CD81 Controls Beige Fat Progenitor Cell Growth and Energy Balance via FAK Signaling.

Adipose tissues dynamically remodel their cellular composition in response to external cues by stimulating beige adipocyte biogenesis; however, the developmental origin and pathways regulating this process remain insufficiently understood owing to adipose tissue heterogeneity. Here, we employed single-cell RNA-seq and identified a unique subset of adipocyte progenitor cells (APCs) that possessed the cell-intrinsic plasticity to give rise to beige fat. This beige APC population is proliferative and marked by cell-surface proteins, including PDGFRα, Sca1, and CD81. Notably, CD81 is not only a beige APC marker but also required for de novo beige fat biogenesis following cold exposure. CD81 forms a complex with αV/ß1 and αV/ß5 integrins and mediates the activation of integrin-FAK signaling in response to irisin. Importantly, CD81 loss causes diet-induced obesity, insulin resistance, and adipose tissue inflammation. These results suggest that CD81 functions as a key sensor of external inputs and controls beige APC proliferation and whole-body energy homeostasis.

Developmental regulation of dermal adipose tissue by BCL11b.

The distinct anatomic environment in which adipose tissues arise during organogenesis is a principle determinant of their adult expansion capacity. Metabolic disease results from a deficiency in hyperplastic adipose expansion within the dermal/subcutaneous depot; thus, understanding the embryonic origins of dermal adipose is imperative. Using single-cell transcriptomics throughout murine embryogenesis, we characterized cell populations, including Bcl11b + cells, that regulate the development of dermal white adipose tissue (dWAT). We discovered that BCL11b expression modulates the Wnt signaling microenvironment to enable adipogenic differentiation in the dermal compartment. Subcutaneous and visceral adipose arises from a distinct population of Nefl + cells during embryonic organogenesis, whereas Pi16 + /Dpp4 + fibroadipogenic progenitors support obesity-stimulated hypertrophic expansion in the adult. Together, these results highlight the unique regulatory pathways used by anatomically distinct adipose depots, with important implications for human metabolic disease.

Functional Interplay between Histone H2B ADP-Ribosylation and Phosphorylation Controls Adipogenesis.

Although ADP-ribosylation of histones by PARP-1 has been linked to genotoxic stress responses, its role in physiological processes and gene expression has remained elusive. We found that NAD+-dependent ADP-ribosylation of histone H2B-Glu35 by small nucleolar RNA (snoRNA)-activated PARP-1 inhibits AMP kinase-mediated phosphorylation of adjacent H2B-Ser36, which is required for the proadipogenic gene expression program. The activity of PARP-1 on H2B requires NMNAT-1, a nuclear NAD+ synthase, which directs PARP-1 catalytic activity to Glu and Asp residues. ADP-ribosylation of Glu35 and the subsequent reduction of H2B-Ser36 phosphorylation inhibits the differentiation of adipocyte precursors in cultured cells. Parp1 knockout in preadipocytes in a mouse lineage-tracing genetic model increases adipogenesis, leading to obesity. Collectively, our results demonstrate a functional interplay between H2B-Glu35 ADP-ribosylation and H2B-Ser36 phosphorylation that controls adipogenesis.

DIDO is necessary for the adipogenesis that promotes diet-induced obesity.

The prevalence of overweight and obesity continues to rise in the population worldwide. Because it is an important predisposing factor for cancer, cardiovascular diseases, diabetes mellitus, and COVID-19, obesity reduces life expectancy. Adipose tissue (AT), the main fat storage organ with endocrine capacity, plays fundamental roles in systemic metabolism and obesity-related diseases. Dysfunctional AT can induce excess or reduced body fat (lipodystrophy). Dido1 is a marker gene for stemness; gene-targeting experiments compromised several functions ranging from cell division to embryonic stem cell differentiation, both in vivo and in vitro. We report that mutant mice lacking the DIDO N terminus show a lean phenotype. This consists of reduced AT and hypolipidemia, even when mice are fed a high-nutrient diet. DIDO mutation caused hypothermia due to lipoatrophy of white adipose tissue (WAT) and dermal fat thinning. Deep sequencing of the epididymal white fat (Epi WAT) transcriptome supported Dido1 control of the cellular lipid metabolic process. We found that, by controlling the expression of transcription factors such as C/EBPα or PPARγ, Dido1 is necessary for adipocyte differentiation, and that restoring their expression reestablished adipogenesis capacity in Dido1 mutants. Our model differs from other lipodystrophic mice and could constitute a new system for the development of therapeutic intervention in obesity.

ADH1B, the adipocyte-enriched alcohol dehydrogenase, plays an essential, cell-autonomous role in human adipogenesis.

Alcohol dehydrogenase 1B (ADH1B) is a primate-specific enzyme which, uniquely among the ADH class 1 family, is highly expressed both in adipose tissue and liver. Its expression in adipose tissue is reduced in obesity and increased by insulin stimulation. Interference with ADH1B expression has also been reported to impair adipocyte function. To better understand the role of ADH1B in adipocytes, we used CRISPR/Cas9 to delete ADH1B in human adipose stem cells (ASC). Cells lacking ADH1B failed to differentiate into mature adipocytes manifested by minimal triglyceride accumulation and a marked reduction in expression of established adipocyte markers. As ADH1B is capable of converting retinol to retinoic acid (RA), we conducted rescue experiments. Incubation of ADH1B-deficient preadipocytes with 9-cis-RA, but not with all-transretinol, significantly rescued their ability to accumulate lipids and express markers of adipocyte differentiation. A homozygous missense variant in ADH1B (p.Arg313Cys) was found in a patient with congenital lipodystrophy of unknown cause. This variant significantly impaired the protein's dimerization, enzymatic activity, and its ability to rescue differentiation in ADH1B-deficient ASC. The allele frequency of this variant in the Middle Eastern population suggests that it is unlikely to be a fully penetrant cause of severe lipodystrophy. In conclusion, ADH1B appears to play an unexpected, crucial and cell-autonomous role in human adipocyte differentiation by serving as a necessary source of endogenous retinoic acid.

miR-26 suppresses adipocyte progenitor differentiation and fat production by targeting Fbxl19.

Fat storage in adult mammals is a highly regulated process that involves the mobilization of adipocyte progenitor cells (APCs) that differentiate to produce new adipocytes. Here we report a role for the broadly conserved miR-26 family of microRNAs (miR-26a-1, miR-26a-2, and miR-26b) as major regulators of APC differentiation and adipose tissue mass. Deletion of all miR-26-encoding loci in mice resulted in a dramatic expansion of adipose tissue in adult animals fed normal chow. Conversely, transgenic overexpression of miR-26a protected mice from high-fat diet-induced obesity. These effects were attributable to a cell-autonomous function of miR-26 as a potent inhibitor of APC differentiation. miR-26 blocks adipogenesis, at least in part, by repressing expression of Fbxl19, a conserved miR-26 target without a previously known role in adipocyte biology that encodes a component of SCF-type E3 ubiquitin ligase complexes. These findings have therefore revealed a novel pathway that plays a critical role in regulating adipose tissue formation in vivo and suggest new potential therapeutic targets for obesity and related disorders.

The P2Y2 receptor mediates terminal adipocyte differentiation and insulin resistance: Evidence for a dual G-protein coupling mode.

Several P2Y nucleotide receptors have been shown to be involved in the early stage of adipocyte differentiation in vitro and insulin resistance in obese mice; however, the exact receptor subtype(s) and its underlying molecular mechanism in relevant human cells are unclear. Here, using human primary visceral preadipocytes as a model, we found that during preadipocyte-to-mature adipocyte differentiation, the P2Y2 nucleotide receptor (P2Y2R) was the most upregulated subtype among the eight known P2Y receptors and the only one further dramatically upregulated after inflammatory TNFα treatment. Functional studies indicated that the P2Y2R induced intracellular Ca2+, ERK1/2, and JNK signaling but not the p38 pathway. In addition, stimulation of the P2Y2R suppressed basal and insulin-induced phosphorylation of AKT, accompanied by decreased GLUT4 membrane translocation and glucose uptake in mature adipocytes, suggesting a role of P2Y2R in insulin resistance. Mechanistically, we found that activation of P2Y2R did not increase lipolysis but suppressed PIP3 generation. Interestingly, activation of P2Y2R triggered Gi-protein coupling, and pertussis toxin pretreatment largely inhibited P2Y2R-mediated ERK1/2 signaling and cAMP suppression. Further, treatment of the cells with AR-C 118925XX, a selective P2Y2R antagonist, significantly inhibited adipogenesis, and P2Y2R knockout decreased mouse body weight gain with smaller eWAT mass infiltrated with fewer macrophages as compared to WT mice in response to a Western diet. Thus, we revealed that terminal adipocyte differentiation and inflammation selectively upregulate P2Y2R expression and that P2Y2R mediates insulin resistance by suppressing the AKT signaling pathway, highlighting P2Y2R as a potential new drug target to combat obesity and type-2 diabetes.

Reorganization of the DNA replication landscape during adipogenesis is closely linked with adipogenic gene expression.

The temporal order of DNA replication along the chromosomes is thought to reflect the transcriptional competence of the genome. During differentiation of mouse 3T3-L1 cells into adipocytes, cells undergo one or two rounds of cell division called mitotic clonal expansion (MCE). MCE is an essential step for adipogenesis; however, little is known about the regulation of DNA replication during this period. Here, we performed genome-wide mapping of replication timing (RT) in mouse 3T3-L1 cells before and during MCE, and identified a number of chromosomal regions shifting toward either earlier or later replication through two rounds of replication. These RT changes were confirmed in individual cells by single-cell DNA-replication sequencing. Coordinate changes between a shift toward earlier replication and transcriptional activation of adipogenesis-associated genes were observed. RT changes occurred before the full expression of these genes, indicating that RT reorganization might contribute to the mature adipocyte phenotype. To support this, cells undergoing two rounds of DNA replication during MCE had a higher potential to differentiate into lipid droplet-accumulating adipocytes, compared with cells undergoing a single round of DNA replication and non-replicating cells.

Metabolome analysis reveals that cyclic adenosine diphosphate ribose contributes to the regulation of differentiation in mice adipocyte.

Adipocytes play a key role in energy storage and homeostasis. Although the role of transcription factors in adipocyte differentiation is known, the effect of endogenous metabolites of low molecular weight remains unclear. Here, we analyzed time-dependent changes in the levels of these metabolites throughout adipocyte differentiation, using metabolome analysis, and demonstrated that there is a positive correlation between cyclic adenosine diphosphate ribose (cADPR) and Pparγ mRNA expression used as a marker of differentiation. We also found that the treatment of C3H10T1/2 adipocytes with cADPR increased the mRNA expression of those marker genes and the accumulation of triglycerides. Furthermore, inhibition of ryanodine receptors (RyR), which are activated by cADPR, caused a significant reduction in mRNA expression levels of the marker genes and triglyceride accumulation in adipocytes. Our findings show that cADPR accelerates adipocytic differentiation via RyR pathway.

Regulatory role of exosome-derived miRNAs and other contents in adipogenesis.

Intramuscular fat (IMF) content significantly impacts meat quality. influenced by complex interactions between skeletal muscle cells and adipocytes. Adipogenesis plays a pivotal role in IMF formation. Exosomes, extracellular membranous nanovesicles, facilitate intercellular communication by transporting proteins, nucleic acids (DNA and RNA), and other biomolecules into target cells, thereby modulating cellular behaviors. Recent studies have linked exosome-derived microRNAs (miRNAs) and other cargo to adipogenic processes. Various cell types, including skeletal muscle cells, interact with adipocytes via exosome secretion and uptake. Exosomes entering adipocytes regulate adipogenesis by modulating key signaling pathways, thereby influencing the extent and distribution of IMF deposition. This review comprehensively explores the origin, formation, and mechanisms of exosome action, along with current research and their applications in adipogenesis. Emphasis is placed on exosome-mediated regulation of miRNAs, non-coding RNAs (ncRNAs), proteins, lipids, and other biomolecules during adipogenesis. Leveraging exosomal contents for genetic breeding and treating obesity-related disorders is discussed. Insights gathered contribute to advancing understanding and potential therapeutic applications of exosome-regulated adipogenesis mechanisms.

Integration of single-cell datasets depicts profiles of macrophages and fibro/adipogenic progenitors in dystrophic muscle.

Single-cell technologies have recently expanded the possibilities for researchers to gain, at an unprecedented resolution level, knowledge about tissue composition, cell complexity, and heterogeneity. Moreover, the integration of data coming from different technologies and sources also offers, for the first time, the possibility to draw a holistic portrait of how cells behave to sustain tissue physiology during the human lifespan and disease. Here, we interrogated and integrated publicly available single-cell RNAseq data to advance the understanding of how macrophages, fibro/adipogenic progenitors, and other cell types establish gene regulatory networks and communicate with each other in the muscle tissue. We identified altered gene signatures and signaling pathways associated with the dystrophic condition, including an enhanced Spp1-Cd44 signaling in dystrophic macrophages. We shed light on the differences among dystrophic muscle aging, considering wild type, mdx, and more severe conditions as in the case of the mdx-2d model. Contextually, we provided details on existing communication relations between muscle niche cell populations, highlighting increased interactions and distinct signaling events that these cells stablish in the dystrophic microenvironment. We believe our findings can help scientists to formulate and test new hypotheses by moving towards a more complete understanding of muscle regeneration and immune system biology.

IGF2BP3-mediated enhanced stability of MYLK represses MSC adipogenesis and alleviates obesity and insulin resistance in HFD mice.

Mesenchymal stem cells (MSCs) hold immense potential as multipotent stem cells and serve as a primary source of adipocytes. The process of MSC adipogenesis plays a crucial role in maintaining systemic metabolic homeostasis and has garnered significant attention in tissue bioengineering. N6-methyladenosine (m6A), the most prevalent RNA modification, is known to regulate cell fate and disease. However, the precise involvement of m6A readers in MSC adipogenesis remains unclear. In this study, we investigated the impact of IGF2BP3, a prominent m6A reader, on MSC adipogenesis. Our findings revealed a decrease in IGF2BP3 expression during the natural adipogenic differentiation of MSCs. Furthermore, IGF2BP3 was found to repress MSC adipogenesis by augmenting the levels of MYLK, a calcium/calmodulin-dependent kinase. Mechanistically, IGF2BP3 interacted with MYLK mRNA in an m6A-dependent manner, extending its half-life and subsequently inhibiting the phosphorylation of the ERK1/2 pathway, thereby impeding the adipogenic differentiation of MSCs. Additionally, we successfully achieved the overexpression of IGF2BP3 through intraperitoneal injection of adeno-associated virus serotype Rec2, which specifically targeted adipose tissue. This intervention resulted in reduced body weight and improved insulin resistance in high-fat diet mice. Overall, our study provides novel insights into the role of IGF2BP3 in MSC adipogenesis, shedding light on adipocyte-related disorders and presenting potential targets for related biomedical applications.

EEF1B2 regulates bone marrow-derived mesenchymal stem cells bone-fat balance via Wnt/ß-catenin signaling.

The pathological advancement of osteoporosis is caused by the uneven development of bone marrow-derived mesenchymal stem cells (BMSCs) in terms of osteogenesis and adipogenesis. While the role of EEF1B2 in intellectual disability and tumorigenesis is well established, its function in the bone-fat switch of BMSCs is still largely unexplored. During the process of osteogenic differentiation, we observed an increase in the expression of EEF1B2, while a decrease in its expression was noted during adipogenesis. Suppression of EEF1B2 hindered the process of osteogenic differentiation and mineralization while promoting adipogenic differentiation. On the contrary, overexpression of EEF1B2 enhanced osteogenesis and strongly inhibited adipogenesis. Furthermore, the excessive expression of EEF1B2 in the tibias has the potential to mitigate bone loss and decrease marrow adiposity in mice with osteoporosis. In terms of mechanism, the suppression of ß-catenin activity occurred when EEF1B2 function was suppressed during osteogenesis. Our collective findings indicate that EEF1B2 functions as a regulator, influencing the differentiation of BMSCs and maintaining a balance between bone and fat. Our finding highlights its potential as a therapeutic target for diseases related to bone metabolism.

AMP-activated protein kinase activation suppresses leptin expression independently of adipogenesis in primary murine adipocytes.

Adipogenesis, defined as the development of mature adipocytes from stem cell precursors, is vital for the expansion, turnover and health of adipose tissue. Loss of adipogenic potential in adipose stem cells, or impairment of adipogenesis is now recognised as an underlying cause of adipose tissue dysfunction and is associated with metabolic disease. In this study, we sought to determine the role of AMP-activated protein kinase (AMPK), an evolutionarily conserved master regulator of energy homeostasis, in adipogenesis. Primary murine adipose-derived stem cells were treated with a small molecule AMPK activator (BI-9774) during key phases of adipogenesis, to determine the effect of AMPK activation on adipocyte commitment, maturation and function. To determine the contribution of the repression of lipogenesis by AMPK in these processes, we compared the effect of pharmacological inhibition of acetyl-CoA carboxylase (ACC). We show that AMPK activation inhibits adipogenesis in a time- and concentration-dependent manner. Transient AMPK activation during adipogenic commitment leads to a significant, ACC-independent, repression of adipogenic transcription factor expression. Furthermore, we identify a striking, previously unexplored inhibition of leptin gene expression in response to both short-term and chronic AMPK activation irrespective of adipogenesis. These findings reveal that in addition to its effect on adipogenesis, AMPK activation switches off leptin gene expression in primary mouse adipocytes independently of adipogenesis. Our results identify leptin expression as a novel target of AMPK through mechanisms yet to be identified.

NOVA1 prevents overactivation of the unfolded protein response and facilitates chromatin access during human white adipogenesis.

The molecular mechanism underlying white adipogenesis in humans has not been fully elucidated beyond the transcriptional level. Here, we found that the RNA-binding protein NOVA1 is required for the adipogenic differentiation of human mesenchymal stem cells. By thoroughly exploring the interactions between NOVA1 and its binding RNA, we proved that NOVA1 deficiency resulted in the aberrant splicing of DNAJC10 with an in-frame premature stop codon, reduced DNAJC10 expression at the protein level and hyperactivation of the unfolded protein response (UPR). Moreover, NOVA1 knockdown abrogated the down-regulation of NCOR2 during adipogenesis and up-regulated the 47b+ splicing isoform, which led to decreased chromatin accessibility at the loci of lipid metabolism genes. Interestingly, these effects on human adipogenesis could not be recapitulated in mice. Further analysis of multispecies genomes and transcriptomes indicated that NOVA1-targeted RNA splicing is evolutionarily regulated. Our findings provide evidence for human-specific roles of NOVA1 in coordinating splicing and cell organelle functions during white adipogenesis.

AFF4 regulates cellular adipogenic differentiation via targeting autophagy.

Transcriptional elongation is a universal and critical step during gene expression. The super elongation complex (SEC) regulates the rapid transcriptional induction by mobilizing paused RNA polymerase II (Pol II). Dysregulation of SEC is closely associated with human diseases. However, the physiological role of SEC during development and homeostasis remains largely unexplored. Here we studied the function of SEC in adipogenesis by manipulating an essential scaffold protein AF4/FMR2 family member 4 (AFF4), which assembles and stabilizes SEC. Knockdown of AFF4 in human mesenchymal stem cells (hMSCs) and mouse 3T3-L1 preadipocytes inhibits cellular adipogenic differentiation. Overexpression of AFF4 enhances adipogenesis and ectopic adipose tissue formation. We further generate Fabp4-cre driven adipose-specific Aff4 knockout mice and find that AFF4 deficiency impedes adipocyte development and white fat depot formation. Mechanistically, we discover AFF4 regulates autophagy during adipogenesis. AFF4 directly binds to autophagy-related protein ATG5 and ATG16L1, and promotes their transcription. Depleting ATG5 or ATG16L1 abrogates adipogenesis in AFF4-overepressing cells, while overexpression of ATG5 and ATG16L1 rescues the impaired adipogenesis in Aff4-knockout cells. Collectively, our results unveil the functional importance of AFF4 in regulating autophagy and adipogenic differentiation, which broaden our understanding of the transcriptional regulation of adipogenesis.

The circadian clock mediates daily bursts of cell differentiation by periodically restricting cell-differentiation commitment.

Most mammalian cells have an intrinsic circadian clock that coordinates metabolic activity with the daily rest and wake cycle. The circadian clock is known to regulate cell differentiation, but how continuous daily oscillations of the internal clock can control a much longer, multiday differentiation process is not known. Here, we simultaneously monitor circadian clock and adipocyte-differentiation progression live in single cells. Strikingly, we find a bursting behavior in the cell population whereby individual preadipocytes commit to differentiate primarily during a 12-h window each day, corresponding to the time of rest. Daily gating occurs because cells irreversibly commit to differentiate within only a few hours, which is much faster than the rest phase and the overall multiday differentiation process. The daily bursts in differentiation commitment result from a differentiation-stimulus driven variable and slow increase in expression of PPARG, the master regulator of adipogenesis, overlaid with circadian boosts in PPARG expression driven by fast, clock-driven PPARG regulators such as CEBPA. Our finding of daily bursts in cell differentiation only during the circadian cycle phase corresponding to evening in humans is broadly relevant, given that most differentiating somatic cells are regulated by the circadian clock. Having a restricted time each day when differentiation occurs may open therapeutic strategies to use timed treatment relative to the clock to promote tissue regeneration.

Regulation of adipogenesis by histone methyltransferases.

Epigenetic regulation is a critical component of lineage determination. Adipogenesis is the process through which uncommitted stem cells or adipogenic precursor cells differentiate into adipocytes, the most abundant cell type of the adipose tissue. Studies examining chromatin modification during adipogenesis have provided further understanding of the molecular blueprint that controls the onset of adipogenic differentiation. Unlike histone acetylation, histone methylation has context dependent effects on the activity of a transcribed region of DNA, with individual or combined marks on different histone residues providing distinct signals for gene expression. Over half of the 42 histone methyltransferases identified in mammalian cells have been investigated in their role during adipogenesis, but across the large body of literature available, there is a lack of clarity over potential correlations or emerging patterns among the different players. In this review, we will summarize important findings from studies published in the past 15 years that have investigated the role of histone methyltransferases during adipogenesis, including both protein arginine methyltransferases (PRMTs) and lysine methyltransferases (KMTs). We further reveal that PRMT1/4/5, H3K4 KMTs (MLL1, MLL3, MLL4, SMYD2 and SET7/9) and H3K27 KMTs (EZH2) all play positive roles during adipogenesis, while PRMT6/7 and H3K9 KMTs (G9a, SUV39H1, SUV39H2, and SETDB1) play negative roles during adipogenesis.

miR-10167-3p targets TCF7L1 to inhibit bovine adipocyte differentiation and promote bovine adipocyte proliferation.

MicroRNAs (miRNAs) are widely involved in various lipogenic processes, including adipocyte proliferation and differentiation, lipid droplet formation, and adipocyte-specific gene activation. The present study aimed to investigate the gene expression profiles of bovine preadipocytes under high miR-10167-3p expression using the RNA-seq technique and to verify the functions of its downstream target genes on the proliferation and differentiation of bovine preadipocytes. First, RNA-seq identified 573 differentially expressed genes (DEGs), of which 243 were downregulated and 330 were upregulated. Then, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that 15.19% of the DEGs were enriched in pathways related to lipid metabolism. Meanwhile, dual-luciferase reporter gene assay verified the target-binding relationship between miR-10167-3p and TCF7L1. The function of TCF7L1 was assessed using several experiments in adipocytes with high TCF7L1 expression and RNA interference. The mRNA and protein expression of proliferation, differentiation, and apoptosis marker genes were detected using qPCR and western blot, respectively; lipid droplet synthesis was detected using oil red O, Nile red, and bodipy staining; adipocyte proliferation was detected by EdU; and apoptosis was detected using flow cytometry. The results revealed that TCF7L1 overexpression inhibited bovine preadipocyte differentiation and apoptosis and promoted their proliferation, with opposite results obtained with its RNA interference. These results may provide a reference for the subsequent investigation of the molecular mechanism of bovine fat deposition.

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.