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CDK7 Mediates the Beta-Adrenergic Signaling in Thermogenic Brown and White Adipose Tissues

Summary Cyclin-dependent kinases (CDKs) are emerging regulators of adipose tissue metabolism. Here we aimed to explore the role of CDK7 in thermogenic fat. We found that CDK7 brown adipose tissue (BAT)-specific knockout mice (Cdk7bKO) have decreased BAT mass and impaired β3-adrenergic signaling and develop hypothermia upon cold exposure. We found that loss of CDK7 in BAT disrupts the induction of thermogenic genes in response to cold. However, Cdk7bKO mice do not show systemic metabolic dysfunction. Increased expression of genes of the creatine metabolism compensates for the heat generation in the BAT of Cdk7bKO mice in response to cold. Finally, we show that CDK7 is required for beta 3-adrenergic agonist-induced browning of white adipose tissue (WAT). Indeed, Cdk7 ablation in all adipose tissues (Cdk7aKO) has impaired browning in WAT. Together, our results demonstrate that CDK7 is an important mediator of beta-adrenergic signaling in thermogenic brown and beige fat.


INTRODUCTION
Brown adipose tissue (BAT) is the main organ for non-shivering thermogenesis, which helps homeothermic vertebrates to maintain constant body temperature in changing nutritional and environmental conditions. Cold exposure leads the release by the sympathetic nervous system of norepinephrine in BAT, which binds to the b-adrenergic receptors, activates downstream signaling pathways, and ultimately leads to the induction of the thermogenic program (Cannon and Nedergaard, 2004). In addition, beige adipocytes, which also participate in non-shivering thermogenesis, emerge within white adipose tissue upon different stimuli, such as chronic cold acclimation or long-term b-adrenergic stimulation (Kajimura et al., 2015). Brown and beige fat activation rely on the action of uncoupling protein 1 (UCP1), which dissipates energy as heat instead of ATP production (Fedorenko et al., 2012). Recently, other UCP-independent alternative thermogenesis mechanisms have been described in brown and beige adipocytes, notably the creatine kinasemediated futile cycling (Kazak et al., 2015(Kazak et al., , 2019, the triglyceride-fatty acid cycling (Granneman et al., 2003), and the SERCA2b-mediated calcium cycling (Ikeda et al., 2017).
Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle progression. During G1 progression, pro-mitotic signals such as growth factors induce synthesis of D-type cyclins (cyclin D1, D2, and D3), which bind and activate CDK4/6 to trigger the phosphorylation of the retinoblastoma protein family, which includes pRB and other pocket proteins (i.e., p107 and p130). RB phosphorylation releases the E2F transcription factors, which promote the transcription of genes required for subsequent cell cycle progression (Malumbres and Barbacid, 2005).
We and others have recently demonstrated that cyclin-dependent kinases (CDKs), key regulators of cell cycle progression and cell proliferation (Murray, 2004), participate in the regulation of metabolic pathways and energy homeostasis (Lopez-Mejia et al., 2018). CDK4 promotes adipocyte differentiation (Lagarrigue et al., 2016), and a mouse model of cdk4 invalidation has increased oxidative metabolism and exercise capacity (Lopez-Mejia et al., 2017). Furthermore, the CDKs target (the retinoblastoma protein, pRB) and downstream effector E2F1 are both directly involved in the BAT activity in response to cold. At basal conditions, the complex pRb-E2F1 sits in the promoters of genes involved in oxidative metabolism and mitochondrial biogenesis, such as PGC1a, and inhibits their transcription BAT. In response to cold, pRB is phosphorylated releasing this repression. Consequently, mice invalidated for E2f1 show higher body temperature upon cold stimulation due to increased oxidative metabolism (Blanchet et al., 2011).
CDK7 and its partners, cyclin H and MAT1, were originally isolated as a CDK-activating kinase (CAK), which was required for full activation of cell-cycle CDKs (Makela et al., 1994;Yee et al., 1995). CDK7 also phosphorylates the RNA polymerase II (Pol II) C-terminal domain (CTD), which initiates transcription (Shiekhattar et al., 1995). Based on this property, the inhibition of CDK7 with small molecules THZ1 has been considered a promising therapy for cancer (Glover-Cutter et al., 2009;Chipumuro et al., 2014;Christensen et al., 2014;Kwiatkowski et al., 2014;Nilson et al., 2015). The mouse model of CDK7 deficiency shows early embryonic lethality, which indicates its importance in development (Ganuza et al., 2012). With heart-specific knockout mice, CDK7/MAT1 complex has been implicated in controlling mitochondrial metabolism gene expression in heart and is important to prevent heart failure (Sano et al., 2007). Since CDK7 is an upstream regulator of the CDK-E2F1-RB pathway, and since these proteins are important modulators of BAT activity, we hypothesized that CDK7 could also be an important regulator of thermogenesis in adipose tissue.
In this study, we show, by analyzing the BAT and white adipose tissue (WAT)-specific Cdk7 bKO and Cdk7 aKO mice, that CDK7 is essential for brown adipose tissue-mediated non-shivering thermogenesis and that CDK7 ablation attenuates beta-adrenergic signaling in brown and beige adipose tissue. In conclusion, CDK7 is an important regulator of beta-adrenergic receptor signaling in thermogenic fat.

CDK7 Brown Fat-Specific Knockout Mice Are Cold Intolerant
To analyze the role of CDK7 specifically in brown adipose tissue, Cdk7 fl/fl mice were crossed with mice expressing the Cre recombinase under the Ucp1 promoter to generate the brown adipose tissue-specific Cdk7 bKO . This resulted in the specific disruption of CDK7 mRNA expression in BAT, whereas the expression of Cdk7 remains intact in both subcutaneous (scWAT) and perigonadal (pgWAT) and other tissues like liver, brain, and muscle ( Figure S1A). Cdk7 fl/fl and Cdk7 bKO mice had similar body weight and fat/lean mass ratio ( Figures 1A-1C). The food intake was indistinguishable between the two groups, as it was the activity at room temperature ( Figures S2A and S2B). Not surprisingly, Cdk7 bKO mice did not demonstrate any difference in glucose tolerance or insulin sensitivity ( Figures S2C and S2D).
To explore the role of CDK7 in thermogenic regulation, Cdk7 bKO and Cdk7 fl/fl control littermates were challenged to acute cold exposure (4 C) and rectal temperatures were monitored. We found that, when food was removed during cold exposure at 4 C, Cdk7 bKO mice developed severe hypothermia (body temperature <30 C) within 3 h, whereas Cdk7 fl/fl mice kept the body temperature over 32 C, indicating that Cdk7 bKO mice were cold intolerant ( Figure 1D). Interestingly, when mice had free access to food during the cold exposure, Cdk7 bKO was able to maintain the body temperature to a similar level as that of the Cdk7 fl/fl mice ( Figure S3A).
To further examine the requirement for CDK7 in regulating BAT function, we next measured whole-body O 2 consumption after acute b3-adrenergic stimulation in anesthetized mice. Although Cdk7 bKO and control littermates showed similar O 2 consumption rates at the baseline, the increased O 2 consumption and energy expenditure triggered by the specific b3-adrenergic receptor agonist CL-316,243 (CL) that was observed in Cdk7 fl/fl mice were blunted in the Cdk7 bKO littermates, which suggested a deficient function of the BAT CDK7 in these mice ( Figure 1E). We did not observe any differences in the respiratory exchange ratio (RER), which suggested no differences in substrate utilization ( Figure 1F).

CDK7 Invalidation Leads to Interscapular BAT Atrophy and Impaired UCP1 Expression
We first observed that the interscapular BAT of Cdk7 bKO mice weighted 60% less than the BAT of control littermates (Figures 2A and 2B). Histologic analysis of BAT sections from Cdk7 bKO mice were heterogeneous in cell size, some adipocytes having increased unilocular lipid droplets and other cells without lipid droplets. In addition, we observed exacerbated eosinophilic (red) staining ( Figure 2C), which indicated an increase in immune cell infiltration in the BAT of the Cdk7 bKO mice.
BAT thermogenesis is mediated by UCP1. We therefore checked its expression in our mouse model to elucidate the molecular basis through which CDK7 controls BAT thermogenesis. Western blotting result ll OPEN ACCESS 2 iScience 23, 101163, June 26, 2020 iScience Article revealed that the expression of both UCP1 and PGC1a was decreased in Cdk7 bKO BAT harvested at room temperature ( Figure 2D and 2E), which was consistent with immunohistochemical analysis ( Figure 2F).

CDK7 Is Required for the Induction of Genes Involved in the Thermogenic and Inflammatory Responses to Cold
To have a global view of the genes that are affected by CDK7 depletion in BAT, we next performed RNA sequencing (RNA-seq) on BAT from control and Cdk7 bKO mice at room temperature or after 6 h of cold exposure with free access to food. Principal-component analysis (PCA) analysis showed a good separation of genotypes and treatments ( Figure 3A). Applying cutoffs of a significance level of false discovery rate (FDR) <0.05, the expression of 1,197 was induced after 6 h cold exposure compared with room temperature conditions in the control group. In contrast, the number of genes whose expression was induced by cold in Cdk7 bKO mice was only 268 ( Figure 3B). Strikingly, only 148 of the 1,197 genes induced by cold exposure in WT mice were significantly induced in Cdk7 bKO BAT, showing that CDK7 is required for the vast majority (87%) of the transcriptional adaptation to acute cold stimulation. On the other hand, in basal conditions at room temperature, 876 genes showed reduced expression in Cdk7 bKO BAT compared with control BAT. Under cold conditions, the expression of 1,382 genes was decreased in Cdk7 bKO BAT compared with control BAT.
The differentially expressed genes in BAT between genotypes was evaluated by GO enrichment analysis. Both at room temperature and upon cold exposure, genes down-regulated in the Cdk7 bKO BAT was enriched in mitochondrial and energy metabolism ( Figures 3D and 3E). Moreover, decreased thermogenic genes in response to cold, like UCP1, PGC1a, and DIO2, was confirmed by qPCR and revealed by heatmap ( Figures 3F and 3G). On the other hand, genes up-regulated in the same conditions in the Cdk7 bKO BAT, compared with the BAT of control mice, were enriched in the inflammation response-related GOs, such as cytokine production ( Figures 3F and 3G). Moreover, the expression of these inflammationrelated genes, like the macrophage markers F4/80 and the cytokine TNF-a, were confirmed by qPCR ( Figure 3H). (B) Fat mass were analyzed by EchoMRI (8-weeks-old male, n = 10 for each group). (C) Lean mass were analyzed by EchoMRI (8-weeks-old male, n = 10 for each group) (D) Acute cold response of Cdk7 bKO and control littermates was performed without food supply to evaluate thermogenic activity in control and Cdk7 bKO mice (male, 6-8 weeks, n = 8 for each group).
(E and F) Loss of CDK7 in BAT leads to a reduced response to acute b3-adrenergic signaling. Mice were anesthetized with pentobarbital and put in the individual chambers at 33 C for basal measurement; after the measurement was stable, CL-316,243 (100 mg/kg) was injected at time indicated by arrows as described in Transparent Methods. Oxygen consumption (E) was measured from a comprehensive laboratory animal monitoring system (CLAMS). (F) The respiratory exchange ratio (RER) is calculated as the ratio between VCO 2 and VO 2 (male, 8-9 weeks, n = 7-8 for each group). Values represent means G SEM. **p < 0.01. See also Figures S1 and S2.

OPEN ACCESS
iScience 23, 101163, June 26, 2020 3 iScience Article Interestingly, RNA pol II phosphorylation was not changed in the BAT of the Cdk7 bKO mice, which suggested that the effects of CDK7 on gene transcription were independent of its function of regulating RNA pol II activity ( Figure 3I). Taken together our data suggested that CDK7 is a critical regulator of brown fat thermogenesis and an inhibitor of adipose tissue inflammation.

Creatine Metabolism Compensates for the Impairment in UCP1-Mediated Thermogenesis in Cdk7 bKO Mice Fed Ad Libitum
Since ''normal'' animal housing conditions (20 C-25 C) represent a chronic mild thermal stress to mice, and our results demonstrated impaired brown adipose tissue function and thermogenesis in Cdk7 bKO mice, we examined the effect of CDK7 invalidation at thermoneutrality. We reasoned that, similar to UCP1 KO mice, Cdk7 bKO mice would become obese owing to the impairment of diet-induced thermogenesis. However, Cdk7 bKO did not develop obesity and showed unaltered oxygen consumption, energy expenditure, and food intake ( Figures S4A-S4F). This suggested that Cdk7 bKO mice, when fed normal diet, developed a mechanism to compensate for the deficient UCP1-dependent energy dissipation in BAT. iScience Article It has been reported that the creatine-driven substrate cycle enhances energy expenditure and thermogenesis in beige and brown fat. Furthermore, a genetic model of mouse with abrogated expression of the key creatine metabolism gene Gatm leads to impairment of diet-induced thermogenesis and mouse become obese. This study provides strong in vivo genetic support for a role of creatine metabolism in energy expenditure. Analysis of our RNA-seq data indicated increased expression of the rate-limiting enzymes of creatine biosynthesis ( Figure 3F). These data were confirmed by qPCR analysis, showing higher expression levels of Gatm, Gamt, the creatine transporter Slc6a8, and the creatine kinase Ckmt1 in Cdk7 bKO BAT, compared with Cdk7 fl/fl control mice ( Figure 4A). These results are in accordance with a compensation of the UCP1-driven thermogenesis impairment by creatine metabolism. To validate this hypothesis, we decided to inhibit creatine metabolism in Cdk7 bKO mice. The Cdk7 bKO and control littermates were daily intraperitoneally injected for 4 days during cold exposure with b-guanidinopropionic acid (b-GPA), a creatine analog that inhibits creatine transport and reduces creatine levels in vivo. We found no difference in body temperature during the first 2 days after b-GPA injection in both Cdk7 bKO mice or littermate controls. The body temperature of Cdk7 bKO mice were, however, significantly lower in mice treated with b-GPA, suggesting that, when UCP1 expression is impaired, creatine metabolism compensates for cold-induced thermogenesis in these mice ( Figure 4C).
Adipose Tissue-Specific Cdk7 aKO Mice Have Impaired Response to b3-Adrenoceptor Agonist in White Adipose Tissue Beige and brown fat share common features in terms of thermogenic properties (Ikeda et al., 2018). Since Cdk7 ablation results in impaired BAT development and function, we wondered how CDK7 could affect white adipose tissue browning. We thus generated pan-adipose tissue-specific Cdk7 knockout mice (Cdk7 aKO ) using the Adipoq promoter-driven Cre recombinase transgenic mice ( Figure S5). The BAT phenotype (in Cdk7 aKO mice, Figure S6) was similar with what we observed with Cdk7 bKO . Eight-week-old control or Cdk7 aKO mice were treated with the b3-adrenoceptor agonist CL-316,243 or an equivalent volume of sterile saline for 7 days. Cdk7 fl/fl control mice exhibited intensive CL-induced browning in scWAT assessed by the appearance of multilocular lipid droplets ( Figure 5A). However, this typical feature of beige in CL-316,243-treated Cdk7 aKO scWAT was less intense compared with Cdk7 fl/fl control mice ( Figure 5A). In pgWAT of control mice, the browning effect of CL-316,243 was milder ( Figure 5B), but in the pgWAT of the Cdk7 aKO mice larger adipocytes were found together with infiltrating non-adipocyte cells. The expression of thermogenesis-related genes in scWAT and pgWAT of control Cdk7 fl/fl mice were increased in response to CL-316,243 treatment, whereas Cdk7 aKO mice did not increase the expression of these genes in the same WAT depots ( Figures 5C-5E), which was consistent with the observed morphological changes (Figures 5A and 5B).
Interestingly, the histology of the pgWAT in Cdk7 aKO mice treated with CL-316,243 resembled an exacerbated inflammatory response ( Figure 5B). Indeed, F4/80 immunostaining confirmed a strong macrophage infiltration ( Figure 5H). Moreover, mRNA expression analysis showed a significant increase in F4/80, IL-6, and TNF-a genes in response to CL-316,243 treatment in pgWAT but not in scWAT ( Figures 5F and 5G). Together, these data indicated that CDK7 is required for the b3-adrenoceptorinduced white adipose tissue browning and that CDK7 ablation triggers an immune response in pgWAT in response to b3-adrenoceptor stimulation.
Induction of the b3-Adrenergic Pathway Triggers a Similar Gene Expression Pattern in the Cdk7 bKO BAT and Cdk7 aKO WAT To identify the specific pathways that CDK7 regulates in WAT, we next performed RNA-seq on pgWAT after 7 days saline or CL-316,243 treatment. PCA analysis result showed a good separation of genotypes and treatments. We did not observe major difference between Cdk7 aKO and Cdk7 fl/fl mice when we analyzed We next performed a cross analysis between the RNA-seq data that we obtained for the BAT of the Cdk7 bKO and Cdk7 fl/fl in response to cold ( Figure 3) and the data of the WAT analysis in Cdk7 aKO and Cdk7 fl/fl mice ( Figures 6A and 6B). We focused on the genes that were commonly up-or down-regulated in the WAT and BAT in response to b3-adrenergic stimulation (CL-316,243 treatment in WAT, and cold in BAT). The Venn diagrams showed that 163 genes were commonly upregulated and 156 genes were inhibited in the intersection of the two datasets ( Figure 6C). The GO Biological Process analysis for the 163 up-and 156 down-regulated genes indicated that the immune response-related pathways were the most representatives in the up-regulated genes. The main down-regulated pathways were fat metabolism, mitochondrial and thermogenic groups ( Figure 6D and Table S1). These results were consistent with the phenotype of both the Cdk7 aKO and Cdk7 bKO mice.

DISCUSSION
We prove here that Cdk7 knockout leads to BAT atrophy, with a consistent decrease in UCP1 expression as well as the expression of oxidative and thermogenic genes in response to cold. Consequently, Cdk7 bKO mice are cold intolerant. The thermogenic property of brown and beige adipocytes can transform stored energy into heat; thus, the manipulation of their activity is a potential therapeutic target to treat obesity and type 2 diabetes (Kajimura et al., 2015). However, the impaired thermogenic function does not necessarily lead to metabolic dysfunction. Cdk7 bKO mice, despite the atrophy of BAT and beige WAT are metabolically normal. This finding is reminiscent of mice lacking ERRg in BAT. ERRg preserves brown fat innate thermogenic activity at thermoneutrality. Similar to Cdk7 bKO mice, ERRg KO mice do not gain more weight than WT mice despite a severely blunted thermogenic capacity (Ahmadian et al., 2018). Moreover, mice with depleted expression of the master regulator of thermogenesis, UCP1, are resistant to diet-induced obesity, which suggests that some compensatory mechanisms take over the energy dissipation processes (Enerback et al., 1997;Liu et al., 2003).We found that in the Cdk7 bKO mice the lack of a metabolic phenotype was explained by a compensatory increase in the expression of genes related to the creatine metabolism. Furthermore, the pharmacological reduction of creatine levels in the Cdk7 bKO mice reduces the core body temperature in response to cold. Indeed, a mitochondrial substrate cycle was found to be regulated by creatine to drive thermogenesis (  iScience Article heat under nutrient availability conditions, whereas under fasting conditions, the creatine cycle cannot compensate. Interestingly, the thermogenic phenotype in the Cdk7 bKO mice was only apparent under fasting conditions, suggesting that the creatine compensatory mechanism is responsible for the slow modulation of the metabolic rate but not for fasting adaptive thermogenesis in brown fat in these mice. The role of CDK7 in adipose tissue biology was not limited to the regulation of BAT function. We show that CDK7 is also required for the b3-adrenergic induced browning of WAT. CDK7 ablation in whole adipose tissue demonstrated reduced expression of oxidative and thermogenic genes after long-term CL-316,243 injection. CDK7 is therefore in the intersection between BAT and WAT biology as a mediator of the b-adrenergic signaling in these tissues. Most interesting, we found that CDK7 ablation triggers an immune reaction in BAT in response to cold and in pgWAT in response to adrenergic stimulation. When we crossed the RNA-seq data in the Cdk7 bKO and Cdk7 aKO mice we evidenced that targeting CDK7 in BAT or WAT causes a cellular increase in the expression of pro-inflammatory cytokines/chemokines in both tissues. Genes involved in the immune response were indeed the first class of genes that were upregulated in the absence of CDK7 both in BAT and in WAT. F4/80 staining confirmed increased macrophage infiltration in both BAT and pgWAT upon CDK7 ablation. In support of our findings is a very recent publication by the group of Wong who demonstrated that CDK7 inhibition using a specific molecule potentiated anti-tumor immunity (Zhang et al., 2020). They showed that the blockade of the activity of CDK1 and CDK2, as a result of CDK7 inhibition, induced DNA replication stress and genome instability, which ultimately triggered the immune response. The resulting phenotype of CDK7 inhibition, i.e., the exacerbated immune response, is similar to the inflammatory reaction that we observed in the adipose tissues of WAT and BAT-specific CDK7 KO mice. Moreover, RNA-seq analysis showed that the major class of genes that were induced by CDK7 inhibition or deletion belonged to the immune response GO in both the cancer and the adipose models. The CDK7-mediated mechanisms that trigger the immune response in BAT and in cancer cells are, however, different. CDK7 functions as both a CAK and TFIIH associated kinase. The CAK function of CDK7 was directly involved in the above-mentioned study in the lung cancer cells (Zhang et al., 2020), whereas this is not likely the mechanism in BAT. Two arguments support this hypothesis. First, in our model, Cdk7 is ablated in mature brown adipocyte, which is considered post mitotic and exempt from cell cycle. Second, another potential CDK7 (CAK) target, CDK6, negatively regulates white fat browning (Hou et al., 2018), which is opposite to our findings with CDK7. Moreover, we show in an independent study (unpublished, in revision) that CDK4, which is a downstream effector of CDK7, has an opposite function in BAT. Cdk4 À/À mice, opposite to Cdk7 knockout mice, have increased BAT activity, proving that the CAK function of CDK7 in thermogenic fat is dispensable.
Upon cold exposure, mice typically increase lipid droplets in BAT, indicative of increased lipogenesis. Cold stimulates the simultaneous induction of fatty acid synthesis and b-oxidation to facilitate both mitochondrial heat production and to preserve a pool of fatty acids (Yu et al., 2002;Sanchez-Gurmaches et al., 2018). Interestingly, the BAT from Cdk7 bKO mice were delipidated after cold exposure ( Figure S3B). Delipidation could be caused, at least in part, by a high ratio of lipolysis, since we observed an increase in the phosphorylation of hormone-sensitive lipase (HSL) at serine 660, which is a marker of active lipolysis. Cdk7 bKO mice may upregulate lipolysis as a compensatory mechanism for their defective thermogenic program (Schreiber et al., 2017;Shin et al., 2017). Moreover, the increased lipolysis and accumulation of free fatty acid could lead to the inflammatory response that we observed in both BAT and pgWAT (Mottillo et al., 2010).
As a component of TFIIH, CDK7 can phosphorylate the RNA polymerase II to regulate transcription. The first characterization of the Cdk7 À/À mice showed, however, that this function is dispensable and can be compensated by other kinases (Ganuza et al., 2012). Our results were consistent with these findings. Indeed, phosphorylation of RNA polymerase II was not different between WT and Cdk7 bKO BAT, which suggested other kinases compensate for the role of CDK7 in transcription initiation also in BAT. Taken together, our data proved that the function of CDK7 in BAT was beyond its canonical role as a CAK or a general transcriptional regulator.  iScience Article CDK7 is an emerging target for cancer therapy. CDK7 inhibitors are efficient in the treatment of several models of cancers, including, but not limited to, the lung, breast, prostate, and ovarian tumors (Sun et al., 2020;Rasool et al., 2019;Kim et al., 2020;Zhang et al., 2020). In our study we show that, in addition to the canonical effects in gene transcription and cell cycle control, CDK7 also regulates the activity of BAT and WAT in response to cold and adrenergic stimulation. Strikingly, BAT activity has been related to cachexia, which is a wasting syndrome suffered by about half of patients with cancer. Cachexia is characterized by high energy expenditure in adipose tissue and skeletal muscle and increased thermogenesis in BAT (Bianchi et al., 1989;Shellock et al., 1986). The mechanisms by which tumors induce thermogenesis in BAT are not fully understood, although the tumor-derived signaling through the parathyroid-hormonerelated protein could explain it (Kir et al., 2014). From our results we can expect that the treatment with CDK7 inhibitors will block the activity of BAT and therefore improve cachexia in patients with cancer.
In conclusion, by combining CDK7 BAT-specific and pan-AT-specific knockout mice model, we demonstrated that CDK7 is an important mediator of b3-adrenergic signaling pathways under catabolic conditions in thermogenic brown and beige fat.

Limitations of the Study
Our study proves that CDK7 regulates BAT activity as well as WAT browning in a cell-cycle-independent manner. We also provide evidence that CDK7 is essential for beta-adrenergic signaling in thermogenic fat. However, further experiments, including phospho-proteomics and in vitro kinase assays, should be employed to identify the targets of CDK7 that mediate the observed effects. These potential targets may include but are not limited to key thermogenic transcriptional factors and possible direct targets on beta-adrenergic signaling pathway. In addition, we found that the compensation by the creatine futile cycle in CDK7 knockout BAT leads to resistance to metabolic dysfunction. Further analyses of metabolites (e.g., creatine, phosphocreatine, ATP, ADP) levels under different conditions (like cold/thermoneutrality, fast/ fed) in CDK7 knockout BAT and control will be helpful to decipher the reciprocal relationship of CDK7 knockout and creatine metabolism.
In addition to the effects of CDK7 in fully differentiated BAT cells, as is shown in this study, we cannot exclude the possibility that CDK7 is also involved in adipocyte differentiation. Specific knockout of CDK7 in the progenitors of brown fat with Myf5-cre during BAT development can be employed to answer this question.
Last but not least, CDK7 ablation triggers immune response in brown adipose tissue and also after CL induced browning in pgWAT. Further experiments need to be done to identify which signal or adipokine secretion could be altered that leads to this immune response, as our mice models are adipocyte specific and any non-adipocyte-related phenotype should originate from adipocyte dysfunction.

Resource Availability
Lead Contact Figure S1. Generation of CDK7 brown fat specific knockout mice model. Related to Figure 1. (A) qPCR analysis of Cdk7 expression in various tissues in Cdk7 bKO and control littermates in fed state (male, n=3, 9-11w). All values represent means ± SEM. **p<0.01. Figure S2. Energy homeostasis in Cdk7 bKO mice. Related to Figure 1. Food intake (A), physical activity (B) were measured during indirect calorimetry tests using a comprehensive laboratory animal monitoring system (CLAMS). (male, 8w, n = 6 for each group). Glucose tolerance test (C) and insulin tolerance test (D) in wild-type and Cdk7 bKO mice (male, 10w, n = 9-11 for each group).  were measured during indirect calorimetry with mice after three weeks at thermoneutrality on HFD. The respiratory exchange ratio (RER) (F) is calculated as the ratio between VCO2 and VO2 (male, 8w, n = 6 for each group).  Values represent means ± SEM. *p<0.05, **p<0.01, ***p<0.001. Table S1. Selected genes expression from the intersection data in Fig. 6C. Related to Figure 6.