Correlation between choline kinase alpha expression and 11C-choline accumulation in breast cancer using positron emission tomography/computed tomography: a retrospective study.

Choline kinase (CK) is reportedly overexpressed in various malignancies. Among its isoforms, CKα overexpression is presumably related to oncogenic change. Choline positron emission tomography (PET) is reportedly useful for detecting and evaluating therapy outcomes in malignancies. In this study, we investigated the correlation between CKα expression and 11C-choline accumulation in breast cancer cells. We also compared the CKα expression level with other pathological findings for investigating tumour activity. Fifty-six patients with breast cancer (mean age: 51 years) who underwent their first medical examination between May 2007 and December 2008 were enrolled. All the patients underwent 11C-choline PET/computed tomography imaging prior to surgery. The maximum standardised uptake value was recorded for evaluating 11C-choline accumulation. The intensity of CKα expression was classified using immunostaining. A significant correlation was observed between CKα expression and 11C-choline accumulation (P < 0.0001). A comparison of breast cancer mortality demonstrated that strong CKα expression was associated with a shorter survival time (P < 0.0001). 11C-choline accumulation was also negatively correlated with survival time (P < 0.0001). Tumours with strong CKα expression are reportedly highly active in breast cancer. A correlation was observed between CKα expression and 11C-choline accumulation, suggesting their role as prognostic indicators of breast cancer.

Megaconial congenital muscular dystrophy due to CHKB gene variants, the first report of thirteen Iranian patients.

Megaconial congenital muscular dystrophy (OMIM: 602,541) related to CHKB gene mutation is a newly defined rare autosomal recessive disorder, with multisystem involvement presenting from the neonatal period to adolescence. Choline kinase beta, lipid transport enzyme, catalyzes the biosynthesis of phosphatidylcholine and phosphatidylethanolamine, two major components of the mitochondrial membrane, on which respiratory enzyme activities are dependent. CHKB gene variants lead to loss-of-function of choline kinase b and lipid metabolism defects and mitochondrial structural changes. To date, many megaconial congenital muscular dystrophy cases due to CHKB gene variants have been reported worldwide. We describe thirteen Iranian megaconial congenital muscular dystrophy cases related to CHKB gene variants, including clinical presentations, laboratory and muscle biopsy findings, and novel CHKB gene variants. The most common symptoms and signs included intellectual disability, delayed gross-motor developmental milestones, language skills problems, muscle weakness, as well as autistic features, and behavioral problems. Muscle biopsy examination showed the striking finding of peripheral arrangements of large mitochondria in muscle fibers and central sarcoplasmic areas devoid of mitochondria. Eleven different CHKB gene variants including six novel variants were found in our patients. Despite the rarity of this disorder, recognition of the multisystem clinical presentations combined with characteristic findings of muscle histology can properly guide to genetic evaluation of CHKB gene.

Large heterozygous deletion and uniparental disomy masquerading as homozygosity in CHKB gene.

BACKGROUND: CHKB mutations have been described in 49 patients with megaconial congenital muscular dystrophy, which is a rare autosomal recessive disorder, of which 40 patients showed homozygosity. METHODS: Peripheral blood genomic DNA samples were extracted from patients and their parents and were tested by whole exome sequencing. Quantitative PCR was performed to detect deletion. Single nucleotide polymorphism analysis was performed to identify uniparental disomy. Quantitative PCR and western blot were used to measure the expression level of CHKB in patient 1-derived immortalized lymphocytes. Mitochondria were observed in lymphocytes by electron microscopy. RESULTS: Two unrelated cases born to non-consanguineous parents were diagnosed with megaconial congenital muscular dystrophy due to apparently homozygous mutations (patient 1: c.225-2A>T; patient 2: c.701C>T) in the CHKB gene using whole exome sequencing. Quantitative PCR revealed that patient 1 had a large deletion encompassing the CHKB gene, inherited from the mother. Single nucleotide polymorphism analysis revealed patient 2 had paternal uniparental isodisomy containing the CHKB gene. In the immortalized lymphocytes from patient 1, decreased expression of CHKB was revealed by quantitative PCR and western blot, and giant mitochondria were observed using electron microscopy. CONCLUSION: We provide a possibility to detect giant mitochondria in other cells when muscle was not available. Moreover, clinicians should be aware that homozygous variants can be masqueraded by uniparental disomy or large deletions in offspring of non-consanguineous parents, and excessive homozygosity may be misdiagnosed.

Twist alters the breast tumor microenvironment via choline kinase to facilitate an aggressive phenotype.

Twist (TWIST1) is a gene required for cell fate specification in embryos and its expression in mammary epithelium can initiate tumorigenesis through the epithelial-mesenchymal transition. To identify downstream target genes of Twist in breast cancer, we performed microarray analysis on the transgenic breast cancer cell line, MCF-7/Twist. One of the targets identified was choline kinase whose upregulation resulted in increased cellular phosphocholine and total choline containing compounds-a characteristic observed in highly aggressive metastatic cancers. To study the interactions between Twist, choline kinase, and their effect on the microenvironment, we used 1H magnetic resonance spectroscopy and found significantly higher phosphocholine and total choline, as well as increased phosphocholine/glycerophosphocholine ratio in MCF-7/Twist cells. We also observed significant increases in extracellular glucose, lactate, and [H +] ion concentrations in the MCF-7/Twist cells. Magnetic resonance imaging of MCF-7/Twist orthotopic breast tumors showed a significant increase in vascular volume and permeability surface area product compared to control tumors. In addition, by reverse transcription-quantitative polymerase chain reaction, we discovered that Twist upregulated choline kinase expression in estrogen receptor negative breast cancer cell lines through FOXA1 downregulation. Moreover, using The Cancer Genome Atlas database, we observed a significant inverse relationship between FOXA1 and choline kinase expression and propose that it could act as a modulator of the Twist/choline kinase axis. The data presented indicate that Twist is a driver of choline kinase expression in breast cancer cells via FOXA1 resulting in the generation of an aggressive breast cancer phenotype.

Choline kinase: An underappreciated rheumatoid arthritis therapeutic target.

Choline kinase (ChoK) has been well documented as a major enzyme involved in the anomalous cellular lipid metabolic profile of chronic inflammatory disorders. However, new research has now been unveiled that helps us to better understand how changes in lipid metabolism influence the transformational phenotype, drug resistance, and antiapoptotic characteristics of invasive cells, leading to rheumatoid arthritis (RA) disease progression. It is still unknown how ChoK modulates the lipid metabolic aberrations that may promote altered cell phenotype and functionality in RA. Herein, we review the current understanding of ChoK's role in altered metabolism in diverse cell types involved in RA progression, and for the first time, we take a step forward to complete the puzzle and summarise striking facts that link choline metabolism to its transformed phenotype, in order to postulate ChoK as a robust therapeutic target in RA. This review forms a foundation on which ChoK can be tackled as a potential biomarker, opening doors for RA diagnosis and prognosis. It frameworks several ChoK inhibitors that rewire the lipid metabolic profile in the inflammatory disease landscape and envisages its being translated to clinics.

DHRS2 inhibits cell growth and metastasis in ovarian cancer by downregulation of CHKα to disrupt choline metabolism.

The short-chain dehydrogenase/reductase (SDR) superfamily has essential roles in lipid metabolism and redox sensing. In recent years, accumulating evidence highlights the emerging association between SDR family enzymes and cancer. Dehydrogenase/reductase member 2(DHRS2) belongs to the NADH/NADPH-dependent SDR family, and extensively participates in the regulation of the proliferation, migration, and chemoresistance of cancer cells. However, the underlying mechanism has not been well defined. In the present study, we have demonstrated that DHRS2 inhibits the growth and metastasis of ovarian cancer (OC) cells in vitro and in vivo. Mechanistically, the combination of transcriptome and metabolome reveals an interruption of choline metabolism by DHRS2. DHRS2 post-transcriptionally downregulates choline kinase α (CHKα) to inhibit AKT signaling activation and reduce phosphorylcholine (PC)/glycerophosphorylcholine (GPC) ratio, impeding choline metabolism reprogramming in OC. These actions mainly account for the tumor-suppressive role of DHRS2 in OC. Overall, our findings establish the mechanistic connection among metabolic enzymes, metabolites, and the malignant phenotype of cancer cells. This could result in further development of novel pharmacological tools against OC by the induction of DHRS2 to disrupt the choline metabolic pathway.

Megaconial congenital muscular dystrophy due to novel CHKB variants: a case report and literature review.

BACKGROUND: Choline kinase beta (CHKB) catalyzes the first step in the de novo biosynthesis of phosphatidyl choline and phosphatidylethanolamine via the Kennedy pathway. Derangement of this pathway might also influence the homeostasis of mitochondrial membranes. Autosomal recessive CHKB mutations cause a rare form of congenital muscular dystrophy known as megaconial congenital muscular dystrophy (MCMD). CASE PRESENTATION: We describe a novel proband presenting MCMD due to unpublished CHKB mutations. The patient is a 6-year-old boy who came to our attention for cognitive impairment and slowly progressive muscular weakness. He was the first son of non-consanguineous healthy parents from Sri Lanka. Neurological examination showed proximal weakness at four limbs, weak osteotendinous reflexes, Gowers' maneuver, and waddling gate. Creatine kinase levels were mildly increased. EMG and brain MRI were normal. Left quadriceps skeletal muscle biopsy showed a myopathic pattern with nuclear centralizations and connective tissue increase. Histological and histochemical staining suggested subsarcolemmal localization and dimensional increase of mitochondria. Ultrastructural analysis confirmed the presence of enlarged ("megaconial") mitochondria. Direct sequencing of CHKB identified two novel defects: the c.1060G > C (p.Gly354Arg) substitution and the c.448-56_29del intronic deletion, segregating from father and mother, respectively. Subcloning of RT-PCR amplicons from patient's muscle RNA showed that c.448-56_29del results in the partial retention (14 nucleotides) of intron 3, altering physiological splicing and transcript stability. Biochemical studies showed reduced levels of the mitochondrial fission factor DRP1 and the severe impairment of mitochondrial respiratory chain activity in patient's muscle compared to controls. CONCLUSIONS: This report expands the molecular findings associated with MCMD and confirms the importance of considering CHKB variants in the differential diagnosis of patients presenting with muscular dystrophy and mental retardation. The clinical outcome of MCMD patients seems to be influenced by CHKB molecular defects. Histological and ultrastructural examination of muscle biopsy directed molecular studies and allowed the identification and characterization of an intronic mutation, usually escaping standard molecular testing.

Mechanism of action and therapeutic route for a muscular dystrophy caused by a genetic defect in lipid metabolism.

CHKB encodes one of two mammalian choline kinase enzymes that catalyze the first step in the synthesis of the membrane phospholipid phosphatidylcholine. In humans and mice, inactivation of the CHKB gene (Chkb in mice) causes a recessive rostral-to-caudal muscular dystrophy. Using Chkb knockout mice, we reveal that at no stage of the disease is phosphatidylcholine level significantly altered. We observe that in affected muscle a temporal change in lipid metabolism occurs with an initial inability to utilize fatty acids for energy via mitochondrial ß-oxidation resulting in shunting of fatty acids into triacyglycerol as the disease progresses. There is a decrease in peroxisome proliferator-activated receptors and target gene expression specific to Chkb-/- affected muscle. Treatment of Chkb-/- myocytes with peroxisome proliferator-activated receptor agonists enables fatty acids to be used for ß-oxidation and prevents triacyglyerol accumulation, while simultaneously increasing expression of the compensatory choline kinase alpha (Chka) isoform, preventing muscle cell injury.

Bi-allelic variants in CHKA cause a neurodevelopmental disorder with epilepsy and microcephaly.

The Kennedy pathways catalyse the de novo synthesis of phosphatidylcholine and phosphatidylethanolamine, the most abundant components of eukaryotic cell membranes. In recent years, these pathways have moved into clinical focus because four of ten genes involved have been associated with a range of autosomal recessive rare diseases such as a neurodevelopmental disorder with muscular dystrophy (CHKB), bone abnormalities and cone-rod dystrophy (PCYT1A) and spastic paraplegia (PCYT2, SELENOI). We identified six individuals from five families with bi-allelic variants in CHKA presenting with severe global developmental delay, epilepsy, movement disorders and microcephaly. Using structural molecular modelling and functional testing of the variants in a cell-based Saccharomyces cerevisiae model, we determined that these variants reduce the enzymatic activity of CHKA and confer a significant impairment of the first enzymatic step of the Kennedy pathway. In summary, we present CHKA as a novel autosomal recessive gene for a neurodevelopmental disorder with epilepsy and microcephaly.

A mouse model of inherited choline kinase ß-deficiency presents with specific cardiac abnormalities and a predisposition to arrhythmia.

The CHKB gene encodes choline kinase ß, which catalyzes the first step in the biosynthetic pathway for the major phospholipid phosphatidylcholine. Homozygous loss-of-function variants in human CHKB are associated with a congenital muscular dystrophy. Dilated cardiomyopathy is present in some CHKB patients and can cause heart failure and death. Mechanisms underlying a cardiac phenotype due to decreased CHKB levels are not well characterized. We determined that there is cardiac hypertrophy in Chkb-/- mice along with a decrease in left ventricle size, internal diameter, and stroke volume compared with wildtype and Chkb+/- mice. Unlike wildtype mice, 60% of the Chkb+/- and all Chkb-/- mice tested displayed arrhythmic events when challenged with isoproterenol. Lipidomic analysis revealed that the major change in lipid level in Chkb+/- and Chkb-/- hearts was an increase in the arrhythmogenic lipid acylcarnitine. An increase in acylcarnitine level is also associated with a defect in the ability of mitochondria to use fatty acids for energy and we observed that mitochondria from Chkb-/- hearts had abnormal cristae and inefficient electron transport chain activity. Atrial natriuretic peptide (ANP) is a hormone produced by the heart that protects against the development of heart failure including ventricular conduction defects. We determined that there was a decrease in expression of ANP, its receptor NPRA, as well as ventricular conduction system markers in Chkb+/- and Chkb-/- mice.

Antisense RNAs Influence Promoter Usage of Their Counterpart Sense Genes in Cancer.

Multiple noncoding natural antisense transcripts (ncNAT) are known to modulate key biological events such as cell growth or differentiation. However, the actual impact of ncNATs on cancer progression remains largely unknown. In this study, we identified a complete list of differentially expressed ncNATs in hepatocellular carcinoma. Among them, a previously undescribed ncNAT HNF4A-AS1L suppressed cancer cell growth by regulating its sense gene HNF4A, a well-known cancer driver, through a promoter-specific mechanism. HNF4A-AS1L selectively activated the HNF4A P1 promoter via HNF1A, which upregulated expression of tumor suppressor P1-driven isoforms, while having no effect on the oncogenic P2 promoter. RNA-seq data from 23 tissue and cancer types identified approximately 100 ncNATs whose expression correlated specifically with the activity of one promoter of their associated sense gene. Silencing of two of these ncNATs ENSG00000259357 and ENSG00000255031 (antisense to CERS2 and CHKA, respectively) altered the promoter usage of CERS2 and CHKA. Altogether, these results demonstrate that promoter-specific regulation is a mechanism used by ncNATs for context-specific control of alternative isoform expression of their counterpart sense genes. SIGNIFICANCE: This study characterizes a previously unexplored role of ncNATs in regulation of isoform expression of associated sense genes, highlighting a mechanism of alternative promoter usage in cancer.

Genome-wide identification of CK gene family suggests functional expression pattern against Cd2+ stress in Gossypium hirsutum L.

Choline kinase (CK) gene plays an important role in plants growth, development and resistance to stress. It mainly regulates the biosynthesis of phosphatidylcholine. This study aims to explore the structure-function relationship, and to provide a framework for functional validation and biochemical characterization of various CK genes. Our analysis showed that 87 CK genes were identified in cotton and 7 diploid plants, of which 43 genes encode CK proteins in 4 cotton species, and 13 genes were identified in Gossypium hirsutum. Most of GhCK genes are affected by the abiotic stress conditions, indicating the importance of CK proteins for plant development and response to abiotic stress. RT-qPCR analysis showed the tissue specificity of GhCK genes in response to Cd2+ and other abiotic stresses. Under Cd2+ stress, the expression level of GhCK gene family members has undergone different changes. The expression level of GhCK5 was enhanced, indicating that Cd2+ stress caused the increase of phosphatidylcholine content, which in turn reacted on the plant cell membrane, finally reached the absorption of Cd2+ into plant cells to repair Cd2+ the purpose of contaminated soil. This study will further broaden our understanding of the association between evolution and function of the GhCK gene family.

DNA Methylation of Human Choline Kinase Alpha Promoter-Associated CpG Islands in MCF-7 Cells.

Choline kinase (CK) is the enzyme catalyzing the first reaction in CDP-choline pathway for the biosynthesis of phosphatidylcholine. Higher expression of the α isozyme of CK has been implicated in carcinogenesis, and inhibition or downregulation of CKα (CHKA) is a promising anticancer approach. This study aimed to investigate the regulation of CKα expression by DNA methylation of the CpG islands found on the promoter of this gene in MCF-7 cells. Four CpG islands have been predicted in the 2000 bp promoter region of ckα (chka) gene. Six CpG island deletion mutants were constructed using PCR site-directed mutagenesis method and cloned into pGL4.10 vectors for promoter activity assays. Deletion of CpG4C region located between -225 and -56 significantly increased the promoter activity by 4-fold, indicating the presence of important repressive transcription factor binding site. The promoter activity of methylated full-length promoter was significantly lower than the methylated CpG4C deletion mutant by 16-fold. The results show that DNA methylation of CpG4C promotes the binding of the transcription factor that suppresses the promoter activity. Electrophoretic mobility shift assay analysis showed that cytosine methylation at MZF1 binding site in CpG4C increased the binding of putative MZF1 in nuclear extract. In conclusion, the results suggest that DNA methylation decreased the promoter activity by promoting the binding of putative MZF1 transcription factor at CpG4C region of the ckα gene promoter.

Gene Expression of CRAL_TRIO Family Proteins modulated by Vitamin E Deficiency in Zebrafish (Danio Rerio).

An evaluation of the impact of vitamin E deficiency on expression of the alpha-tocopherol transfer protein (α-TTP) and related CRAL_TRIO genes was undertaken using livers from adult zebrafish based on the hypothesis that increased lipid peroxidation would modulate gene expression. Zebrafish were fed either a vitamin E sufficient (E+) or deficient (E-) diet for 9 months, then fish were euthanized, and livers were harvested. Livers from the E+ relative to E- fish contained 40-times more α-tocopherol (P <0.0001) and one fourth the malondialdehyde (P = 0.0153). RNA was extracted from E+ and E- livers, then subject to evaluation of gene expression of ttpa and other genes of the CRAL_TRIO family, genes of antioxidant markers, and genes related to lipid metabolism. Ttpa expression was not altered by vitamin E status. However, one member of the CRAL_TRIO family, tyrosine-protein phosphatase non-receptor type 9 gene (ptpn9a), showed a 2.4-fold increase (P = 0.029) in E- relative to E+ livers. Further, we identified that the gene for choline kinase alpha (chka) showed a 3.0-fold increase (P = 0.010) in E- livers. These outcomes are consistent with our previous findings that show vitamin E deficiency increased lipid peroxidation causing increases in phospholipid turnover.

Choline kinase alpha 2 acts as a protein kinase to promote lipolysis of lipid droplets.

Lipid droplets are important for cancer cell growth and survival. However, the mechanism underlying the initiation of lipid droplet lipolysis is not well understood. We demonstrate here that glucose deprivation induces the binding of choline kinase (CHK) α2 to lipid droplets, which is sequentially mediated by AMPK-dependent CHKα2 S279 phosphorylation and KAT5-dependent CHKα2 K247 acetylation. Importantly, CHKα2 with altered catalytic domain conformation functions as a protein kinase and phosphorylates PLIN2 at Y232 and PLIN3 at Y251. The phosphorylated PLIN2/3 dissociate from lipid droplets and are degraded by Hsc70-mediated autophagy, thereby promoting lipid droplet lipolysis, fatty acid oxidation, and brain tumor growth. In addition, levels of CHKα2 S279 phosphorylation, CHKα2 K247 acetylation, and PLIN2/3 phosphorylation are positively correlated with one another in human glioblastoma specimens and are associated with poor prognosis in glioblastoma patients. These findings underscore the role of CHKα2 as a protein kinase in lipolysis and glioblastoma development.

Megaconial congenital muscular dystrophy secondary to novel CHKB mutations resemble atypical Rett syndrome.

Megaconial congenital muscular dystrophy (CMD)(OMIM #602541), related to CHKB mutation, is a rare autosomal recessive disorder. To date, only 35 confirmed patients are recorded. We present a detailed description of the clinical, histopathological, imaging, and genetic findings of five children from four Indian families. The children had moderate-to-severe autistic behavior, hand stereotypies, and global developmental delay mimicking atypical Rett syndrome. In addition, generalized hypotonia was a common initial finding. The progression of muscle weakness was variable, with two patients having a milder phenotype and three having a severe form. Interestingly, the majority did not attain sphincter control. Only patient 1 had classical ichthyotic skin changes. Muscle biopsy in two patients showed a myopathic pattern with characteristic peripherally placed enlarged mitochondria on modified Gomori trichrome stain and electron microscopy. Genetic analysis in these patients identified three novel null mutations in CHKB [c.1027dupA (p.Ser343LysfsTer86);c.224 + 1G > T (5' splice site); c.1123C > T (p.Gln375Ter)] and one reported missense mutation, c.581G > A (p.Arg194Gln), all in the homozygous state. Megaconial CMD, although rare, forms an important group with a complex phenotypic presentation and accounted for 5.5% of our genetically confirmed CMD patients. Atypical Rett syndrome-like presentation may be a clue towards CHKB-related disorder.

Comparison of Lesional Juvenile Myositis and Lupus Skin Reveals Overlapping Yet Unique Disease Pathophysiology.

OBJECTIVE: Skin inflammation heralds systemic disease in juvenile myositis, yet we lack an understanding of pathogenic mechanisms driving skin inflammation in this disease. We undertook this study to define cutaneous gene expression signatures in juvenile myositis and identify key genes and pathways that differentiate skin disease in juvenile myositis from childhood-onset systemic lupus erythematosus (SLE). METHODS: We used formalin-fixed paraffin-embedded skin biopsy samples from 15 patients with juvenile myositis (9 lesional, 6 nonlesional), 5 patients with childhood-onset SLE, and 8 controls to perform transcriptomic analysis and identify significantly differentially expressed genes (DEGs; q ≤ 5%) between patient groups. We used Ingenuity Pathway Analysis (IPA) to highlight enriched biologic pathways and validated DEGs by immunohistochemistry and quantitative real-time polymerase chain reaction. RESULTS: Comparison of lesional juvenile myositis to control samples revealed 221 DEGs, with the majority of up-regulated genes representing interferon (IFN)-stimulated genes. CXCL10, CXCL9, and IFI44L represented the top 3 DEGs (fold change 23.2, 13.3, and 13.0, respectively; q < 0.0001). IPA revealed IFN signaling as the top canonical pathway. When compared to childhood-onset SLE, lesional juvenile myositis skin shared a similar gene expression pattern, with only 28 unique DEGs, including FBLN2, CHKA, and SLURP1. Notably, patients with juvenile myositis who were positive for nuclear matrix protein 2 (NXP-2) autoantibodies exhibited the strongest IFN signature and also demonstrated the most extensive Mx-1 immunostaining, both in keratinocytes and perivascular regions. CONCLUSION: Lesional juvenile myositis skin demonstrates a striking IFN signature similar to that previously reported in juvenile myositis muscle and peripheral blood. Further investigation into the association of a higher IFN score with NXP-2 autoantibodies may provide insight into disease endotypes and pathogenesis.

Emergent Coordination of the CHKB and CPT1B Genes in Eutherian Mammals: Implications for the Origin of Brown Adipose Tissue.

Mitochondrial fatty acid oxidation (FAO) contributes to the proton motive force that drives ATP synthesis in many mammalian tissues. In eutherian (placental) mammals, brown adipose tissue (BAT) can also dissipate this proton gradient through uncoupling protein 1 (UCP1) to generate heat, but the evolutionary events underlying the emergence of BAT are unknown. An essential step in FAO is the transport of cytoplasmic long chain acyl-coenzyme A (acyl-CoA) into the mitochondrial matrix, which requires the action of carnitine palmitoyltransferase 1B (CPT1B) in striated muscle and BAT. In eutherians, the CPT1B gene is closely linked to the choline kinase beta (CHKB) gene, which is transcribed from the same DNA strand and terminates just upstream of CPT1B. CHKB is a rate-limiting enzyme in the synthesis of phosphatidylcholine (PC), a predominant mitochondrial membrane phospholipid, suggesting that the coordinated expression of CHKB and CPT1B may cooperatively enhance mitochondrial FAO. The present findings show that transcription of the eutherian CHKB and CPT1B genes is linked within a unitary epigenetic domain targeted to the CHKB gene, and that that this regulatory linkage appears to have resulted from an intergenic deletion in eutherians that significantly altered the distribution of CHKB and CPT1B expression. Informed by the timing of this event relative to the emergence of BAT, the phylogeny of CHKB-CPT1B synteny, and the insufficiency of UCP1 to account for eutherian BAT, these data support a mechanism for the emergence of BAT based on the acquisition of a novel capacity for adipocyte FAO in a background of extant UCP1.