iLIN28B Tg mice are likewise resistant to diabetes

Although Lin28a and Lin28b both block let-7 miRNAs, they are differentially regulated, resulting in distinct expression patterns during normal development and malignant transformation (Guo et al., 2006; Viswanathan et al., 2009). Given that LIN28B is overexpressed more frequently than LIN28A in human cancer, we sought to determine if LIN28B exerts a similar effect on glucose metabolism. Thus, we generated a mouse strain with human LIN28B driven by a tetracycline transactivator rtTA placed under the control of the Rosa26 locus (iLIN28B mouse) (See Experimental Procedures). After 14 days of treatment with the tetracycline analogue doxycycline (dox), high levels of LIN28B were induced and mature let-7’s were repressed in metabolically important organs (Figure 1H,I), resulting in hypoglycemia with an average fasting glucose of <50 mg/dL in induced mice compared to >150 mg/dL in control mice (p < 0.01). To determine the kinetics of this effect, we measured fed state glucose daily and noted falling glucose levels after 5 days (Figure 1J). Glucose and insulin tolerance tests on dox-induced animals on normal diets showed considerable improvements in glucose tolerance and insulin sensitivity (Figure 1K,L). In checking for islet β cell hyperactivity, we found that iLIN28B mice produced no more insulin than control littermates during glucose challenge (data not shown). Under HFD, we found that induced iLIN28B mice were surprisingly resistant to weight gain (Figure 1M) despite a trend towards increased food intake (9.9 vs. 4.8 g/mouse/day; p = 0.075). These mice continued to exhibit superior glucose tolerance after 14 days of dox induction under HFD (Figure 1N), when average weights were 34.5 ± 1.05 grams for controls and 27.1 ± 0.99 grams for iLIN28B mice, demonstrating that HFD had a strong obesogenic and diabetogenic effect on control but not on LIN28B induced animals. Unlike the Lin28a Tg mice, expression was not leaky in the iLIN28B mice (Figure 1H and ​and3F)3F) and uninduced mice exhibited no growth or glucose phenotypes (Figure S1C,D), making this a better model for inducible Lin28 hyperactivation. These data show that both Lin28 homologues have similar effects on glucose metabolism and obesity, suggesting that these effects are mediated through common mRNA or miRNA targets of the Lin28 family.

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Figure 3

Insulin-PI3K-mTOR signaling is activated by Lin28a/b and suppressed by let-7

(A) Western blot analysis of Lin28a protein expression in C2C12 myoblasts infected with control pBabe or Lin28a overexpression vector, and mouse ESCs, with tubulin as the loading control.

(B) Quantitative PCR for let-7 isoforms in C2C12 myoblasts, normalized to sno142, after Lin28a overexpression.

(C) 2-deoxy-D-[³H] glucose uptake assay on 3-day-differentiated C2C12 myotubes with and without Lin28a overexpression, treated with DMSO, the PI3K inhibitor LY294002, and the mTOR inhibitor rapamycin for 24 hrs.

(D) Western blot analysis of the effects of Lin28a overexpression on PI3K-mTOR signaling in C2C12 myoblasts, under serum-fed (fed), 18 hr serum starved (SS) or insulin-stimulated (Ins) conditions. Insulin stimulation was performed in serum-starved myoblasts with 10 μg/mL insulin for 5 min. Prior to insulin stimulation, serum-starved myoblasts were treated with either DMSO or 20 ng/mL rapamycin for 1 hr.

(E) Western blot analysis of the effects of let-7f or control miRNA on PI3K-mTOR signaling in C2C12 myoblasts under serum-fed (fed), 18 hr serum starved (SS) or insulin-stimulated (Ins) conditions.

(F) Western blot analysis of the effects of LIN28B induction by dox on PI3K-mTOR signaling in quadriceps muscles in vivo (n = 3 iLIN28B Tg mice and 3 LIN28B Tg only mice).

(G) Insr and p-4EBP1 protein levels in wild-type and Lin28a muscle-specific knockout adults.

Lin28a is physiologically required for normal glucose homeostasis

We then asked if Lin28a is physiologically required for normal glucose metabolism in one specific adult tissue compartment, skeletal muscle, since previous studies have found low but significant levels of Lin28a expression in the muscle tissues of mice (Yang and Moss, 2003; Zhu et al, 2010). We generated a skeletal muscle-specific knockout of Lin28a by crossing the mouse myogenic factor 5 Cre recombinase strain (Myf5-Cre) to the Lin28a^fl/fl conditional loss of function mouse (See Experimental Procedures). These muscle-specific knockout mice showed impaired glucose tolerance (Figure 1O) and insulin resistance (Figure 1P) relative to wild-type littermates, demonstrating that Lin28a activity in skeletal muscles is required for normal glucose homeostasis. We analyzed miRNA expression in muscle tissue by qRT-PCR and found no significant difference in let-7 levels during adult (data not shown) or embryonic stages (Figure S1E), suggesting that Lin28a loss of function affects glucose homeostasis either through let-7-independent mRNA binding or through changes in the spatiotemporal distribution of let-7 miRNA. Together, these data show that Lin28 isoforms are important and essential regulators of glucose homeostasis.

iLet-7 mice are glucose intolerant

In addition to their ability to suppress let-7 biogenesis, Lin28a and Lin28b also regulate mRNA targets such as Igf2, HMGA1, OCT4, histones and cyclins through non-let-7 dependent mechanisms of mRNA binding and enhanced translation (Polesskaya et al., 2007; Xu and Huang, 2009; Xu et al., 2009; Peng et al., 2011). To test if altered let-7 expression might produce the opposite phenotypes of Lin28a/b gain of function, we generated a mouse strain in which let-7g can be induced with dox under the control of the Rosa26 locus (iLet-7 mouse, See Experimental Procedures). To ensure that endogenous Lin28 would not block pri- or pre-let-7g biogenesis, we used a chimeric let-7g species called let-7S21L (let-7g Stem, mir-21 Loop), in which the loop region of the precursor miRNA derives from mir-21 and cannot be bound by Lin28, thus allowing for let-7 processing despite Lin28 expression (Piskounova et al., 2008). Global transgene induction from three weeks of age onwards increases mature let-7g levels in liver (>50-fold), skin (>20-fold), fat (~4-fold) and muscle (~4-fold) (Figure 2A). This level of let-7 overexpression led to reduced body size and growth rates in induced animals (Figure 2B,C). Growth retardation was proportional and not manifested as preferential size reduction in any particular organs (Figure S2A). Similar to the iLIN28B mice, leaky expression was not detected and uninduced male mice exhibited no growth or glucose phenotypes (Figure S2B–D).

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Figure 2

iLet-7 mice are glucose intolerant

(A) let-7g and let-7a qRT-PCR in tissues of dox induced iLet-7 mice (n = 3) and controls (n = 3).

(B) Reduced size of induced animals.

(C) iLet-7 growth curve for males and females.

(D) Fed state glucose in iLet-7 mice induced for 5 days.

GTTs performed on mice fed with either (E) normal diet or (F) HFD.

(G) ITT on normal diet.

(H) Insulin production during a glucose challenge.

(I) GTT of LIN28B/Let-7 compound heterozygote mice before (blue) and after (red) induction with dox.

(J) Area under the curve (AUC) analysis for this GTT.

Controls for iLet-7 Tg mice carry either the Let-7 or Rosa26-M2rtTa transgene only. The numbers of experimental animals are listed within the charts.

After 5 days of let-7 induction, these iLet-7 mice produced an increase in fed state glucose (Figure 2D). GTT revealed glucose intolerance in mice fed normal (Figure 2E) or HFD (Figure 2F). Surprisingly, ITT failed to detect a difference in insulin sensitivity (Figure 2G). The decreased glucose tolerance in the setting of comparable insulin sensitivity suggested either decreased insulin production from islet β cells in response to glucose, or higher insulin secretion to compensate for peripheral insulin resistance. Thus, we measured insulin production following glucose challenge, and found that iLet-7 mice produced more insulin than controls (Figure 2H). These results demonstrated that broad overexpression of let-7 results in peripheral glucose intolerance and compensatory overproduction of insulin from islet β cells.

To test if let-7 induction could abrogate the glucose uptake phenotype of LIN28B overexpression, we crossed the iLIN28B to the iLet-7 inducible mice. After 10 days of induction, simultaneous induction of LIN28B and let-7g did not result in any differences in glucose tolerance (Figure 2I,J), in contrast to LIN28B or let-7g induction alone. Taken together, the opposing effects of Lin28 and let-7 expression on glucose regulation show that Lin28 overexpression influences metabolism in part by suppressing let-7, and that let-7 alone is sufficient to regulate glucose metabolism in vivo.

Insulin-PI3K-mTOR signaling is activated by Lin28a/b and suppressed by let-7

To dissect the molecular mechanism of the effects of Lin28 and let-7 on glucose regulation, we turned to the C2C12 cell culture system. Overexpression of Lin28a in C2C12 myoblasts resulted in protein levels of Lin28a similar to that observed in mouse embryonic stem cells (ESCs) (Figure 3A), and led to robust let-7 suppression (Figure 3B). In C2C12 myotubes differentiated for 3 days, Lin28a promoted Ser473 phosphorylation of Akt and Ser235/236 phosphorylation of S6 ribosomal protein, suggesting activation of the PI3K-mTOR pathway (Figure S3A). In this setting, Lin28a increased myotube glucose uptake by 50% (Figure 3C). Lin28a-dependent glucose uptake was abrogated by 24hr treatment with the PI3K/mTOR inhibitor LY294002 or the mTOR inhibitor rapamycin (Figure 3C), but not the MAPK/ERK inhibitor PD98059 (Figure S3B), demonstrating that Lin28a-dependent glucose uptake requires the PI3K-mTOR pathway.

To exclude myotube differentiation-dependent phenomena, we tested the effects of Lin28a on PI3K-mTOR signaling in undifferentiated myoblasts under serum-fed, serum-starved, and insulin-stimulated conditions (Figure 3D). In the serum-fed state, we found that Lin28a promoted the activation of PI3K/Akt signaling by increasing Akt phosphorylation at both Ser473 and Thr308, compared to the pBabe control. Furthermore, we found that Lin28a robustly increased the phosphorylation of mTORC1 signaling targets S6 and 4EBP1 in the serum-fed state. Serum-starvation for 18 hours abrogated the phosphorylation of Akt, S6 and 4EBP1, indicating that Lin28a-induction of PI3K-mTOR signaling requires exogenous growth factor stimulation. Upon insulin stimulation, Akt phosphorylation increased dramatically and, both phospho-S6 and phospho-4EBP1 levels were increased even further by Lin28a overexpression, suggesting that Lin28a increases the insulin-sensitivity of C2C12 myoblasts. Importantly, we found that rapamycin abrogated the Lin28a-induction of phospho-S6 and phospho-4EBP1 upon insulin stimulation, but did not affect let-7 levels (Figure S3C) or Lin28a itself (Figure 3D), indicating that the mTOR dependence is occurring downstream of Lin28a.

To test if the effects of Lin28a on insulin-PI3K-mTOR signaling are let-7-dependent, we transfected either mature let-7f duplex or a negative control miRNA into both Lin28a-overexpressing and pBabe control myoblasts (Figure S3D and Figure 3E). Because mature let-7 duplexes cannot be bound and inhibited by Lin28a protein, this experiment tests if PI3K-mTOR activation is occurring downstream of let-7. Transfection with control miRNA did not affect Lin28a-induction of the phosphorylation of Akt, S6, or 4EBP1 in serum-starved myoblasts upon insulin stimulation. Transfection with let-7f, however, attenuated the Lin28a-induction of phospho-Akt (Ser473), and abrogated the increase in S6 and 4EBP1 phosphorylation upon insulin stimulation in Lin28a-overexpressing myoblasts (Figure 3E). In pBabe control myoblasts, let-7 duplex still suppressed S6 and 4EBP1 phosphorylation in the serum-fed state, serum-starved, and insulin-stimulated conditions, relative to total S6 and 4EBP1 protein. The suppression of mTOR signaling by let-7 even in the absence of Lin28a implies that let-7 can act independently downstream of Lin28a. Together with data indicating that let-7 abrogates Lin28a-specific induction of p-Akt, p-S6 and p-4EBP1 upon insulin stimulation, this demonstrates that the effects of Lin28a on PI3K-mTOR signaling are at least in part due to let-7 and that Lin28 and let-7 exert opposing effects on PI3K-mTOR signaling.

To test if these effects of Lin28 on insulin-PI3K-mTOR signaling are also relevant in vivo, we examined the quadriceps muscles of iLIN28B mice and found that dox-induction led to increases in the phosphorylation of Akt (S473), S6 and 4EBP1, the targets of PI3K-mTOR signaling (Figure 3F). Furthermore, the Insulin-like growth factor 1 receptor (Igf1r) and the Insulin receptor (Insr) proteins were also upregulated in the muscles upon LIN28B induction, reinforcing the fact that Lin28a/b drives insulin-PI3K-mTOR signaling in C2C12 myoblasts and within mouse tissues. On the other hand, similar analysis of the Lin28a muscle-specific knockout mice revealed reduced Insr and p-4EBP1 expression (Figure 3G), demonstrating that Lin28a is both necessary and sufficient to influence glucose metabolism through the regulation of insulin-PI3K-mTOR signaling in vivo.

Lin28a/b and let-7 regulate genes in the insulin-PI3K-mTOR pathway

On the RNA level, Lin28a overexpression in C2C12 myoblasts leads to an increase in mRNA levels of multiple genes in the insulin-PI3K-mTOR signaling pathway (Figure S4A). Although both Lin28a suppression of let-7 and direct Lin28a binding to mRNAs could increase mRNA stability and thus increase mRNA levels, it is possible that these increases do not reflect direct interactions. To find direct targets, we performed a bioinformatic screen using the TargetScan 5.1 algorithm (Grimson et al., 2007), and found that 16 genes in the insulin-PI3K-mTOR pathway contained evolutionarily conserved let-7 binding sites in their respective 3′UTRs (Figure 4A,B). Next, we performed 3′ UTR luciferase reporter assays to determine if these genes were bona fide and direct targets of let-7. To do this, we generated luciferase reporters with twelve human 3′UTR fragments containing conserved let-7 sites. Luciferase reporter expression in human HEK293T cells after transfection of either mature let-7f duplex or a negative control miRNA demonstrated that the 3′ UTRs of INSR, IGF1R, IRS2, PIK3IP1, AKT2, TSC1 and RICTOR were targeted by let-7 for suppression (Figure 4C). Three-base mismatch mutations in the seed region of the let-7 binding sites abrogated let-7’s suppression of INSR, IGF1R and IRS2. To confirm that the luciferase reporters predicted actual changes in protein expression mediated by let-7, we assayed the endogenous expression of some of these proteins upon Lin28a/b overexpression. We found that an increase in Lin28a upregulated Irs2 (Figure 4D) in vitro, and that an increase in LIN28B upregulated Igf1r and Insr protein in skeletal muscles in vivo (Figure 3F). Conversely, INSR and IRS2 are reduced upon both let-7f transfection and LIN28B shRNA knockdown in HEK293T, demonstrating that these regulatory mechanisms hold in both mouse and human cells, and in the setting of both LIN28B gain and loss of function (Figure 4E). This establishes a direct mechanism for let-7’s repression and Lin28’s derepression of multiple components in the insulin-PI3K-mTOR signaling cascade.

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Figure 4

Lin28a/b and let-7 regulate genes in the insulin-PI3K-mTOR pathway

(A) Shown are the numbers of conserved let-7 binding sites within 3′UTRs found using the TargetScan algorithm.

(B) Putative let-7 binding sites in 16 genes of the insulin-PI3K-mTOR pathway and in Lin28a/b.

(C) 3′UTR luciferase reporter assays performed to determine functional let-7 binding sites. Bar graphs show relative luciferase reporter expression in human HEK293T cells after transfection of mature let-7f duplex normalized to negative control miRNA. Shown also are mutations in the seed sequence of the let-7 binding sites for INSR, IGF1R and IRS2.

(D) Western blot analysis of Lin28a, Irs2, and tubulin in C2C12 myoblasts with and without Lin28a overexpression.

(E) Western blot analysis of LIN28B, total IRS2, INSR and TUBULIN in HEK293T cells with either let-7f transfection or shRNA knockdown of LIN28B.

Previously, Lin28a has been shown to enhance Igf2 translation independently of let-7 (Polesskaya et al., 2007), offering an alternative mechanism by which Lin28a might activate the insulin-PI3K-mTOR pathway. To determine the relative contribution of this mechanism, we performed in vitro and in vivo loss of function experiments. Following siRNA knockdown of Igf2 in C2C12 (the efficacy of knockdown is shown in Figure S4B), we found only minimal changes in S6 and 4EBP1 phosphorylation (Figure S4C). In these C2C12 myotubes, glucose uptake was unaffected by Igf2 knockdown, but significantly decreased by let-7a (Figure S4D). In addition, we crossed the Lin28a Tg mice with Igf2 knockout mice and found that the absence of Igf2 did not abrogate enhanced glucose uptake, insulin sensitivity, or the anti-obesity effect mediated by Lin28a (Figure S4E–H). Taken together, these data indicate that the metabolic phenotypes we have observed are not solely due to the ability of Lin28a/b to promote translation of Igf02 mRNA, but do not rule out the possibility that Lin28a/b might modulate other mRNAs in the insulin-PI3K-mTOR signaling pathway.

mTOR mediates Lin28a’s enhancement of growth and glucose metabolism in vivo

Given that Lin28a activates the insulin-PI3K-mTOR pathway both in vitro and in vivo, we asked whether the metabolic effects of Lin28a in vivo could be abrogated by pharmacological inhibition of the mTOR pathway. To do this, we injected Lin28a Tg and wild-type littermates with rapamycin 3 times per week beginning when mice were 18 days old. Rapamycin abrogated the growth enhancement in Lin28a Tg mice at doses that had minimal growth suppressive effects on wild-type mice (Figure 5A,B), suggesting that Lin28a promotes growth in an mTOR-dependent manner. Selective suppression of Lin28a-driven growth was observed using several parameters: weight (Figure 5B,C), crown-rump length (Figure 5D), and tail width (Figure 5E). We also tested if the enhanced glucose uptake phenotype in vivo was likewise dependent on mTOR. Indeed, glucose tolerance testing showed that short-term rapamycin reversed the enhanced glucose uptake effect of Lin28a (Figure 5F,G) and reduced the insulin-sensitivity of Lin28a Tg mice to wild-type levels (Figure 5H,I). These data indicate that the glucose uptake, insulin sensitivity and animal growth phenotypes of Lin28a overexpression in vivo are dependent on mTOR signaling.

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Figure 5

mTOR is required for Lin28a’s effects on growth and glucose metabolism in vivo

(A) Rapamycin (left 2 mice) and vehicle (right 2 mice) treated wild-type and Lin28a Tg mice shows relative size differences.

(B) Curves showing relative growth (normalized to weight on first day of treatment) for mice treated from 3 weeks to 6.5 weeks of age. Blue and red represent wild-type and Lin28a Tg mice, respectively. Solid and dotted lines represent vehicle and rapamycin treated mice, respectively. Growth was measured by several other parameters:

(C) weight, (D) crown-rump length or height, and (E) tail width.

(F) GTT performed after 2 doses of vehicle or (G) rapamycin.

(H) ITT performed after 1 dose of vehicle or (I) rapamycin.

Controls for Lin28a Tg mice are WT. The numbers of experimental animals are listed within the charts.

Lin28a/b and let-7 influence glucose metabolism through the insulin-PI3K-mTOR pathway

We have shown that Lin28a/b and let-7 regulates insulin-PI3K-mTOR signaling, a highly conserved pathway that regulates growth and glucose metabolism throughout evolution. PI3K/Akt signaling is known to promote Glut4 translocation to upregulate glucose uptake, while mTOR signaling can promote glucose uptake and glycolysis by changing gene expression independently of Glut4 translocation (Brugarolas et al., 2003; Buller et al., 2008; Duvel et al., 2010). Previous studies have shown that Lin28a directly promotes Igf2 (Polesskaya et al., 2007) and HMGA1 translation (Peng et al., 2011), and that let-7 suppresses IGF1R translation in hepatocellular carcinoma cells (Wang et al., 2010). Consistent with these findings, our results define a model whereby Lin28a/b and let-7 coordinately regulate the insulin-PI3K-mTOR pathway at multiple points (Figure 6E), a concept that is consistent with the hypotheses that miRNAs and RNA binding proteins regulate signaling pathways by tuning the production of a broad array of proteins rather than switching single components on or off (Kennell et al., 2008; Hatley et al., 2010; Small and Olson, 2011). Coordinated regulation is important because negative feedback loops exist within the insulin-PI3K-mTOR pathway. Loss-of-function and pharmacological inhibition studies have shown that the mTOR target S6K1, for instance, inhibits and desensitizes insulin-PI3K signaling by phosphorylating IRS1 protein and suppressing IRS1 gene transcription (Harrington et al., 2004; Shah et al., 2004; Tremblay et al., 2007; Um et al., 2004). Conversely, TSC1–2 promotes insulin-PI3K signaling by suppressing mTOR signaling (Harrington et al., 2004; Shah et al., 2004). Although the effects of let-7 and Lin28a/b on the expression of individual genes are modest, simultaneous regulation of multiple components such as IGF2, IGF1R, INSR, IRS2, PIK3IP1, AKT2, TSC1, RICTOR in the insulin-PI3K-mTOR signaling pathway could explain how this RNA processing pathway coordinately regulates insulin sensitivity and glucose metabolism by effectively bypassing these negative feedback loops.

Whereas our work has implicated let-7 as a regulator of insulin-PI3K-mTOR signaling, we do not exclude a parallel role for direct mRNA targets of Lin28a/b in glucose metabolism, a hypothesis supported by the recent findings that HMGA1 is translationally regulated by LIN28A and mutated in 5–10% of T2D patients (Peng et al., 2011; Chiefari et al., 2011). Such non-let-7 functions are also suggested by the fact that muscle-specific loss of Lin28a results in glucose derangement without significant let-7 changes. Nevertheless, it remains likely that during other developmental stages or in other tissues, let-7 suppression by Lin28a or Lin28b is required for normal glucose homeostasis. The effects of the Lin28/let-7 pathway on glucose metabolism in our murine models, together with our observation that genes regulated by let-7 are associated with T2D risk in humans, indicates important functional roles for both Lin28a/b and let-7 in human metabolism.

Indirect Calorimetry

The apparatus used was a set of 16 OxyMax® Metabolic Activity Monitoring chambers (Columbus Instruments; Columbus, OH, USA). Each chamber consisted of a self-contained unit capable of providing continuous measurements of an individual mouse’s total activity and feeding behavior. Monitoring occurred over a 3-day period. Each subject was placed into an individual chamber on day 1, with free access to food and water during the course of the experiment. Subjects were maintained under a normal 12:12 h light:dark cycle. All measurements were sampled periodically (at approximately 12-min intervals) and automatically recorded via the OXYMAX Windows V3.22 software. Activity measures over the final 24-h period were parceled into 2-h bins and these were used to express diurnal activity levels.

Quantitative RT-PCR

Performed with standard methods, which are described in detail in the Extended Experimental Procedures.

Histology

Tissue samples were fixed in 10% buffered formalin or Bouin’s solution and embedded in paraffin.

Glucose and insulin tolerance tests

Overnight-fasted mice were given i.p. glucose (2 mg/g body weight). For insulin tolerance test, 5 hour fasted mice were given 0.75 U insulin/kg body weight by i.p. injection (Humulin). Blood glucose was determined with a Lifescan One Touch glucometer. Insulin, GH, and Igf1 levels were measured by ELISA (Crystal Chem).

Cloning

Murine Lin28a and human LIN28B cDNA was subcloned into pBabe.Puro and pMSCV.Neo retroviral vectors. LIN28B and Control shRNA in lentiviral plasmids were purchased from Sigma-Aldrich and previously reported in Viswanathan et al., 2009. UTR cloning for luciferase reporters is described in Supplemental Table S2.

Cell culture, viral production, and transfection

Performed using standard methods as described in the Extended Experimental Procedures.

Glucose uptake assay

In vitro glucose uptake assays were performed as described in Berti and Gammeltoft, 1999.

Drug treatments

Rapamycin was injected i.p. 3 times a week for mouse experiments. For cell culture, C2C12 myotubes differentiated for 3 days were incubated with inhibitors for 1 day prior to glucose uptake assays. See Extended Experimental Procedures for further details.

Western blot assay

Performed using standard methods. Detailed methods and reagents used are described in the Extended Experimental Procedures.

Luciferase reporter assay

10 ng of each construct was co-transfected with 10 nM miRNA duplexes or into HEK-293T cells in a 96-well plate using lipofectamin-2000 (Invitrogen). After 48 hours, the cell extract was obtained; firefly and Renilla luciferase activities were measured with the Promega Dual-Luciferase^® reporter system.

MAGENTA analysis

See Results, Table 1 Legend, and Extended Experimental Procedures for detailed methods.

We thank John Powers, Harith Rajagopalan, Jason Locasale, Abdel Saci, Akash Patnaik, Charles Kaufman, Christian Mosimann and Lewis Cantley for invaluable discussions and advice, Roderick Bronson and the Harvard Medical School Rodent Histopathology Core for mouse tissue pathology, and the Harvard Neurobehavior Laboratory for CLAMS experiments. This work was supported by grants from the US NIH to G.Q.D., a Graduate Training in Cancer Research Grant and a American Cancer Society Postdoctoral Fellowship to H.Z., the NSS Scholarship from the Agency for Science, Technology and Research, Singapore for N.S.C, an NIH NIDDK Diseases Career Development Award to M.G.K, and an American Diabetes Association Postdoctoral Fellowship for A.V.S. J.M.E. was supported by the National Human Genome Research Institute (NHGRI). R.I.G. was supported by US National Institute of General Medical Sciences (NIGMS) and is a Pew Research Scholar. D.A. is a Distinguished Clinical Scholar of the Doris Duke Charitable Foundation. G.Q.D. is a recipient of Clinical Scientist Awards in Translational Research from the Burroughs Wellcome Fund and the Leukemia and Lymphoma Society, and an investigator of the Howard Hughes Medical Institute and the Manton Center for Orphan Disease Research.