Dietary Folate and Cofactors Accelerate Age-dependent p16 Epimutation to Promote Intestinal Tumorigenesis.

The extent to which non-genetic environmental factors, such as diet, contribute to carcinogenesis has been long debated. One potential mechanism for the effects of environmental factors is through epigenetic modifications that affect gene expression without changing the underlying DNA sequence. However, the functional cooperation between dietary factors and cancer-causing epigenetic regulation is largely unknown. Here, we use a mouse model of age-dependent p16 epimutation, in which the p16 gene activity is directly controlled by promoter DNA methylation. We show p16 epimutation is modulated by folate and cofactors in dietary supplementation, which leads to increased colon cancer risk. Importantly, our findings provide functional evidence concerning the safety of folate fortification in the general population. SIGNIFICANCE: Our study demonstrates that dietary folate and cofactors modulate tumor-suppressor gene methylation to increase intestinal tumorigenesis. Our findings highlight the need for monitoring the long-term safety of folate fortification in high-risk individuals.

Functional characterization of age-dependent p16 epimutation reveals biological drivers and therapeutic targets for colorectal cancer.

BACKGROUND: Methylation of the p16 promoter resulting in epigenetic gene silencing-known as p16 epimutation-is frequently found in human colorectal cancer and is also common in normal-appearing colonic mucosa of aging individuals. Thus, to improve clinical care of colorectal cancer (CRC) patients, we explored the role of age-related p16 epimutation in intestinal tumorigenesis. METHODS: We established a mouse model that replicates two common genetic and epigenetic events observed in human CRCs: Apc mutation and p16 epimutation. We conducted long-term survival and histological analysis of tumor development and progression. Colonic epithelial cells and tumors were collected from mice and analyzed by RNA sequencing (RNA-seq), quantitative PCR, and flow cytometry. We performed single-cell RNA sequencing (scRNA-seq) to characterize tumor-infiltrating immune cells throughout tumor progression. We tested whether anti-PD-L1 immunotherapy affects overall survival of tumor-bearing mice and whether inhibition of both epigenetic regulation and immune checkpoint is more efficacious. RESULTS: Mice carrying combined Apc mutation and p16 epimutation had significantly shortened survival and increased tumor growth compared to those with Apc mutation only. Intriguingly, colon tumors with p16 epimutation exhibited an activated interferon pathway, increased expression of programmed death-ligand 1 (Pdl1), and enhanced infiltration of immune cells. scRNA-seq further revealed the presence of Foxp3+ Tregs and γδT17 cells, which contribute to an immunosuppressive tumor microenvironment (TME). Furthermore, we showed that a combined therapy using an inhibitor of DNA methylation and a PD-L1 immune checkpoint inhibitor is more effective for improving survival in tumor-bearing mice than blockade of either pathway alone. CONCLUSIONS: Our study demonstrated that age-dependent p16 epimutation creates a permissive microenvironment for malignant transformation of polyps to colon cancer. Our findings provide a mechanistic rationale for future targeted therapy in patients with p16 epimutation.

Discovery of benzo[d]isothiazole derivatives as novel scaffold inhibitors targeting the programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) interaction through "ring fusion" strategy.

The inhibition of programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) interaction by monoclonal antibodies (mAbs) has achieved promising outcomes in cancer immunotherapy. Due to the inherent deficiencies of mAbs drugs, such as high cost of treatment, immunogenicity, poor pharmacokinetics and penetration of solid tumors, researchers are encouraged to develop small molecule inhibitors, to overcome mAbs drugs' deficiencies and change the situation where small molecule drugs are not available on the market. Herein, we reported a series of benzo[d]isothiazole derivatives targeting the PD-1/PD-L1 interaction through "ring fusion" strategy using BMS-202 as a starting point. Among them, compound D7 exhibited the best inhibitory activity with an IC50 value of 5.7 nM by homogeneous time-resolved fluorescence (HTRF) binding assay. In immunotoxicity analysis, D7 showed low cytotoxicity to Jurkat T cells in CCK-8 assay compared to BMS-202. The binding mode between D7 and PD-L1 protein was explored by molecular docking and molecular dynamics (MD) simulations, which revealed crucial chemical groups, such as biphenyl group interacting with Ile54A, Tyr56A, Met115A, Ala121A, Ile54B, Met115B, Ala121B and Tyr123B by hydrophobic interactions, bromobenzene moiety forming π-π stacking interaction with Tyr56B, as well as l-serine moiety forming hydrogen bond (H-bond) and salt bridge interactions with Asp122A and Lys124A. Furthermore, molecular modeling studies showed that D7 is likely to bind to the FA8 (fatty acid 8) binding site of human serum albumin (HSA). Taken together, D7 significantly inhibits the PD-1/PD-L1 interaction with low cytotoxicity, indicating that D7 is a promising starting point for further drug development in cancer immunotherapy.

The concurrence of DNA methylation and demethylation is associated with transcription regulation.

The mammalian DNA methylome is formed by two antagonizing processes, methylation by DNA methyltransferases (DNMT) and demethylation by ten-eleven translocation (TET) dioxygenases. Although the dynamics of either methylation or demethylation have been intensively studied in the past decade, the direct effects of their interaction on gene expression remain elusive. Here, we quantify the concurrence of DNA methylation and demethylation by the percentage of unmethylated CpGs within a partially methylated read from bisulfite sequencing. After verifying 'methylation concurrence' by its strong association with the co-localization of DNMT and TET enzymes, we observe that methylation concurrence is strongly correlated with gene expression. Notably, elevated methylation concurrence in tumors is associated with the repression of 40~60% of tumor suppressor genes, which cannot be explained by promoter hypermethylation alone. Furthermore, methylation concurrence can be used to stratify large undermethylated regions with negligible differences in average methylation into two subgroups with distinct chromatin accessibility and gene regulation patterns. Together, methylation concurrence represents a unique methylation metric important for transcription regulation and is distinct from conventional metrics, such as average methylation and methylation variation.

Drug discovery targeting p21-activated kinase 4 (PAK4): a patent review.

Introduction: The Ser/Thr protein kinase PAK4 is a downstream regulator of Cdc42, mediating cytoskeleton remodeling, and cell motility, and inhibiting apoptosis and transcriptional regulation. Nowadays, efforts in PAK4 inhibitor development are focusing on improving inhibitory selectivity, cellular potency, and in vivo pharmacokinetic properties, and identifying the feasibility of immunotherapy combination in oncology therapy.Areas covered: This review summarized the development of PAK4 inhibitors that reported on patents in the past two decades. According to their binding features, these inhibitors were classified into type I, type I 1/2, and PAMs. Their designing ideas and SAR were elucidated in this review. Moreover, synergistic therapy of PAK4 inhibitors with PD-1/PD-L1 or CAR-T were also summarized .Expert opinion: In the past years, preclinical and clinical studies of PAK4 inhibitors ended in failure due to poor selectivity, cellular activity, or pharmacokinetic issues. There are researchers questioning the reliability of PAK4 as a drug target, particularly PAK4-related therapy is concerned with the distinguishment of the non-kinase functions and catalytic functions triggered by PAK4 phosphorylation. Meanwhile, synergistic effects of PAK4 inhibitors with PD-1/PD-L1 and CAR-T immunotherapy shed light for the development of PAK4 inhibitors.

Epigenome - A mediator for host-microbiome crosstalk.

The interaction between the gut and its eventual trillions of microbe inhabitants during microbial colonization, represents a critical time period for establishing the overall health and wellbeing of an individual. The gut microbiome represents a diverse community of microbes that are critical for many physiological roles of the host including host metabolism. These processes are controlled by a fine-tuned chemical cross talk between the host and microbiota. Although the exact mechanisms behind this cross talk remains elusive, microbiota induced epigenetic mechanisms like DNA methylation and histone modifications may be key. This review presents our perspective on the epigenome as a mediator for host-microbiota cross talk, as well as methodology to study epigenetics, the role of dysbiosis in disease, and how the gut microbiome-host axis may be used in personal medicine.

Predict multicategory causes of death in lung cancer patients using clinicopathologic factors.

BACKGROUND: Random forests (RF) is a widely used machine-learning algorithm, and outperforms many other machine learning algorithms in prediction-accuracy. But it is rarely used for predicting causes of death (COD) in cancer patients. On the other hand, multicategory COD are difficult to classify in lung cancer patients, largely because they have multiple labels (versus binary labels). METHODS: We tuned RF algorithms to classify 5-category COD among the lung cancer patients in the surveillance, epidemiology and end results-18, whose lung cancers were diagnosed in 2004, for the completeness in their follow-up. The patients were randomly divided into training and validation sets (1:1 and 4:1 sample-splits). We compared the prediction accuracy of the tuned RF and multinomial logistic regression (MLR) models. RESULTS: We included 42,257 qualified lung cancers in the database. The COD were lung cancer (72.41%), other causes or alive (14.43%), non-lung cancer (6.85%), cardiovascular disease (5.35%), and infection (0.96%). The tuned RF model with 300 iterations and 10 variables outperformed the MLR model (accuracy = 69.8% vs 64.6%, 1:1 sample-split), while 4:1 sample-split produced lower prediction-accuracy than 1:1 sample-split. The top-10 important factors in the RF model were sex, chemotherapy status, age (65+ vs < 65 years), radiotherapy status, nodal status, T category, histology type and laterality, all of which except T category and laterality were also important in MLR model. CONCLUSION: We tuned RF models to predict 5-category CODs in lung cancer patients, and show RF outperforms MLR in prediction accuracy. We also identified the factors associated with these COD.

A freestanding electrochromic copolymer for multicolor smart window.

Electrochromic devices with low-cost, energy-saving advantages, and controllable color switching have gained widely attention. Yet, electrochromic materials are limited for smart window due to challenges such as difficulty freestanding, monotonous color change, slow switching capability, and low optical contrast. In this work, a freestanding copolymers based on Poly(N-vinylcarbazole) (PVK) and 3, 4-ethoxylenedioxythiophene (EDOT) are designed. The copolymer as-synthesized by the good secondary film-forming of PVK not only contains the freestanding property of PVK, but also possesses the excellent electrical and electrochemical properties of poly(3, 4-ethoxylenedioxythiophene) (PEDOT). The freestanding copolymer was used to create the multicolor: brown, dark brown, purple, and blue. A high optical contrast of up to 39.1% and a color efficiency of up to 107.00 cm 2C-1 prove a significant coloration and bleaching effect, which is satisfactory for the application of electrochromic devices. Further, an electrochromic device based on P(PVK-co-EDOT) as coloring materials is constructed. This work contributes new ideas into the design of electrochromic smart materials.

Classify multicategory outcome in patients with lung adenocarcinoma using clinical, transcriptomic and clinico-transcriptomic data: machine learning versus multinomial models.

Classification of multicategory survival-outcome is important for precision oncology. Machine learning (ML) algorithms have been used to accurately classify multi-category survival-outcome of some cancer-types, but not yet that of lung adenocarcinoma. Therefore, we compared the performances of 3 ML models (random forests, support vector machine [SVM], multilayer perceptron) and multinomial logistic regression (Mlogit) models for classifying 4-category survival-outcome of lung adenocarcinoma using the TCGA. Mlogit model overall performed similar to SVM and multilayer perceptron models (micro-average area under curve=0.82), while random forests model was inferior. Surprisingly, transcriptomic data alone and clinico-transcriptomic data appeared sufficient to accurately classify the 4-category survival-outcome in these patients, but no models using clinical data alone performed well. Notably, NDUFS5, P2RY2, PRPF18, CCL24, ZNF813, MYL6, FLJ41941, POU5F1B, and SUV420H1 were the top-ranked genes that were associated with alive without disease and inversely linked to other outcomes. Similarly, BDKRB2, TERC, DNAJA3, MRPL15, SLC16A13, CRHBP and ACSBG2 were associated with alive with progression and GAL3ST3, AD2, RAB41, HDC, and PLEKHG1 associated with dead with disease, respectively, while also inversely linked other outcomes. These cross-linked genes may be used for risk-stratification and future treatment development.

Study of Environmental Enteropathy and Malnutrition (SEEM) in Pakistan: protocols for biopsy based biomarker discovery and validation.

BACKGROUND: Environmental Enteropathy (EE), characterized by alterations in intestinal structure, function, and immune activation, is believed to be an important contributor to childhood undernutrition and its associated morbidities, including stunting. Half of all global deaths in children < 5 years are attributable to under-nutrition, making the study of EE an area of critical priority. METHODS: Community based intervention study, divided into two sub-studies, 1) Longitudinal analyses and 2) Biopsy studies for identification of EE features via omics analyses. Birth cohorts in Matiari, Pakistan established: moderately or severely malnourished (weight for height Z score (WHZ) < - 2) children, and well-nourished (WHZ > 0) children. Blood, urine, and fecal samples, for evaluation of potential biomarkers, will be collected at various time points from all participants (longitudinal analyses). Participants will receive appropriate educational and nutritional interventions; non-responders will undergo further evaluation to determine eligibility for further workup, including upper gastrointestinal endoscopy. Histopathological changes in duodenal biopsies will be compared with duodenal biopsies obtained from USA controls who have celiac disease, Crohn's disease, or who were found to have normal histopathology. RNA-Seq will be employed to characterize mucosal gene expression across groups. Duodenal biopsies, luminal aspirates from the duodenum, and fecal samples will be analyzed to define microbial community composition (omic analyses). The relationship between histopathology, mucosal gene expression, and community configuration will be assessed using a variety of bioinformatic tools to gain better understanding of disease pathogenesis and to identify mechanism-based biomarkers. Ethical review committees at all collaborating institutions have approved this study. All results will be made available to the scientific community. DISCUSSION: Operational and ethical constraints for safely obtaining intestinal biopsies from children in resource-poor settings have led to a paucity of human tissue-based investigations to understand and reverse EE in vulnerable populations. Furthermore, EE biomarkers have rarely been correlated with gold standard histopathological confirmation. The Study of Environmental Enteropathy and Malnutrition (SEEM) is designed to better understand the pathophysiology, predictors, biomarkers, and potential management strategies of EE to inform strategies to eradicate this debilitating pathology and accelerate progress towards the 2030 Sustainable Development Goals. TRIAL REGISTRATION: Retrospectively registered; clinicaltrials.gov ID NCT03588013 .

Maternal folate genes and aberrant DNA hypermethylation in pediatric acute lymphoblastic leukemia.

BACKGROUND: There is evidence that maternal genotypes in folate-related genes are associated with pediatric acute lymphoblastic leukemia (ALL) independent of offspring genotype. We evaluated the relationship between maternal genotypes in methionine synthase (MTR) and DNA methylation status in ALL to better characterize the molecular mechanism underlying this association. PROCEDURE: We obtained bone marrow samples from 51 patients with ALL at diagnosis and from 6 healthy donors. Mothers of patients provided a saliva sample and were genotyped at 11 tagSNPs in MTR. DNA methylation was measured in bone marrow mononuclear cells of patients and six healthy marrow donors. We used hierarchical clustering to identify patients with a hypermethylator phenotype based on 281 differentially methylated promoter CpGs. We used logistic regression to estimate the effects of maternal genotype on the likelihood of DNA hypermethylation in ALL and Ingenuity Pathway Analysis to identify networks enriched for differentially methylated genes. RESULTS: Twenty-two cases (43%) demonstrated promoter hypermethylation, which was more frequent among those with ETV6-RUNX1 fusion and initial white blood cell count < 50 x 109/L. Maternal rs12759827 was associated with aberrant DNA methylation (odds ratio [OR] 4.67, 95% confidence interval 1.46-16.31); non-significantly elevated ORs were observed for all other SNPs. Aberrantly methylated promoter CpGs aligned to genes with known cancer-related functions. DISCUSSION: Maternal folate metabolic genotype may be associated with DNA methylation patterns in ALL in their offspring. Therefore, the effect of maternal genotypes on ALL susceptibility may act through aberrant promoter methylation, which may contribute to the in utero origins of ALL.

Enhanced inflammation and attenuated tumor suppressor pathways are associated with oncogene-induced lung tumors in aged mice.

Aging is often accompanied by a dramatic increase in cancer susceptibility. To gain insights into how aging affects tumor susceptibility, we generated a conditional mouse model in which oncogenic KrasG12D was activated specifically in lungs of young (3-5 months) and old (19-24 months) mice. Activation of KrasG12D in old mice resulted in shorter survival and development of higher-grade lung tumors. Six weeks after KrasG12D activation, old lung tissues contained higher numbers of adenomas than their young tissue counterparts. Lung tumors in old mice displayed higher proliferation rates, as well as attenuated DNA damage and p53 tumor suppressor responses. Gene expression comparison of lung tumors from young and old mice revealed upregulation of extracellular matrix-related genes in young tumors, indicative of a robust cancer-associated fibroblast response. In old tumors, numerous inflammation-related genes such as Ccl7, IL-1ß, Cxcr6, and IL-15ra were consistently upregulated. Increased numbers of immune cells were localized around the periphery of lung adenomas from old mice. Our experiments indicate that more aggressive lung tumor formation in older KrasG12D mice may be in part the result of subdued tumor suppressor and DNA damage responses, an enhanced inflammatory milieu, and a more accommodating tissue microenvironment.

[An Incoherent Broadband Optical Cavity Spectroscopy for Measuring Weak Absorption Cross Section of Sulfur Dioxide].

As a highly sensitive detection technology, incoherent broadband cavity enhanced absorption spectroscopy (IBBCEAS) have successfully measured a variety of trace gases. According to the principle of cavity enhanced absorption spectroscopy, if the accurate concentration of the target gas, the curve of the mirror reflectance, effective absorption path length, the light intensity of the absorbing gas and non-absorbing gas are known, the absorption cross section of the absorption gas can be measured. The accurate measurements of absorption cross section are necessary for satellite retrievals of atmospheric trace gases and the atmospheric research. This paper describes an incoherent broadband cavity enhanced absorption spectroscopy(IBBCEAS) instrument with 365 nm LED as the light source for measuring absorption cross section of SO2 from 357 to 385 nm which is arising from the spin-forbidden a ³B1--X¹A1 transition. In comparison to the literature absorption cross section of SO2, and correlation coefficient r is 0.997 3. The result shows the potential of the IBBCEAS system for measuring weak absorption cross section.

[The Detection of Atmospheric HONO and NO2 with Fiber Coupling Long-Path Differential Optical Absorption Spectroscopy System].

A new type of fiber coupling Long-Path Differential Optical Absorption Spectroscopy System(LP-DOAS) based on schmidt - cassegrain telescope was introduced in detail in this paper and it was applied to the accurate measurement of the actual atmospheric HONO and NO2. This measuring system simplified the structure of traditional LP-DOAS system, combining with the design of optical fiber coupling.It made full use of the telescope primary mirror's effective area. The effects of the offset, dark current and telescope stray light to the new LP-DOAS system were discussed in this paper; On a clear day, the ratio between telescope stray light and the optical intensities was less than 1%. To verify the accurate of the new LP-DOAS system, the atmospheric NO2 were simultaneously measured with the new LP-DOAS system and traditional LP-DOAS system. The correlation coefficient R2 was up to 0.968. The observation of atmospheric HONO was carried out by using the fiber coupling LP-DOAS in Gucheng, Hebei Province, China, and the detection limit (2σ) of HONO and NO2 was 84.2 and 144.6 ppt , respectively, with 2 490 m path length and the average time resolution of about 30 s. In the whole measurement in Gucheng, the maximum of HONO and NO2 were 3.2 and 37.8 ppb, respectively, and the minimum were both under the detection limits; the ratio of HONO/NO2 at night was calculated.

[Cruise Observation of SO2, NO2 and Benzene with Mobile Portable DOAS in the Industrial Park].

The self-developed portable DOAS instrument based on differential optical absorption spectroscopy(DOAS) and composed of optical fiber spectrograph and multiple-pass cell was introduced. The standard gases of SO2 and NO2 were employed to test the accuracy and stability of the system, and then cruise observation of SO2, NO2 and benzene was carried out using the system in Tongling industrial park. During the entire period, the polluted gases showed high concentrations near the contaminated areas and the maximum concentrations of SO2, NO2 and benzene were 5 023.2, 2 195.2 and 162.5 µg·m-3, respectively. With 12.6 m optical path, the detection limits of SO2, NO2 and benzene were 67.0, 169.9, 30.6 µg·m-3, respectively. The portable DOAS system provides a convenient and effective technique for industrial park about emergency and supervisory monitoring and evaluation of gas leakage and fugitive emissions of gaseous pollutants.

Postnatal epigenetic regulation of intestinal stem cells requires DNA methylation and is guided by the microbiome.

BACKGROUND: DNA methylation is an epigenetic mechanism central to development and maintenance of complex mammalian tissues, but our understanding of its role in intestinal development is limited. RESULTS: We use whole genome bisulfite sequencing, and find that differentiation of mouse colonic intestinal stem cells to intestinal epithelium is not associated with major changes in DNA methylation. However, we detect extensive dynamic epigenetic changes in intestinal stem cells and their progeny during the suckling period, suggesting postnatal epigenetic development in this stem cell population. We find that postnatal DNA methylation increases at 3' CpG islands (CGIs) correlate with transcriptional activation of glycosylation genes responsible for intestinal maturation. To directly test whether 3' CGI methylation regulates transcription, we conditionally disrupted two major DNA methyltransferases, Dnmt1 or Dnmt3a, in fetal and adult intestine. Deficiency of Dnmt1 causes severe intestinal abnormalities in neonates and disrupts crypt homeostasis in adults, whereas Dnmt3a loss was compatible with intestinal development. These studies reveal that 3' CGI methylation is functionally involved in the regulation of transcriptional activation in vivo, and that Dnmt1 is a critical regulator of postnatal epigenetic changes in intestinal stem cells. Finally, we show that postnatal 3' CGI methylation and associated gene activation in intestinal epithelial cells are significantly altered by germ-free conditions. CONCLUSIONS: Our results demonstrate that the suckling period is critical for epigenetic development of intestinal stem cells, with potential important implications for lifelong gut health, and that the gut microbiome guides and/or facilitates these postnatal epigenetic processes.

Regulation of AURKC expression by CpG island methylation in human cancer cells.

AURKC, a member of the Aurora kinase gene family, is highly expressed in testis but is either moderately expressed or repressed in most somatic cells. Varying expression of AURKC has been observed in human cancers, but the underlying mechanisms of differential expression have been investigated only to a limited extent. We investigated the role of promoter CpG methylation in the regulation of AURKC gene expression in human cancer cells, in relation to a recently reported AURKC transcription repressor PLZF/ZBTB16, implicated in transformation and tumorigenesis. AURKC and PLZF/ZBTB16 expression profiles were investigated in reference to CpG methylation status on the AURKC promoter experimentally, and also in The Cancer Genome Atlas (TCGA) dataset involving multiple cancer types. AURKC promoter showed dense to moderate hypermethylation correlating with low to moderate expression of the gene in normal somatic cells and cancer cell lines, while testis with high expression revealed marked hypo-methylation. Treatment with the demethylating agent, 5-aza-dC, but not the histone deacetylase (HDAC) inhibitor, TSA, led to elevated expression in cancer cell lines, indicating that promoter DNA methylation negatively regulates AURKC expression. High expression of PLZF in PLZF-transfected cells treated with 5-aza-dC only partially repressed expression of AURKC despite 5-aza-dC also inducing elevated PLZF expression. Analyses of the TCGA data showed differential expression of AURKC in multiple cancer types and stronger correlation of AURKC expression with CpG methylation compared to PLZF levels. These findings demonstrate that differential promoter CpG methylation is an important mechanism regulating AURKC expression in cancer cells.

Phase-change properties of GeSbTe thin films deposited by plasma-enchanced atomic layer depositon.

Phase-change access memory (PCM) appears to be the strongest candidate for next-generation high-density nonvolatile memory. The fabrication of ultrahigh-density PCM depends heavily on the thin-film growth technique for the phase-changing chalcogenide material. In this study, Ge2Sb2Te5 (GST) and GeSb8Te thin films were deposited by plasma-enhanced atomic layer deposition (ALD) method using Ge [(CH3)2 N]4, Sb [(CH3)2 N]3, Te(C4H9)2 as precursors and plasma-activated H2 gas as reducing agent of the metallorganic precursors. Compared with GST-based device, GeSb8Te-based device exhibits a faster switching speed and reduced reset voltage, which is attributed to the growth-dominated crystallization mechanism of the Sb-rich GeSb8Te films. These results show that ALD is an attractive method for preparation of phase-change materials.

Cell-autonomous activation of Hedgehog signaling inhibits brown adipose tissue development.

Although recent studies have shown that brown adipose tissue (BAT) arises from progenitor cells that also give rise to skeletal muscle, the developmental signals that control the formation of BAT remain largely unknown. Here, we show that brown preadipocytes possess primary cilia and can respond to Hedgehog (Hh) signaling. Furthermore, cell-autonomous activation of Hh signaling blocks early brown-preadipocyte differentiation, inhibits BAT formation in vivo, and results in replacement of neck BAT with poorly differentiated skeletal muscle. Finally, we show that Hh signaling inhibits BAT formation partially through up-regulation of chicken ovalbumin upstream promoter transcription factor II (COUP-TFII). Taken together, our studies uncover a previously unidentified role for Hh as an inhibitor of BAT development.