Metformin Resistance Is Associated with Expression of Inflammatory and Invasive Genes in A549 Lung Cancer Cells.

Metformin, the most commonly used drug for type 2 diabetes, has recently been shown to have beneficial effects in patients with cancer. Despite growing evidence that metformin can inhibit tumor cell proliferation, invasion, and metastasis, studies on drug resistance and its side effects are lacking. Here, we aimed to establish metformin-resistant A549 human lung cancer cells (A549-R) to determine the side effects of metformin resistance. Toward this, we established A549-R by way of prolonged treatment with metformin and examined the changes in gene expression, cell migration, cell cycle, and mitochondrial fragmentation. Metformin resistance is associated with increased G1-phase cell cycle arrest and impaired mitochondrial fragmentation in A549 cells. We demonstrated that metformin resistance highly increased the expression of proinflammatory and invasive genes, including BMP5, CXCL3, VCAM1, and POSTN, using RNA-seq analysis. A549-R exhibited increased cell migration and focal adhesion formation, suggesting that metformin resistance may potentially lead to metastasis during anti-cancer therapy with metformin. Taken together, our findings indicate that metformin resistance may lead to invasion in lung cancer cells.

MLL3/MLL4 methyltransferase activities control early embryonic development and embryonic stem cell differentiation in a lineage-selective manner.

H3K4me1 methyltransferases MLL3 (KMT2C) and MLL4 (KMT2D) are critical for enhancer activation, cell differentiation and development. However, roles of MLL3/4 enzymatic activities and MLL3/4-mediated enhancer H3K4me1 in these processes remain unclear. Here we report that constitutive elimination of both MLL3 and MLL4 enzymatic activities prevents initiation of gastrulation and leads to early embryonic lethality in mice. However, selective elimination of MLL3/4 enzymatic activities in embryonic, but not extraembryonic, lineages leaves gastrulation largely intact. Consistent with this, embryonic stem cells (ESCs) lacking MLL3/4 enzymatic activities can differentiate toward the three embryonic germ layers but show aberrant differentiation to extraembryonic endoderm (ExEn) and trophectoderm. The failure in ExEn differentiation can be attributed to markedly reduced enhancer-binding of the lineage-determining transcription factor GATA6. Furthermore, we show that MLL3/4-catalyzed H3K4me1 is largely dispensable for enhancer activation during ESC differentiation. Together, our findings suggest a lineage-selective, but enhancer activation-independent, role of MLL3/4 methyltransferase activities in early embryonic development and ESC differentiation.

Transcriptome and computational approaches highlighting the molecular regulation of Zacco platypus induced by mesocosm exposure to common disinfectant chlorine.

Chlorine (Cl2) is a disinfectant often used in swimming pools and water treatment facilities. However, it is released into aquatic ecosystems, where it may harm non-targeted organisms. Here, we performed a mesocosm experiment exposing Zacco platypus (Z. platypus) to biocide Cl2 for 30 days (30 d) at two days' time points 15 days (15 d) and 30 d samples were collected. Here, Z. platypus was exposed to a sublethal concentration (0.1 mg/L) of Cl2, and comparative transcriptomics analyses were performed to determine their response mechanisms at the molecular level. According to RNA sequencing of the whole-body transcriptome, 860 and 1189 differentially expressed genes (DEGs) were identified from the 15 d and 30 d responses to Cl2, respectively. After enrichment analysis of GO (Gene Ontology) functions and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, identified DEGs were demonstrated to be associated with a variety of functions, including "ion binding and transmembrane transporters". Cl2 also induced oxidative stress in Z. platypus by increasing the levels of reactive oxygen species (ROS) while decreasing the catalase (CAT) content and the levels of solute carrier family 22 member 11 (slc22a11), Caspase-8 (casp-8), inducible nitric oxide synthase (NOS2), cytosolic phospholipase A2 gamma (PLA2G4). However, Z. platypus still allows recovery during stress suspension by increasing the expression levels of solute carrier family proteins. The GO and KEGG annotation results revealed that the expression of DEGs were related to the detoxification process, immune response, and antioxidant mechanism. Additionally, protein-protein interaction networks (PPI) and cytoHubba analyses identified sixteen hub genes and their interaction. These findings elucidate the regulation of various DEGs and signaling pathways in response to Cl2 exposure, which will improve our knowledge and laid foundation for further investigation of the toxicity of Cl2 to Z. platypus.

α-ketoglutarate suppresses immediate early gene expression in cancer cells.

Cancer cells exhibit increased glutamine consumption compared to normal cells, supporting cell survival and proliferation. Glutamine is converted to α-ketoglutarate (αKG), which then enters the tricarboxylic acid cycle to generate ATP. Recently, therapeutic modulation of glutamine metabolism has become an attractive metabolic anti-cancer strategy. However, how synergistic combination therapy is required to overcome glutamine metabolism drug resistance remains elusive. To address this issue, we first investigated the role of αKG in regulating gene expression in several cancer cell lines. Using RNA-seq analysis and histone modification screening, we demonstrated that αKG reduced the expression of the immediate early gene (IEG) in cancer cells in an H3K27 acetylation-dependent manner. Conversely, glutaminase (GLS) inhibitors induce IEG expression in cancer cells. Furthermore, we showed that siRNA knockdown of orphan nuclear receptor subfamily 4 group A member 1 (NR4A1) induces IEG expression. Notably, the NR4A1 agonist cytosporone B sensitizes GLS inhibitor resistance to cancer cell death. Together, these findings indicate that therapeutic targeting of IEG dysregulation by αKG can be a potentially effective anti-cancer therapeutic strategy for glutamine metabolism inhibitors.

Cathepsin D as a potential therapeutic target to enhance anticancer drug-induced apoptosis via RNF183-mediated destabilization of Bcl-xL in cancer cells.

Cathepsin D (Cat D) is well known for its roles in metastasis, angiogenesis, proliferation, and carcinogenesis in cancer. Despite Cat D being a promising target in cancer cells, effects and underlying mechanism of its inhibition remain unclear. Here, we investigated the plausibility of using Cat D inhibition as an adjuvant or sensitizer for enhancing anticancer drug-induced apoptosis. Inhibition of Cat D markedly enhanced anticancer drug-induced apoptosis in human carcinoma cell lines and xenograft models. The inhibition destabilized Bcl-xL through upregulation of the expression of RNF183, an E3 ligase of Bcl-xL, via NF-κB activation. Furthermore, Cat D inhibition increased the proteasome activity, which is another important factor in the degradation of proteins. Cat D inhibition resulted in p62-dependent activation of Nrf2, which increased the expression of proteasome subunits (PSMA5 and PSMB5), and thereby, the proteasome activity. Overall, Cat D inhibition sensitized cancer cells to anticancer drugs through the destabilization of Bcl-xL. Furthermore, human renal clear carcinoma (RCC) tissues revealed a positive correlation between Cat D and Bcl-xL expression, whereas RNF183 and Bcl-xL expression indicated inverse correlation. Our results suggest that inhibition of Cat D is promising as an adjuvant or sensitizer for enhancing anticancer drug-induced apoptosis in cancer cells.

Proactive Handover Decision for UAVs with Deep Reinforcement Learning.

The applications of Unmanned Aerial Vehicles (UAVs) are rapidly growing in domains such as surveillance, logistics, and entertainment and require continuous connectivity with cellular networks to ensure their seamless operations. However, handover policies in current cellular networks are primarily designed for ground users, and thus are not appropriate for UAVs due to frequent fluctuations of signal strength in the air. This paper presents a novel handover decision scheme deploying Deep Reinforcement Learning (DRL) to prevent unnecessary handovers while maintaining stable connectivity. The proposed DRL framework takes the UAV state as an input for a proximal policy optimization algorithm and develops a Received Signal Strength Indicator (RSSI) based on a reward function for the online learning of UAV handover decisions. The proposed scheme is evaluated in a 3D-emulated UAV mobility environment where it reduces up to 76 and 73% of unnecessary handovers compared to greedy and Q-learning-based UAV handover decision schemes, respectively. Furthermore, this scheme ensures reliable communication with the UAV by maintaining the RSSI above -75 dBm more than 80% of the time.

CityGML urban model generation using national public datasets for flood damage simulations: A case study in Korea.

Managing information at city level has become increasingly important owing to the introduction of smart cities and the increasing severity of disasters due to climate change. A data collection framework, model construction, and information management must be established to systematically manage information at the city level. This study developed an urban model generation method using detailed attributes within the City Geography Markup Language (CityGML), a standard data schema for 3D representation of cities based on different types of publicly available information within Korea. The generated model was used to develop a method for simulating flooding status, degree of flooding, and level of building damage after heavy rainfall, in Korea. Furthermore, we developed a method to estimate the loss of human life and property damage by combining the results of the flood analysis with the city model. The proposed methodology supports the creation of standard-based models for flood analysis and exhibits strong interoperability for application to different areas of analysis.

MED1 is a lipogenesis coactivator required for postnatal adipose expansion.

MED1 often serves as a surrogate of the general transcription coactivator complex Mediator for identifying active enhancers. MED1 is required for phenotypic conversion of fibroblasts to adipocytes in vitro, but its role in adipose development and expansion in vivo has not been reported. Here, we show that MED1 is not generally required for transcription during adipogenesis in culture and that MED1 is dispensable for adipose development in mice. Instead, MED1 is required for postnatal adipose expansion and the induction of fatty acid and triglyceride synthesis genes after pups switch diet from high-fat maternal milk to carbohydrate-based chow. During adipogenesis, MED1 is dispensable for induction of lineage-determining transcription factors (TFs) PPARγ and C/EBPα but is required for lipid accumulation in the late phase of differentiation. Mechanistically, MED1 controls the induction of lipogenesis genes by facilitating lipogenic TF ChREBP- and SREBP1a-dependent recruitment of Mediator to active enhancers. Together, our findings identify a cell- and gene-specific regulatory role of MED1 as a lipogenesis coactivator required for postnatal adipose expansion.

Deletion of phospholipase D1 decreases bone mass and increases fat mass via modulation of Runx2, ß-catenin-osteoprotegerin, PPAR-γ and C/EBPα signaling axis.

In osteoporosis, mesenchymal stem cells (MSCs) prefer to differentiate into adipocytes at the expense of osteoblasts. Although the balance between adipogenesis and osteogenesis has been closely examined, the mechanism of commitment determination switch is unknown. Here we demonstrate that phospholipase D1 (PLD1) plays a key switch in determining the balance between bone and fat mass. Ablation of Pld1 reduced bone mass but increased fat in mice. Mechanistically, Pld1/- MSCs inhibited osteoblast differentiaion with diminished Runx2 expression, while osteoclast differentiation was accelerated in Pld1-/- bone marrow-derived macrophages. Pld1-/- osteoblasts showed decreased expression of osteogenic makers. Increased number and resorption activity of osteoclasts in Pld1-/- mice were corroborated with upregulation of osteoclastogenic markers. Moreover, Pld1-/- osteoblasts reduced ß-catenin mediated-osteoprotegerin (OPG) with increased RANKL/OPG ratio which resulted in accelerated osteoclast differentiation. Thus, low bone mass with upregulated osteoclasts could be due to the contribution of both osteoblasts and osteoclasts during bone remodeling. Moreover, ablation of Pld1 further increased bone loss in ovariectomized mice, suggesting that PLD1 is a negative regulator of osteoclastogenesis. Furthermore, loss of PLD1 increased adipogenesis, body fat mass, and hepatic steatosis along with upregulation of PPAR-γ and C/EBPα. Interestingly, adipocyte-specific Pld1 transgenic mice rescued the compromised phenotypes of fat mass and adipogenesis in Pld1 knockout mice. Collectively, PLD1 regulated the bifurcating pathways of mesenchymal cell lineage into increased osteogenesis and decreased adipogenesis, which uncovered a previously unrecognized role of PLD1 in homeostasis between bone and fat mass.

Phospholipase D1 is upregulated by vorinostat and confers resistance to vorinostat in glioblastoma.

Glioblastoma (GBM) is an aggressive brain tumor and drug resistance remains a major barrier for therapeutics. Epigenetic alterations are implicated in GBM pathogenesis, and epigenetic modulators including histone deacetylase (HDAC) inhibitors are exploited as promising anticancer therapies. Here, we demonstrate that phospholipase D1 (PLD1) is a transcriptional target of HDAC inhibitors and confers resistance to HDAC inhibitor in GBM. Treatment of vorinostat upregulates PLD1 through PKCζ-Sp1 axis. Vorinostat induces dynamic changes in the chromatin structure and transcriptional machinery associated with PLD1 promoter region. Cotreatment of vorinostat with PLD1 inhibitor further attenuates invasion, angiogenesis, colony-forming capacity, and self-renewal capacity, compared with those of either treatment. PLD1 inhibitor overcomes resistance to vorinostat in GBM cells intracranial GBM tumors. Our finding provides new insight into the role of PLD1 as a target of resistance to vorinostat, and PLD1 inhibitor might provide the basis for therapeutic combinations with improved efficacy of HDAC inhibitor.

Inhibition of phospholipase D2 augments histone deacetylase inhibitor-induced cell death in breast cancer cells.

BACKGROUND: Histone deacetylase (HDAC) inhibitors are promising anticancer drugs but their effect on tumor treatment has been disappointing mainly due to the acquisition of HDAC inhibitor resistance. However, the mechanisms underlying such resistance remain unclear. METHODS: In this study, we performed Western blot, q-PCR, and promoter assay to examine the expression of HDAC inhibitor-induced phospholipase D2 (PLD2) in MDA-MB231and MDA-MB435 breast cancer cells. Apoptosis and proliferation were analyzed by flow cytometry. In addition to invasion and migration assay, angiogenesis was further measured using in vitro tube formation and chick embryo chorioallantoic membrane model. RESULTS: HDAC inhibitors including suberoylanilide hydroxamic acid (SAHA), trichostatin, and apicidin, induce expression of PLD2 in a transcriptional level. SAHA upregulates expression of PLD2 via protein kinase C-ζ in breast cancer cells and increases the enzymatic activity of PLD. The combination treatment of SAHA with PLD2 inhibitor significantly enhances cell death in breast cancer cells. Phosphatidic acid, a product of PLD activity, prevented apoptosis promoted by cotreatment with SAHA and PLD2 inhibitor, suggesting that SAHA-induced PLD2 expression and subsequent activation of PLD2 might confers resistance of breast cancer cells to HDAC inhibitor. The combinational treatment of the drugs significantly suppressed invasion, migration, and angiogenesis, compared with that of either treatment. CONCLUSION: These findings provide further insight into elucidating the advantages of combination therapy with HDAC and PLD2 inhibitors over single-agent strategies for the treatment of cancer.

Selective binding of the PHD6 finger of MLL4 to histone H4K16ac links MLL4 and MOF.

Histone methyltransferase MLL4 is centrally involved in transcriptional regulation and is often mutated in human diseases, including cancer and developmental disorders. MLL4 contains a catalytic SET domain that mono-methylates histone H3K4 and seven PHD fingers of unclear function. Here, we identify the PHD6 finger of MLL4 (MLL4-PHD6) as a selective reader of the epigenetic modification H4K16ac. The solution NMR structure of MLL4-PHD6 in complex with a H4K16ac peptide along with binding and mutational analyses reveal unique mechanistic features underlying recognition of H4K16ac. Genomic studies show that one third of MLL4 chromatin binding sites overlap with H4K16ac-enriched regions in vivo and that MLL4 occupancy in a set of genomic targets depends on the acetyltransferase activity of MOF, a H4K16ac-specific acetyltransferase. The recognition of H4K16ac is conserved in the PHD7 finger of paralogous MLL3. Together, our findings reveal a previously uncharacterized acetyllysine reader and suggest that selective targeting of H4K16ac by MLL4 provides a direct functional link between MLL4, MOF and H4K16 acetylation.

Somatic Mutations Increase Hepatic Clonal Fitness and Regeneration in Chronic Liver Disease.

Normal tissues accumulate genetic changes with age, but it is unknown if somatic mutations promote clonal expansion of non-malignant cells in the setting of chronic degenerative diseases. Exome sequencing of diseased liver samples from 82 patients revealed a complex mutational landscape in cirrhosis. Additional ultra-deep sequencing identified recurrent mutations in PKD1, PPARGC1B, KMT2D, and ARID1A. The number and size of mutant clones increased as a function of fibrosis stage and tissue damage. To interrogate the functional impact of mutated genes, a pooled in vivo CRISPR screening approach was established. In agreement with sequencing results, examination of 147 genes again revealed that loss of Pkd1, Kmt2d, and Arid1a promoted clonal expansion. Conditional heterozygous deletion of these genes in mice was also hepatoprotective in injury assays. Pre-malignant somatic alterations are often viewed through the lens of cancer, but we show that mutations can promote regeneration, likely independent of carcinogenesis.

H3.3K4M destabilizes enhancer H3K4 methyltransferases MLL3/MLL4 and impairs adipose tissue development.

Histone 3 lysine 4 (H3K4) methyltransferases MLL3 and MLL4 (MLL3/4) are required for enhancer activation during cell differentiation, though the mechanism is incompletely understood. We have attempted to address this issue by generating two mouse lines: one expressing H3.3K4M, a lysine-4-to-methionine (K4M) mutation of histone H3.3 that inhibits H3K4 methylation, and the other carrying conditional double knockout of MLL3/4 enzymatic SET domain. Expression of H3.3K4M in lineage-specific precursor cells depletes H3K4 methylation and impairs adipose tissue and muscle development. Mechanistically, H3.3K4M prevents enhancer activation in adipogenesis by destabilizing MLL3/4 proteins but not other Set1-like H3K4 methyltransferases MLL1, MLL2, SET1A and SET1B. Notably, deletion of the enzymatic SET domain in lineage-specific precursor cells mimics H3.3K4M expression, destabilizes MLL3/4 proteins, and prevents adipose tissue and muscle development. Interestingly, destabilization of MLL3/4 by H3.3K4M in adipocytes does not affect adipose tissue maintenance and thermogenic function. Together, our findings indicate that expression of H3.3K4M, or deletion of the enzymatic SET domain, destabilizes enhancer H3K4 methyltransferases MLL3/4 and impairs adipose tissue and muscle development.

HOXA9 Reprograms the Enhancer Landscape to Promote Leukemogenesis.

Aberrant expression of HOXA9 is a prominent feature of acute leukemia driven by diverse oncogenes. Here we show that HOXA9 overexpression in myeloid and B progenitor cells leads to significant enhancer reorganizations with prominent emergence of leukemia-specific de novo enhancers. Alterations in the enhancer landscape lead to activation of an ectopic embryonic gene program. We show that HOXA9 functions as a pioneer factor at de novo enhancers and recruits CEBPα and the MLL3/MLL4 complex. Genetic deletion of MLL3/MLL4 blocks histone H3K4 methylation at de novo enhancers and inhibits HOXA9/MEIS1-mediated leukemogenesis in vivo. These results suggest that therapeutic targeting of HOXA9-dependent enhancer reorganization can be an effective therapeutic strategy in acute leukemia with HOXA9 overexpression.

Depletion of Nsd2-mediated histone H3K36 methylation impairs adipose tissue development and function.

The epigenetic mechanisms regulating adipose tissue development and function are poorly understood. In this study, we show that depletion of histone H3K36 methylation by H3.3K36M in preadipocytes inhibits adipogenesis by increasing H3K27me3 to prevent the induction of C/EBPα and other targets of the master adipogenic transcription factor peroxisome proliferator-activated receptor-γ (PPARγ). Depleting H3K36 methyltransferase Nsd2, but not Nsd1 or Setd2, phenocopies the effects of H3.3K36M on adipogenesis and PPARγ target expression. Consistently, expression of H3.3K36M in progenitor cells impairs brown adipose tissue (BAT) and muscle development in mice. In contrast, depletion of histone H3K36 methylation by H3.3K36M in adipocytes in vivo does not affect adipose tissue weight, but leads to profound whitening of BAT and insulin resistance in white adipose tissue (WAT). These mice are resistant to high fat diet-induced WAT expansion and show severe lipodystrophy. Together, these results suggest a critical role of Nsd2-mediated H3K36 methylation in adipose tissue development and function.

Brd4 binds to active enhancers to control cell identity gene induction in adipogenesis and myogenesis.

The epigenomic reader Brd4 is an important drug target for cancers. However, its role in cell differentiation and animal development remains largely unclear. Using two conditional knockout mouse strains and derived cells, we demonstrate that Brd4 controls cell identity gene induction and is essential for adipogenesis and myogenesis. Brd4 co-localizes with lineage-determining transcription factors (LDTFs) on active enhancers during differentiation. LDTFs coordinate with H3K4 mono-methyltransferases MLL3/MLL4 (KMT2C/KMT2D) and H3K27 acetyltransferases CBP/p300 to recruit Brd4 to enhancers activated during differentiation. Brd4 deletion prevents the enrichment of Mediator and RNA polymerase II transcription machinery, but not that of LDTFs, MLL3/MLL4-mediated H3K4me1, and CBP/p300-mediated H3K27ac, on enhancers. Consequently, Brd4 deletion prevents enhancer RNA production, cell identity gene induction and cell differentiation. Interestingly, Brd4 is dispensable for maintaining cell identity genes in differentiated cells. These findings identify Brd4 as an enhancer epigenomic reader that links active enhancers with cell identity gene induction in differentiation.

MLL4 prepares the enhancer landscape for Foxp3 induction via chromatin looping.

MLL4 is an essential subunit of the histone H3 Lys4 (H3K4)-methylation complexes. We found that MLL4 deficiency compromised the development of regulatory T cells (Treg cells) and resulted in a substantial decrease in monomethylated H3K4 (H3K4me1) and chromatin interaction at putative gene enhancers, a considerable portion of which were not direct targets of MLL4 but were enhancers that interacted with MLL4-bound sites. The decrease in H3K4me1 and chromatin interaction at the enhancers not bound by MLL4 correlated with MLL4 binding at distant interacting regions. Deletion of an upstream MLL4-binding site diminished the abundance of H3K4me1 at the regulatory elements of the gene encoding the transcription factor Foxp3 that were looped to the MLL4-binding site and compromised both the thymic differentiation and the inducible differentiation of Treg cells. We found that MLL4 catalyzed methylation of H3K4 at distant unbound enhancers via chromatin looping, which identifies a previously unknown mechanism for regulating the T cell enhancer landscape and affecting Treg cell differentiation.

Histone H3 lysine 4 methyltransferase KMT2D.

Histone-lysine N-methyltransferase 2D (KMT2D), also known as MLL4 and MLL2 in humans and Mll4 in mice, belongs to a family of mammalian histone H3 lysine 4 (H3K4) methyltransferases. It is a large protein over 5500 amino acids in size and is partially functionally redundant with KMT2C. KMT2D is widely expressed in adult tissues and is essential for early embryonic development. The C-terminal SET domain is responsible for its H3K4 methyltransferase activity and is necessary for maintaining KMT2D protein stability in cells. KMT2D associates with WRAD (WDR5, RbBP5, ASH2L, and DPY30), NCOA6, PTIP, PA1, and H3K27 demethylase UTX in one protein complex. It acts as a scaffold protein within the complex and is responsible for maintaining the stability of UTX. KMT2D is a major mammalian H3K4 mono-methyltransferase and co-localizes with lineage determining transcription factors on transcriptional enhancers. It is required for the binding of histone H3K27 acetyltransferases CBP and p300 on enhancers, enhancer activation and cell-type specific gene expression during differentiation. KMT2D plays critical roles in regulating development, differentiation, metabolism, and tumor suppression. It is frequently mutated in developmental diseases, such as Kabuki syndrome and congenital heart disease, and various forms of cancer. Further understanding of the mechanism through which KMT2D regulates gene expression will reveal why KMT2D mutations are so harmful and may help generate novel therapeutic approaches.

MLL3/MLL4 are required for CBP/p300 binding on enhancers and super-enhancer formation in brown adipogenesis.

Histone H3K4me1/2 methyltransferases MLL3/MLL4 and H3K27 acetyltransferases CBP/p300 are major enhancer epigenomic writers. To understand how these epigenomic writers orchestrate enhancer landscapes in cell differentiation, we have profiled genomic binding of MLL4, CBP, lineage-determining transcription factors (EBF2, C/EBPß, C/EBPα, PPARγ), coactivator MED1, RNA polymerase II, as well as epigenome (H3K4me1/2/3, H3K9me2, H3K27me3, H3K36me3, H3K27ac), transcriptome and chromatin opening during adipogenesis of immortalized preadipocytes derived from mouse brown adipose tissue (BAT). We show that MLL4 and CBP drive the dynamic enhancer epigenome, which correlates with the dynamic transcriptome. MLL3/MLL4 are required for CBP/p300 binding on enhancers activated during adipogenesis. Further, MLL4 and CBP identify super-enhancers (SEs) of adipogenesis and that MLL3/MLL4 are required for SE formation. Finally, in brown adipocytes differentiated in culture, MLL4 identifies primed SEs of genes fully activated in BAT such as Ucp1. Comparison of MLL4-defined SEs in brown and white adipogenesis identifies brown-specific SE-associated genes that could be involved in BAT functions. These results establish MLL3/MLL4 and CBP/p300 as master enhancer epigenomic writers and suggest that enhancer-priming by MLL3/MLL4 followed by enhancer-activation by CBP/p300 sequentially shape dynamic enhancer landscapes during cell differentiation. Our data also provide a rich resource for understanding epigenomic regulation of brown adipogenesis.