-burning thermogenic gene program of brown and beige adipocytes. Here, we reveal that the protein-protein interaction between the transcriptional coregulator ZFP423 and brown fat determination factor EBF2 is essential for restraining the thermogenic phenotype of white adipose tissue (WAT). Disruption of the ZFP423-EBF2 protein interaction through CRISPR-Cas9 gene editing triggers widespread "browning" of WAT in adult mice. Mechanistically, ZFP423 recruits the NuRD corepressor complex to EBF2-bound thermogenic gene enhancers. Loss of adipocyte Zfp423 induces an EBF2 NuRD-to-BAF coregulator switch and a shift in PPARγ occupancy to thermogenic genes. This shift in PPARγ occupancy increases the antidiabetic efficacy of the PPARγ agonist rosiglitazone in obesity while diminishing the unwanted weight-gaining effect of the drug. These data indicate that ZFP423 controls EBF2 coactivator recruitment and PPARγ occupancy to determine the thermogenic plasticity of adipocytes and highlight the potential of therapeutically targeting transcriptional brakes to induce beige adipocyte biogenesis in obesity.

Pioneering EBF2 remodels the brown fat chromatin landscape.

In this issue of Genes & Development, Shapira and colleagues (pp. 660-673) outline mechanisms by which the brown fat transcription factor early B-cell factor 2 (EBF2) selectively activates brown lineage-specific gene expression. The investigators show that EBF2 interacts with and recruits a tissue-specific BAF chromatin remodeling complex to brown fat gene enhancers, thereby regulating chromatin accessibility. Their findings provide important insight into epigenetic regulation of adipocyte fate and thermogenic gene expression.

EBF2 transcriptionally regulates brown adipogenesis via the histone reader DPF3 and the BAF chromatin remodeling complex.

The transcription factor early B-cell factor 2 (EBF2) is an essential mediator of brown adipocyte commitment and terminal differentiation. However, the mechanisms by which EBF2 regulates chromatin to activate brown fat-specific genes in adipocytes were unknown. ChIP-seq (chromatin immunoprecipitation [ChIP] followed by deep sequencing) analyses in brown adipose tissue showed that EBF2 binds and regulates the activity of lineage-specific enhancers. Mechanistically, EBF2 physically interacts with the chromatin remodeler BRG1 and the BAF chromatin remodeling complex in brown adipocytes. We identified the histone reader protein DPF3 as a brown fat-selective component of the BAF complex that was required for brown fat gene programming and mitochondrial function. Loss of DPF3 in brown adipocytes reduced chromatin accessibility at EBF2-bound enhancers and led to a decrease in basal and catecholamine-stimulated expression of brown fat-selective genes. Notably, Dpf3 is a direct transcriptional target of EBF2 in brown adipocytes, thereby establishing a regulatory module through which EBF2 activates and also recruits DPF3-anchored BAF complexes to chromatin. Together, these results reveal a novel mechanism by which EBF2 cooperates with a tissue-specific chromatin remodeling complex to activate brown fat identity genes.

The RING E3 ligase SDIR1 destabilizes EBF1/EBF2 and modulates the ethylene response to ambient temperature fluctuations in Arabidopsis.

The gaseous phytohormone ethylene mediates numerous aspects of plant growth and development as well as stress responses. The F-box proteins EIN3-binding F-box protein 1 (EBF1) and EBF2 are key components that ubiquitinate and degrade the master transcription factors ethylene insensitive 3 (EIN3) and EIN3-like 1 (EIL1) in the ethylene response pathway. Notably, EBF1 and EBF2 themselves undergo the 26S proteasome-mediated proteolysis induced by ethylene and other stress signals. However, despite their importance, little is known about the mechanisms regulating the degradation of these proteins. Here, we show that a really interesting new gene (RING)-type E3 ligase, salt- and drought-induced ring finger 1 (SDIR1), positively regulates the ethylene response and promotes the accumulation of EIN3. Further analyses indicate that SDIR1 directly interacts with EBF1/EBF2 and targets them for ubiquitination and proteasome-dependent degradation. We show that SDIR1 is required for the fine tuning of the ethylene response to ambient temperature changes by mediating temperature-induced EBF1/EBF2 degradation and EIN3 accumulation. Thus, our work demonstrates that SDIR1 functions as an important modulator of ethylene signaling in response to ambient temperature changes, thereby enabling plant adaptation under fluctuating environmental conditions.

Role of microRNA-19b-3p on osteoporosis after experimental spinal cord injury in rats.

Osteoporosis is a common complication accompanied by spinal cord injury (SCI) occurrence. MicroRNAs (miRNAs) have been proved to play a crucial role in the progression of osteoporosis, but their regulating mechanism is unclear. The present study investigated miRNA-19b-3p level in SCI rats induced by modified Allen method, as well as the role of miRNA-19b-3p in osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). MiRNA-19b-3p expression and bone mineral density (BMD) of femurs were measured at day 21 and day 60 after SCI in rats. Obvious miRNA-19b-3p up-regulation and aggravated bone loss were observed. MiRNA-19b-3p overexpression suppressed BMSC-derived osteoblast differentiation, which was confirmed by the decrease in alkaline phosphatase (ALP) activity, EBF2 expression, osteoprotegrin (OPG) to receptor activator of nuclear factor kappa-B ligand (RANKL) ratio and cell mineralization degree. Besides, MiRNA-19b-3p knockdown could reverse this phenomenon. Dual-luciferase reporter assays verified the targeting relationship between miRNA-19b-3p and EBF2. The in vivo experiments confirmed miRNA-19b-3p down-regulation could significantly attenuate osteoporosis after SCI, which was verified by increased BMD, collagen content, femur mineralization degree, EBF2 protein, and OPG-to-RANKL ratio. The results show that miRNA-19b-3p plays an important role in the osteoporosis process after SCI through regulating EBF2 expression.

Age-Progressive and Gender-Dependent Bone Phenotype in Mice Lacking Both Ebf1 and Ebf2 in Prrx1-Expressing Mesenchymal Cells.

Ebfs are a family of transcription factors regulating the differentiation of multiple cell types of mesenchymal origin, including osteoblasts. Global deletion of Ebf1 results in increased bone formation and bone mass, while global loss of Ebf2 leads to enhanced bone resorption and decreased bone mass. Targeted deletion of Ebf1 in early committed osteoblasts leads to increased bone formation, whereas deletion in mature osteoblasts has no effect. To study the effects of Ebf2 specifically on long bone development, we created a limb bud mesenchyme targeted Ebf2 knockout mouse model by using paired related homeobox gene 1 (Prrx1) Cre. To investigate the possible interplay between Ebf1 and Ebf2, we deleted both Ebf1 and Ebf2 in the cells expressing Prrx1. Mice with Prrx1-targeted deletion of Ebf2 had a very mild bone phenotype. However, deletion of both Ebf1 and Ebf2 in mesenchymal lineage cells lead to significant, age progressive increase in bone volume. The phenotype was to some extent gender dependent, leading to an increase in both trabecular and cortical bone in females, while in males a mild cortical bone phenotype and a growth plate defect was observed. The phenotype was observed at both 6 and 12 weeks of age, but it was more pronounced in older female mice. Our data suggest that Ebfs modulate bone homeostasis and they are likely able to compensate for the lack of each other. The roles of Ebfs in bone formation appear to be complex and affected by multiple factors, such as age and gender.

PIF3 is a negative regulator of the CBF pathway and freezing tolerance in Arabidopsis.

Light and temperature are major environmental factors that coordinately control plant growth and survival. However, how plants integrate light and temperature signals to better adapt to environmental stresses is poorly understood. PHYTOCHROME-INTERACTING FACTOR 3 (PIF3), a key transcription factor repressing photomorphogenesis, has been shown to play a pivotal role in mediating plants' responses to various environmental signals. In this study, we found that PIF3 functions as a negative regulator of Arabidopsis freezing tolerance by directly binding to the promoters of C-REPEAT BINDING FACTOR (CBF) genes to down-regulate their expression. In addition, two F-box proteins, EIN3-BINDING F-BOX 1 (EBF1) and EBF2, directly target PIF3 for 26S proteasome-mediated degradation. Consistently, ebf1 and ebf2 mutants were more sensitive to freezing than were the wild type, and the pif3 mutation suppressed the freezing-sensitive phenotype of ebf1 Furthermore, cold treatment promoted the degradation of EBF1 and EBF2, leading to increased stability of the PIF3 protein and reduced expression of the CBF genes. Together, our study uncovers an important role of PIF3 in Arabidopsis freezing tolerance by negatively regulating the expression of genes in the CBF pathway.

The tumor secretory factor ZAG promotes white adipose tissue browning and energy wasting.

Cachexia is a complex tissue-wasting syndrome characterized by inflammation, hypermetabolism, increased energy expenditure, and anorexia. Browning of white adipose tissue (WAT) is one of the significant factors that contribute to energy wasting in cachexia. By utilizing a cell implantation model, we demonstrate here that the lipid mobilizing factor zinc-α2-glycoprotein (ZAG) induces WAT browning in mice. Increased circulating levels of ZAG not only induced lipolysis in adipose tissues but also caused robust browning in WAT. Stimulating WAT progenitors with ZAG recombinant protein or expression of ZAG in mouse embryonic fibroblasts (MEFs) strongly enhanced brown-like differentiation. At the molecular level, ZAG stimulated peroxisome proliferator-activated receptor γ (PPARγ) and early B cell factor 2 expression and promoted their recruitment to the PR/SET domain 16 (Prdm16) promoter, leading to enhanced expression of Prdm16, which determines brown cell fate. In brown adipose tissue, ZAG stimulated the expression of PPARγ and PPARγ coactivator 1α and promoted recruitment of PPARγ to the uncoupling protein 1 (Ucp1) promoter, leading to increased expression of Ucp1. Overall, our results reveal a novel function of ZAG in WAT browning and highlight the targeting of ZAG as a potential therapeutic application in humans with cachexia.-Elattar, S., Dimri, M., Satyanarayana, A. The tumor secretory factor ZAG promotes white adipose tissue browning and energy wasting.

Dynamic Expression and New Functions of Early B Cell Factor 2 in Cerebellar Development.

The collier/Olf1/EBF family genes encode helix-loop-helix transcription factors (TFs) highly conserved in evolution, initially characterized for their roles in the immune system and in various aspects of neural development. The Early B cell Factor 2 (Ebf2) gene plays an important role in the establishment of cerebellar cortical topography and in Purkinje cell (PC) subtype specification. In the adult cerebellum, Ebf2 is expressed in zebrin II (ZII)-negative PCs, where it suppresses the ZII+ molecular phenotype. However, it is not clear whether Ebf2 is restricted to a PC subset from the onset of its expression or is initially distributed in all PCs and silenced only later in the prospective ZII+ subtype. Moreover, the dynamic distribution and role of Ebf2 in the differentiation of other cerebellar cells remain unclarified. In this paper, by genetic fate mapping, we determine that Ebf2 mRNA is initially found in all PC progenitors, suggesting that unidentified upstream factors silence its expression before completion of embryogenesis. Moreover we show Ebf2 activation in an early born subset of granule cell (GC) precursors homing in the anterior lobe. Conversely, Ebf2 transcription is repressed in other cerebellar cortex interneurons. Last, we show that, although Ebf2 only labels the medial cerebellar nuclei (CN) in the adult cerebellum, the gene is expressed prenatally in projection neurons of all CN. Importantly, in Ebf2 nulls, fastigial nuclei are severely hypocellular, mirroring the defective development of anterior lobe PCs. Our findings further clarify the roles of this terminal selector gene in cerebellar development.

Layer 4 pyramidal neurons exhibit robust dendritic spine plasticity in vivo after input deprivation.

Pyramidal neurons in layers 2/3 and 5 of primary somatosensory cortex (S1) exhibit somewhat modest synaptic plasticity after whisker input deprivation. Whether neurons involved at earlier steps of sensory processing show more or less plasticity has not yet been examined. Here, we used longitudinal in vivo two-photon microscopy to investigate dendritic spine dynamics in apical tufts of GFP-expressing layer 4 (L4) pyramidal neurons of the vibrissal (barrel) S1 after unilateral whisker trimming. First, we characterize the molecular, anatomical, and electrophysiological properties of identified L4 neurons in Ebf2-Cre transgenic mice. Next, we show that input deprivation results in a substantial (∼50%) increase in the rate of dendritic spine loss, acutely (4-8 d) after whisker trimming. This robust synaptic plasticity in L4 suggests that primary thalamic recipient pyramidal neurons in S1 may be particularly sensitive to changes in sensory experience. Ebf2-Cre mice thus provide a useful tool for future assessment of initial steps of sensory processing in S1.

EBF2 Links KMT2D-Mediated H3K4me1 to Suppress Pancreatic Cancer Progression via Upregulating KLLN.

Mono-methylation of histone H3 on Lys 4 (H3K4me1), which is catalyzed by histone-lysine N-methyltransferase 2D (KMT2D), serves as an important epigenetic regulator in transcriptional control. In this study, the authors identify early B-cell factor 2 (EBF2) as a binding protein of H3K4me1. Combining analyses of RNA-seq and ChIP-seq data, the authors further identify killin (KLLN) as a transcriptional target of KMT2D and EBF2 in pancreatic ductal adenocarcinoma (PDAC) cells. KMT2D-dependent H3K4me1 and EBF2 are predominantly over-lapped proximal to the transcription start site (TSS) of KLLN gene. Comprehensive functional assays show that KMT2D and EBF2 cooperatively inhibit PDAC cells proliferation, migration, and invasion through upregulating KLLN. Such inhibition on PDAC progression is also achieved through increasing H3K4me1 level by GSK-LSD1, a selective inhibitor of lysine-specific demethylase 1 (LSD1). Taken together, these findings reveal a new mechanism underlying PDAC progression and provide potential therapeutic targets for PDAC treatment.

Bmp4 regulates chick Ebf2 and Ebf3 gene expression in somite development.

The chick Early B-cell Factor-2 and 3 (cEbf2 and cEbf3) genes are members of EBF family of helix loop helix transcription factors. The expression, regulation and importance of these genes have been extensively studied in lymphatic, nervous and muscular tissues. Recently, a new role for some members of EBF in bone development has been investigated. However, the expression profile and regulation in the axial skeleton precursor, the somite, have yet to be elucidated. Therefore, this study was aimed to investigate the expression and regulation of cEbf2 and cEbf3 genes in the developing chick embryo somite from HH4 to HH28. The spatiotemporal expression study revealed predominant localization of cEbf2 and cEbf3 in the lateral sclerotomal domains and later around vertebral cartilage anlagen of the arch and the proximal rib. Subsequently, microsurgeries, ectopic gene expression experiments were performed to analyze which tissues and factors regulate cEbf2 and cEbf3 expression. Lateral barriers experiments indicated the necessity for lateral signal(s) in the regulation of cEbf2 and cEbf3 genes. Results from tissue manipulations and ectopic gene expression experiments indicate that lateral plate-derived Bmp4 signals are necessary for the initiation and maintenance of cEbf2 and cEbf3 genes in somites. In conclusion, cEbf2 and cEbf3 genes are considered as lateral sclerotome markers which their expression is regulated by Bmp4 signals from the lateral plate mesoderm.

Control of murine brown adipocyte development by GATA6.

Brown adipose tissue (BAT) is a thermogenic organ that protects animals against hypothermia and obesity. BAT derives from the multipotent paraxial mesoderm; however, the identity of embryonic brown fat progenitor cells and regulators of adipogenic commitment are unclear. Here, we performed single-cell gene expression analyses of mesenchymal cells during mouse embryogenesis with a focus on BAT development. We identified cell populations associated with the development of BAT, including Dpp4+ cells that emerge at the onset of adipogenic commitment. Immunostaining and lineage-tracing studies show that Dpp4+ cells constitute the BAT fascia and contribute minorly as adipocyte progenitors. Additionally, we identified the transcription factor GATA6 as a marker of brown adipogenic progenitor cells. Deletion of Gata6 in the brown fat lineage resulted in a striking loss of BAT. Together, these results identify progenitor and transitional cells in the brown adipose lineage and define a crucial role for GATA6 in BAT development.

Seedling morphogenesis: when ethylene meets high ambient temperature.

Unlike animals, plant development is plastic and sensitive to environmental changes. For example, Arabidopsis thaliana seedlings display distinct growth patterns when they are grown under different light or temperature conditions. Moreover, endogenous plant hormone such as ethylene also impacts seedling morphology. Ethylene induces hypocotyl elongation in light-grown seedlings but strongly inhibits hypocotyl elongation in etiolated (dark-grown) seedlings. Another characteristic ethylene response in etiolated seedlings is the formation of exaggerated apical hooks. Although it is well known that high ambient temperature promotes hypocotyl elongation in light-grown seedlings (thermomorphogenesis), ethylene suppresses thermomorphogenesis. On another side, high ambient temperature also inhibits the ethylene-responsive hypocotyl shortening and exaggerated hook formation in etiolated seedlings. Therefore, the simplest phytohormone ethylene exhibits almost the most complicated responses, depending on temperature and/or light conditions. In this review, we will focus on two topics related to the main theme of this special issue (response to high temperature): (1) how does high temperature suppress ethylene-induced seedling morphology in dark-grown seedlings, and (2) how does ethylene inhibit high temperature-induced seedling growth in light-grown seedlings. Controlling ethylene biosynthesis through antisense technology was the hallmark event in plant genetic engineering in 1990, we assume that manipulations on plant ethylene signaling in agricultural plants may pave the way for coping with climate change in future.

Early B Cell Factor Activity Controls Developmental and Adaptive Thermogenic Gene Programming in Adipocytes.

Brown adipose tissue (BAT) activity protects animals against hypothermia and represents a potential therapeutic target to combat obesity. The transcription factor early B cell factor-2 (EBF2) promotes brown adipocyte differentiation, but its roles in maintaining brown adipocyte fate and in stimulating BAT recruitment during cold exposure were unknown. We find that the deletion of Ebf2 in adipocytes of mice ablates BAT character and function, resulting in cold intolerance. Unexpectedly, prolonged exposure to cold restores the thermogenic profile and function of Ebf2 mutant BAT. Enhancer profiling and genetic assays identified EBF1 as a candidate regulator of the cold response in BAT. Adipocyte-specific deletion of both Ebf1 and Ebf2 abolishes BAT recruitment during chronic cold exposure. Mechanistically, EBF1 and EBF2 promote thermogenic gene transcription through increasing the expression and activity of ERRα and PGC1α. Together, these studies demonstrate that EBF proteins specify the developmental fate and control the adaptive cold response of brown adipocytes.

MiR-204-5p promotes apoptosis and inhibits migration of osteosarcoma via targeting EBF2.

Osteosarcoma is one of the most malignant cancer adolescents and young adults and metastatic osteosarcoma is a huge life threat with a 5-year survival lower than 20%. However, the mechanisms through which localized osteosarcoma turned metastatic are not fully understood. Here, we studied the role of miR-204-5p in osteosarcoma and found that miR-204-5p is downregulated in both osteosarcoma patients and osteosarcoma cell lines. In addition, overexpression of miR-204-5p resulted in increase of osteosarcoma cell apoptosis and decrease of osteosarcoma cell migration and invasion. Besides, our in vivo xenograft data showed strong inhibitory role of miR-204-5p in tumor growth. Importantly, our data showed that miR-204-5p regulates the mRNA stability of Early B Cell Factor 2 (EBF2), a crucial regulator in osteosarcoma apoptosis, by directly binding to 3' UTR of EBF2. Besides, our data further revealed that overexpressed EBF2 inhibited apoptosis and facilitated migration and invasion of osteosarcoma cells. Additionally, EBF2 overexpression rescued the phenotype caused by miR-204-5p.Our data indicated that miR-204-5p is an anti-oncogenic miRNA in osteosarcoma which functions through inhibiting oncogenic transcription factor EBF2. These results provided new therapeutic targets for metastatic osteosarcoma and insights into molecular regulation of EBF2.

Variants in the Gene EBF2 Are Associated with Kawasaki Disease in a Korean Population.

PURPOSE: Kawasaki disease (KD) is a mucocutaneous lymph node syndrome. It is mainly seen in young children under the age of five. KD is a multifactorial disorder that includes genetic variants. The present study investigated the association between KD and single nucleotide polymorphisms (SNPs) in the candidate gene early B cell factor 2 (EBF2), which is associated with inflammation markers. MATERIALS AND METHODS: An SNP analysis was performed by whole exon sequencing of the EBF2 gene. Our study comprised a total of 495 subjects (295 KD patients and 200 unrelated normal controls) from a Korean population. Tag SNPs were discovered using the Haploview program. Genotyping of the EBF2 gene was performed with the TaqMan® assay with real-time PCR methods. RESULTS: Polymorphism of rs10866845 showed a significant difference in allele frequency between KD patients and controls (p=0.040). The EBF2 gene polymorphisms were significantly associated with KD on logistic regression analysis. CONCLUSION: EBF2 gene variants can contribute to KD in the Korean population.

Stabilizing the Transcription Factors by E3 Ligase COP1.

Photomorphogenesis is oppositely regulated by two groups of transcription factors. CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) degrades the positive factors but stabilizes the negative ones to predominantly repress photomorphogenesis. It is known that COP1 degrades substrates as an E3 ligase. Two recent studies unraveled the long-sought mechanisms of how COP1 stabilizes the negative transcription factors.

Conserved function of the long noncoding RNA Blnc1 in brown adipocyte differentiation.

OBJECTIVE: Long noncoding RNAs (lncRNAs) are emerging as important regulators of diverse biological processes. Recent work has demonstrated that the inducible lncRNA Blnc1 stimulates thermogenic gene expression during brown and beige adipocyte differentiation. However, whether Blnc1 is functionally conserved in humans has not been explored. In addition, the molecular basis of the Blnc1 ribonucleoprotein complex in thermogenic gene induction remains incompletely understood. The aims of the current study were to: i) investigate functional conservation of Blnc1 in mice and humans and ii) elucidate the molecular mechanisms by which Blnc1 controls the thermogenic gene program in brown adipocytes. METHODS: Full-length human Blnc1 was cloned and examined for its ability to stimulate brown adipocyte differentiation. Different truncation mutants of Blnc1 were generated to identify functional RNA domains responsible for thermogenic gene induction. RNA-protein interaction studies were performed to delineate the molecular features of the Blnc1 ribonucleoprotein complex. RESULTS: Blnc1 is highly conserved in mice and humans at the sequence and function levels, both capable of stimulating brown adipocyte gene expression. A conserved RNA domain was identified to be required and sufficient for the biological activity of Blnc1. We identified hnRNPU as an RNA-binding protein that facilitates the assembly and augments the transcriptional function of the Blnc1/EBF2 ribonucleoprotein complex. CONCLUSIONS: Blnc1 is a conserved lncRNA that promotes thermogenic gene expression in brown adipocytes through formation of the Blnc1/hnRNPU/EBF2 ribonucleoprotein complex.