Zc3h10 Acts as a Transcription Factor and Is Phosphorylated to Activate the Thermogenic Program.

Brown adipose tissue harbors UCP1 to dissipate chemical energy as heat. However, the transcriptional network that governs the thermogenic gene program is incompletely understood. Zc3h10, a CCCH-type zinc finger protein, has recently been reported to bind RNA. However, we report here that Zc3h10 functions as a transcription factor to activate UCP1 not through the enhancer region, but by binding to a far upstream region of the UCP1 promoter. Upon sympathetic stimulation, Zc3h10 is phosphorylated at S126 by p38 mitogen-activated protein kinase (MAPK) to increase binding to the distal region of the UCP1 promoter. Zc3h10, as well as mutant Zc3h10, which cannot bind RNA, enhances thermogenic capacity and energy expenditure, protecting mice from diet-induced obesity. Conversely, Zc3h10 ablation in UCP1+ cells in mice impairs thermogenic capacity and lowers oxygen consumption, leading to weight gain. Hence, Zc3h10 plays a critical role in the thermogenic gene program and may present future targets for obesity therapeutics

New Transcriptional Regulators of Non-shivering Thermogenesis

Unlike white adipose tissue (WAT), which stores excess energy as triglycerides, brown adipose tissue (BAT) burns fatty acids and glucose to produce heat. The thermogenic ability of BAT is due to the specialized inner mitochondrial proton transporter named uncoupling protein 1 (UCP1), which dissipates the proton motive force generated by the electron transport chain to create heat instead of ATP. Despite data suggesting that increasing BAT activity may be a promising antiobesity therapy, an inclusive model of the transcriptional regulation of thermogenic genes remain unclear. The aim of this dissertation work was to identify and characterize novel regulators of the UCP1 promoter and nonshivering thermogenesis. Chapter 1 reviews BAT in both mice and human, profiling the basic mechanism of uncoupled respiration and cold-induced nonshivering thermogenesis. Unlike classical BAT, which has constitutive UCP1 expression, brown adipocyte-like cells arise in WAT depots following prolonged cold exposure and contribute to whole body thermogenic capacity. While having similar functions, these cells arise from different precursor populations, having unique gene signatures and potentially depot specific regulation. Human BAT resembles either classical BAT or brown adipocyte-like cells in a depot specific manner, with differing levels of basal UCP1 expression and expression profiles. Finally, known transcriptional and hormonal regulators of BAT are discussed. Chapter 2 profiles my screening efforts to identify novel transcriptional regulators of the UCP1. Briefly, a library of over 1100 transcription factors was screened for activation of the UCP1 promoter. Expression profiling of the positive factors identified 6 novel, brown fat enriched transcriptional activators of UCP1. The first such transcription factor identified was the previously uncharacterized C2H2 type zinc-finger protein, Zfp516. Zfp516 is induced by cold where it binds and activates a brown fat gene program. Zfp516 ablation is embryonic lethal, but Zfp516 knockout embryos have little to no UCP1 expression and aberrant morphology. On the other hand, adipose specific transgenic overexpression in aP2-Zfp516 resulted in marked browning of inguinal WAT, increased body temperature and whole body energy expenditure, and prevention of diet-induced obesity. Chapter 3 profiles a second transcription factor identified in my screening efforts, the CCCH-type zinc finger protein, Zc3h10. Zc3h10, together with multiple cofactors, binds and activates the distal UCP1 promoter. Ablation of Zc3h10 results in defective BAT differentiation in cells, while adipose specific transgenic overexpression in aP2-Zc3h10 mice results in a lean phenotype. Finally, chapter 4 concludes this work, discussing my findings in the context of the field of brown adipocyte biology and presents future directions and remaining questions. This study has identified novel transcriptional regulators of UCP1, contributing significantly to the understanding of brown adipocyte biology and nonshivering thermogenesis, and providing new targets for future antiobesity therapeutics

aP2 Zfp516 SubQ Adipocyte

<p>Raw microarray data from subq adipocyte of aP2-Zfp516 mice.</p

Genetic and epigenetic control of adipose development

White adipose tissue (WAT) is the primary energy storage organ and its excess contributes to obesity, while brown adipose tissue (BAT) and inducible thermogenic (beige/brite) adipocytes in WAT dissipate energy via Ucp1 to maintain body temperature. BAT and subcutaneous WAT develop perinatally while visceral WAT forms after birth from precursors expressing distinct markers, such as Myf5, Pref-1, Wt1, and Prx1, depending on the anatomical location. In addition to the embryonic adipose precursors, a pool of endothelial cells or mural cells expressing Pparγ, Pdgfrβ, Sma and Zfp423 may become adipocytes during WAT expansion in adults. Several markers, such as Cd29, Cd34, Sca1, Cd24, Pdgfrα and Pref-1 are detected in adult WAT SVF cells that can be differentiated into adipocytes. However, potential heterogeneity and differences in developmental stage of these cells are not clear. Beige cells form in a depot- and condition-specific manner by de novo differentiation of precursors or by transdifferentiation. Thermogenic gene activation in brown and beige adipocytes relies on common transcriptional machinery that includes Prdm16, Zfp516, Pgc1α and Ebf2. Moreover, through changing the chromatin landscape, histone methyltransferases, such as Mll3/4 and Ehmt1, as well as demethylases, such as Lsd1, play an important role in regulating the thermogenic gene program. With the presence of BAT and beige/brite cells in human adults, increasing thermogenic activity of BAT and BAT-like tissues may help promote energy expenditure to combat obesity

Epigenetic Regulation of the Thermogenic Adipose Program

In contrast to white adipose tissue (WAT), which stores energy in the form of triglycerides, brown adipose tissue (BAT) dissipates energy by producing heat to maintain body temperature by burning glucose and fatty acids in a process called adaptive thermogenesis. The presence of an inducible thermogenic adipose tissue, and its beneficial effects for maintaining body weight and glucose and lipid homeostasis, has raised intense interest in understanding the regulation of thermogenesis. Elucidating the regulatory mechanisms underlying the thermogenic adipose program may provide excellent targets for therapeutics against obesity and diabetes. Here we review recent research on the role of epigenetics in the thermogenic gene program, focusing on DNA methylation and histone modifications

LSD1 Interacts with Zfp516 to Promote UCP1 Transcription and Brown Fat Program

Zfp516, a brown fat (BAT)-enriched and cold-inducible transcription factor, promotes transcription of UCP1 and other BAT-enriched genes for non-shivering thermogenesis. Here, we identify lysine-specific demethylase 1 (LSD1) as a direct binding partner of Zfp516. We show that, through interaction with Zfp516, LSD1 is recruited to UCP1 and other BAT-enriched genes, such as PGC1α, to function as a coactivator by demethylating H3K9. We also show that LSD1 is induced during brown adipogenesis and that LSD1 and its demethylase activity is required for the BAT program. Furthermore, we show that LSD1 ablation in mice using Myf5-Cre alters embryonic BAT development. Moreover, BAT-specific deletion of LSD1 via the use of UCP1-Cre impairs the BAT program and BAT development, making BAT resemble WAT, reducing thermogenic activity and promoting obesity. Finally, we demonstrate an in vivo requirement of the Zfp516-LSD1 interaction for LSD1 function in BAT gene activation

Zc3h10 Acts as a Transcription Factor and Is Phosphorylated to Activate the Thermogenic Program.

Brown adipose tissue harbors UCP1 to dissipate chemical energy as heat. However, the transcriptional network that governs the thermogenic gene program is incompletely understood. Zc3h10, a CCCH-type zinc finger protein, has recently been reported to bind RNA. However, we report here that Zc3h10 functions as a transcription factor to activate UCP1 not through the enhancer region, but by binding to a far upstream region of the UCP1 promoter. Upon sympathetic stimulation, Zc3h10 is phosphorylated at S126 by p38 mitogen-activated protein kinase (MAPK) to increase binding to the distal region of the UCP1 promoter. Zc3h10, as well as mutant Zc3h10, which cannot bind RNA, enhances thermogenic capacity and energy expenditure, protecting mice from diet-induced obesity. Conversely, Zc3h10 ablation in UCP1+ cells in mice impairs thermogenic capacity and lowers oxygen consumption, leading to weight gain. Hence, Zc3h10 plays a critical role in the thermogenic gene program and may present future targets for obesity therapeutics

LSD1 Interacts with Zfp516 to Promote UCP1 Transcription and Brown Fat Program

Zfp516, a brown fat (BAT)-enriched and cold-inducible transcription factor, promotes transcription of UCP1 and other BAT-enriched genes for non-shivering thermogenesis. Here, we identify lysine-specific demethylase 1 (LSD1) as a direct binding partner of Zfp516. We show that, through interaction with Zfp516, LSD1 is recruited to UCP1 and other BAT-enriched genes, such as PGC1α, to function as a coactivator by demethylating H3K9. We also show that LSD1 is induced during brown adipogenesis and that LSD1 and its demethylase activity is required for the BAT program. Furthermore, we show that LSD1 ablation in mice using Myf5-Cre alters embryonic BAT development. Moreover, BAT-specific deletion of LSD1 via the use of UCP1-Cre impairs the BAT program and BAT development, making BAT resemble WAT, reducing thermogenic activity and promoting obesity. Finally, we demonstrate an in vivo requirement of the Zfp516-LSD1 interaction for LSD1 function in BAT gene activation

Cold-Inducible Zfp516 Activates UCP1 Transcription to Promote Browning of White Fat and Development of Brown Fat

Uncoupling protein 1 (UCP1) mediates nonshivering thermogenesis and, upon cold exposure, is induced in brown adipose tissue (BAT) and subcutaneous white adipose tissue (iWAT). Here, by high-throughput screening using the UCP1 promoter, we identify Zfp516 as a transcriptional activator of UCP1 as well&nbsp;as PGC1α, thereby promoting a BAT program. Zfp516 itself is induced by cold and sympathetic stimulation through the cAMP-CREB/ATF2 pathway. Zfp516 directly binds to the proximal region of the UCP1 promoter, not to the enhancer region where other transcription factors bind, and interacts with PRDM16 to activate the UCP1 promoter. Although ablation of Zfp516 causes embryonic lethality, knockout embryos still show drastically reduced BAT mass. Overexpression of Zfp516 in adipose tissue promotes browning of iWAT even at room temperature, increasing body temperature and energy expenditure and preventing diet-induced obesity. Zfp516 may represent a future target for obesity therapeutics