Cidea is an essential transcriptional coactivator regulating mammary ...

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Cidea is an essential transcriptional coactivator regulating mammary gland secretion of milk lipids

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© 2012 Nature America, Inc. All rights reserved.

Wenshan Wang1, Na Lv1, Shasha Zhang1, Guanghou Shui2, Hui Qian1, Jingfeng Zhang1, Yuanying Chen1, Jing Ye3, Yuansheng Xie4, Yuemao Shen5, Markus R Wenk2 & Peng Li1 Adequate lipid secretion by mammary glands during lactation is essential for the survival of mammalian offspring. However, the mechanism governing this process is poorly understood. Here we show that Cidea is expressed at high levels in lactating mammary glands and its deficiency leads to premature pup death as a result of severely reduced milk lipids. Furthermore, the expression of xanthine oxidoreductase (XOR), an essential factor for milk lipid secretion, is markedly lower in Cideadeficient mammary glands. Conversely, ectopic Cidea expression induces the expression of XOR and enhances lipid secretion in vivo. Unexpectedly, as Cidea has heretofore been thought of as a cytoplasmic protein, we detected it in the nucleus and found it to physically interact with transcription factor CCAAT/enhancer-binding protein b (C/EBPb) in mammary epithelial cells. We also observed that Cidea induces XOR expression by promoting the association of C/EBPb onto, and the dissociation of HDAC1 from, the promoter of the Xdh gene encoding XOR. Finally, we found that Fsp27, another CIDE family protein, is detected in the nucleus and interacts with C/EBPb to regulate expression of a subset of C/EBPb downstream genes in adipocytes. Thus, Cidea acts as a previously unknown transcriptional coactivator of C/EBPb in mammary glands to control lipid secretion and pup survival. The establishment of normal lactation requires the proliferation and differentiation of mammary epithelial cells to form a lobuloalveolar structure, marked upregulation of protein and lipid synthesis and, finally, the secretion of milk1–3. In the lactating mammary gland, lipid secretion consists of two steps: the envelopment of cytosolic lipid droplets (CLDs) within the apical plasma membrane, followed by budding out to the lumen of mammary alveoli as milk fat globules (MFGs). XOR, which is usually diffused in the cytoplasm of mammary epithelial cells, is localized to the apical membrane to interact with the cytoplasmic domain of butyrophilin (BTN) when secretion is activated. The interaction between XOR, BTN and some unknown proteins on CLDs promotes the envelopment of CLDs and their budding off as MFGs4–7. Reduced milk lipid secretion in lactating mammary glands has been linked to poor newborn survival in several genetically modified mouse models5,8–10. As a crucial mediator of milk lipid secretion, XOR expression is markedly upregulated during pregnancy and lactation under the control of the transcription factor C/EBPβ11–13. However, little is known about the molecular mechanism underlying mammary gland–specific expression of the gene encoding XOR. CIDE-family proteins, including Cidea, Cideb and Fsp27 (Cidec), are lipid droplet–associated proteins important in various aspects of lipid metabolism14–17. Cidea is highly enriched in adult brown adipose tissue (BAT), and its deficiency results in the accumulation of smaller lipid droplets, improved insulin sensitivity and resistance to diet-induced obesity18–21.

Here we show that Cidea is expressed at high levels in lactating mammary glands and that its deficiency results in reduced milk lipid secretion and poor newborn survival. In addition, we show that Cidea is localized to the nucleus and interacts with C/EBPβ to enhance the expression of a subset of C/EBPβ downstream genes, including that encoding XOR, that controls milk lipid secretion. RESULTS Cidea expression in lactating mammary glands In the course of breeding Cidea−/− mice, we unexpectedly found that both Cidea−/− and wild-type pups fostered by Cidea−/− females had lower body weight and died within 3 d postpartum. However, when they were cross-fostered by wild-type or heterozygous females, Cidea−/− pups gained weight steadily and thrived (Fig. 1a,b). These observations led us to suspect that Cidea has a role in lactation. Indeed, we observed that Cidea began to be highly expressed in mammary glands at day 14.5 of pregnancy, and its expression was maintained at high levels throughout lactation and declined during post-lactational involution (Fig. 1c,d). This expression profile coincides well with that of XOR (Fig. 1c). In contrast, Fsp27 and perilipin (Plin), two adipocyte-specific proteins, were barely detectable in the mammary glands of either pregnant or lactating mice (Fig. 1d). Immunohistochemical staining of sections of lactating mammary glands showed that Cidea was expressed in epithelial cells but not in the surrounding adipose tissue (Fig. 1e). Cidea protein was also

1Tsinghua-Peking

Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China. 2Yong Loo Lin School of Medicine, Department of Biochemistry, and Department of Biological Sciences, National University of Singapore, Singapore. 3Department of Pathology, The Fourth Military Medical University, Xi’an, Shaanxi, China. 4The State Key Laboratory of Kidney Disease, Institute of Nephrology, Chinese PLA General Hospital, Beijing, China. 5School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong, China. Correspondence should be addressed to P.L. ([email protected]). Received 17 May 2011; accepted 30 November 2011; published online 15 January 2012; doi:10.1038/nm.2614

nature medicine VOLUME 18 | NUMBER 2 | FEBRUARY 2012

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Casein Figure 1 Cidea is expressed at Cidea–/– Cidea Bodipy Merge β-actin WAT higher levels in mammary glands of Plin pregnant and lactating mice. (a,b) Survival Fsp27 rates (a) and average body weights (b) of Cidea−/− litters nursed by wild-type or Cidea−/− females (n = 8 each), and wild-type litters nursed by wild-type or Cidea−/− females (n = 7 each). Six litters were nursed by each strain of female mice. (c,d) Levels of Cidea mRNA (c) and protein (d) during mammary gland development. PD, pregnancy day; LD, lactation day. R2 is the Pearson correlation coefficient for the expression of Cidea (blue) and XOR (red). β-actin was used as a loading control for western blotting; Fsp27 and Plin are markers for stromal adipose tissue. These data were obtained from whole-tissue lysates of mammary glands. (e) Immunohistochemical staining of sections of lactating (L2D) mammary glands from wildtype and Cidea−/− mice with antibody to Cidea. Scale bars, 200 µm. (f) Western blotting showing the expression of Cidea protein in MECs isolated on L2D. Casein is a marker for MECs. (g,h) Western blotting (g) and immunofluorescence staining (h) showing expression of Cidea in the fraction of MFGs. MFGs were labeled with Bodipy 493/503 fatty acids (green). Scale bars, 2 µm. (i,j) Cidea and XOR mRNA (i) and protein (j) levels in differentiated mammary epithelial cells (HC11). The expression of Cidea mRNA was strongly correlated with that of XOR (R2 = 0.992). XOR, Plin2 and casein are markers for mammary gland development. Fsp27 and Plin are specific markers for white adipose tissue (WAT). Error bars show means ± s.e.m. 200 µm

detected in isolated lactating mammary epithelial cells (MECs; Fig. 1f) and in isolated MFGs (Fig. 1g,h), indicating that Cidea is secreted into milk via MFGs. Furthermore, Cidea expression was detected on day 4 of differentiation in mammary epithelial HC11 cells and increased over the course of differentiation, similarly to the expression of XOR (Fig. 1i,j). Impaired milk lipid secretion in lactating female Cidea−/− mice To investigate how the high expression of Cidea in the mammary glands of pregnant and lactating females affects the survival of newborn pubs, we collected milk from lactating females and performed high-resolution lipidomics analyses. The concentrations of tri- and diacylglycerols (TAGs and DAGs), which together make up 98% of the total milk lipids 22, were markedly lower in milk taken from Cidea−/− mice compared with wild-type mice (Fig. 2a). The lower fat concentrations could not have been caused by an overall defect in milk nutrient secretion because the volume of milk and the concentration of total milk proteins were similar in wild-type and Cidea−/− females (Fig. 2a). Gas chromatography–mass spectrometry analysis showed that the concentrations of most fatty acid species were markedly lower in Cidea−/− mice (Supplementary Fig. 1a). Liquid chromatography–mass spectrometry analysis further showed that concentrations of most individual TAG or DAG species, including those containing linoleic acid (FA18:2), α-linoleic

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acid (FA18:3) or docosahexaenoic acid (FA22:6), were markedly lower in the milk of Cidea−/− mice (Supplementary Fig. 1b,c). The amount of phosphoserine, one of the major components of the plasma membrane, was also substantially lower in the milk of Cidea−/− mice (Fig. 2b), whereas the concentrations of other phospholipids, such as phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin, ceramide and glucosylceramide, were similar in the milk of Cidea−/− and wild-type mice (Supplementary Fig. 1d). Because milk lipids are secreted as MFGs, with lipid droplets wrapped by a plasma membrane bilayer, we measured the sizes of MFGs from wild-type and Cidea−/− mice and found that MFGs from Cidea−/− mice were substantially smaller (Fig. 2c), having an average diameter of 1.12 ± 0.03 µm compared with 7.94 ± 0.30 µm for wildtype mice (Fig. 2d). The number of MFGs from Cidea−/− mice was also fewer by 70% (Fig. 2e). Consistent with the reduced milk lipid secretion, cytosolic TAG and free fatty acid (FFA) concentrations in MECs were higher in Cidea−/− than in wild-type mice (Fig. 2f ). Notably, the number of dark particles in lumens, representing casein micelles23, was similar in wild-type and mutant mice (Fig. 2g). The lipid-secretion defect could not have been caused by an impairment in lobuloalveolar development because the number and organization of lobuloalveolar structures in the mammary glands of both pregnant (day 16.5 of pregnancy, P16.5D) and early lactating (day 2

VOLUME 18 | NUMBER 2 | FEBRUARY 2012 nature medicine

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Figure 2 Impaired milk lipid secretion in lactating Cidea−/− female mice. (a,b) Analysis of lipid components of milk (a) and total milk protein concentrations and milk volume (b) collected from the mammary glands of female mice. n = 5 for each genotype. PS, phosphoserine. (c) Immunofluorescence staining showing the morphology of MFGs (green, stained with Bodipy 493/503) from wild-type and Cidea−/− mice. Scale bars, 5 µm. (d,e) Size and number of MFGs (LD, lipid droplets) from wild-type and Cidea−/− mice, analyzed using Image-Pro Plus 5.0. (f) Cellular TAG and FFA concentrations in wild-type and Cidea−/− MECs at L2D (n = 6 for each genotype). (g) Electron microscopic images of mammary gland sections from wild-type and Cidea−/− mice at L2D. Scale bars, 4 µm. Arrow indicates small lipid droplets in the lumen of mammary glands of Cidea −/− mice. (h) Morphology of mammary glands from wild-type and Cidea−/− mice at P16.5D and L2D, visualized by H&E staining. Scale bars, 50 µm. (i,j) TAG secretion in Cidea-KD HC11 cells differentiated for 10 d (i), and in Cidea-overexpressing HC11 cells differentiated for 2 d (j). Ad, infection with adenovirus expressing scrambled short hairpin RNA (control for knockdown), Cidea-KD sequence, GFP (control for overexpression) or Cidea. DPM, disintegrations per minute, a measure of radioactivity. Error bars show means ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; NS, no statistical significance.

of lactation, L2D) females were similar in Cidea−/− and wild-type mice (Fig. 2h and Supplementary Fig. 2a,b). Electron microscopic analysis found very few lipid droplets in the lumens of lactating Cidea−/− mammary gland alveoli, whereas abundant lipid droplets had accumulated in the lumens of wild-type mammary gland alveoli (Fig. 2g). In accordance with the established role of Cidea in controlling lipid droplet size, Cidea−/− MECs contained many smaller lipid droplets in the cytoplasm, whereas wild-type cells contained only a few large lipid droplets, most of which were located near the lumen face (Fig. 2g). Overall, these data indicate that Cidea deficiency results in reduced milk lipid secretion and the accumulation of smaller lipid droplets.


Next, we measured the actual rates of lipid secretion in differentiated wild-type and Cidea-knockdown (Cidea-KD) HC11 cells. Compared with undifferentiated wild-type cells, the rate of TAG secretion was approximately tenfold higher in the differentiated wild-type HC11 cells (Supplementary Fig. 2c). The rate of TAG secretion in differentiated Cidea-KD HC11 cells was 70% lower than in differentiated wild-type HC11 cells (Fig. 2i), despite their similar rates of TAG synthesis and fatty acid uptake (Supplementary Fig. 2d,f). In contrast, the overexpression of Cidea in early differentiated HC11 cells markedly enhanced TAG secretion, although no substantial difference was observed in their rates of TAG synthesis or fatty acid uptake (Fig. 2j and Supplementary Fig. 2e,g).

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Figure 3 Cidea controls lipid secretion by regulating XOR expression. (a,b) Levels of mRNAs (a) and proteins (b) in wild-type and Cidea−/− mammary glands. Wap, whey acidic protein. (c–f) Western blots (c,e; quantified by TOTAL-LAB software in d,f) showing XOR expression in differentiated Cidea-KD HC11 cells (c,d) or Cidea-overexpressing HC11 cells (e,f). Cells were infected with adenoviruses described in Figure 2i,j. The relative protein level in control cells (Ad-scramble or Ad-GFP) was designated as 1. (g) TAG secretion in HC11 cells infected with indicated adenoviruses. Ad-HA-XOR expresses HA-tagged XOR. (h) Western blotting showing the expression of XOR in HC11 cells in g. Error bars show means ± s.e.m. *P < 0.05, **P < 0.01; NS, no statistical significance.

Overall, these data strongly support the notion that Cidea is crucial in controlling lipid secretion in MECs. Cidea controls lipid secretion by regulating XOR expression To explore the molecular process by which Cidea controls milk lipid secretion, we examined the expression of XOR, BTN and perilipin 2 (Plin2), which mediate milk lipid secretion24,25. The mRNA and protein levels of XOR in mammary glands of pregnant and lactating Cidea−/− mice were markedly reduced, whereas expression of BTN and Plin2 remained relatively unchanged (Fig. 3a,b and Supplementary Fig. 3a). Consistent with this, XOR protein levels in the milk of Cidea−/− mice were also reduced (Supplementary Fig. 3b). The reduced XOR expression in Cidea−/− mice could not have been caused by lower levels of C/EBPβ because C/EBPβ expression was similar in wild-type and Cidea−/− lactating mammary glands (Supplementary Fig. 3c,d). Knockdown of Cidea in differentiated HC11 cells also led to substantially lower expression of XOR but not of Plin2 or casein (Fig. 3c,d). In contrast, the overexpression of Cidea resulted in higher XOR expression but similar levels of Plin2 and casein (Fig. 3e,f). Cidea-dependent induction of XOR expression was also observed in MCF7 cells (a human mammary epithelial cell line) but not in nonmammary epithelial cells, such as 293T or HeLa cells (data not shown). To determine whether XOR is a mediator of Cidea-controlled lipid secretion, we introduced XOR back into differentiated CideaKD HC11 cells. The rate of TAG secretion was threefold higher in Cidea-KD HC11 mice expressing XOR than in Cidea-KD HC11 mice without added XOR, indicating that the reintroduction of XOR restored lipid secretion in Cidea-KD cells (Fig. 3g,h). Moreover, the overexpression of XOR did not affect the size of cytoplasmic lipid droplets in Cidea-KD HC11 cells (Supplementary Fig. 3e). Cidea interacts with C/EBPb and regulates Xdh promoter activity To delineate the regulatory pathway from Cidea to XOR expression, we generated luciferase reporters fused to the promoter region of Xdh (the gene encoding XOR; the promoter region, which we term XORP, comprises positions −1100 to +100), which contains a typical C/EBPβ

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binding element (CBE; Fig. 4a). We then tested whether Cidea induced expression of this wild-type XORP–fused luciferase reporter (XORP-WT). When Cidea was overexpressed, the activity of XORPWT was markedly higher (eightfold excess; Fig. 4b). Stimulation by Cidea was not observed with a luciferase reporter fused to a mutant XORP carrying an alteration in the CBE (XORP-mutant)12 (Fig. 4b) or when C/EBPβ was knocked down (Supplementary Fig. 4a). Consistent with this, the induction of endogenous XOR expression by the overexpression of Cidea was abrogated by knockdown of C/EBPβ (Fig. 4c). Furthermore, Cidea-induced TAG secretion was blocked when C/EBPβ was knocked down (Fig. 4d). Therefore, Cidea-induced XOR expression and lipid secretion are dependent on C/EBPβ in mammary epithelial cells. Next, we used coimmunoprecipitation to assess whether the ability of Cidea to enhance C/EBPβ-dependent XOR expression is linked to a physical interaction between Cidea and C/EBPβ. When a nuclear fraction isolated from lactating mammary glands was immuno precipitated with an antibody against Cidea, C/EBPβ was detected in the immunoprecipitate (Fig. 4e). C/EBPβ did not coprecipitate with the control antibody to IgG. In the reciprocal experiment, endogenous C/EBPβ was also able to coprecipitate Cidea in differentiated MECs (Supplementary Fig. 4b). When overexpressed in 293T cells, Flagtagged Cidea and Fsp27 (another CIDE-family protein) were also able to pull down C/EBPβ (Fig. 4f). In reciprocal experiment, Flag-tagged C/EBPβ coimmunoprecipitated HA-tagged Cidea but not HA-tagged Plin2 (Supplementary Fig. 4c). We further mapped the interaction interfaces between Cidea and C/EBPβ and found that Cidea proteins containing amino acid residues 1–163 (Cidea1–163), residues 118–217 (Cidea118–217) or a deletion of the lipid droplet–association domain (residues 164–200; Cidea∆164–200) were able to coprecipitate C/EBPβ (Fig. 4g). In contrast, deletion of residues 118–163 from Cidea (Cidea∆118–163) completely eliminated the interaction between Cidea and C/EBPβ (Fig. 4g). GST pull-down assays with bacterially generated C/EBPβ and GST-Cidea fusion proteins showed that GST-Cidea110–163, but not GST-Cidea33–110, was sufficient to pull down C/EBPβ (Fig. 4h), suggesting that the Cidea and C/EBPβ interaction is direct and not


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mediated by other factor(s). When overexpressed, full-length Cidea and a deletion mutant (Cidea∆164–200) induced XORP-luciferase activity and endogenous XOR expression (Fig. 4i and Supplementary Fig. 4d,e). In addition, deletion of the C/EBPβ interaction interface from Cidea (Cidea∆118–163) resulted in a complete loss of Cideainduced TAG secretion (Fig. 4j). Overall, these data indicate that Cidea and C/EBPβ form a complex in cells. This interaction is mediated by residues 118–163 in Cidea, which are required for Cideainduced XOR expression and lipid secretion in lactating mammary glands. Consistent with a previous analysis in brown adipocytes15,20, the overexpression of full-length Cidea in HC11 cells increased the size of lipid droplets, whereas deletion of its lipid droplet-association domain (residues 164–200) or deletion of residues 118–163 rendered Cidea inactive in promoting formation of large lipid droplets (Supplementary Fig. 4f). Nuclear distribution of Cidea and Fsp27 Next, we examined by biochemical fractionation whether any portion of Cidea is localized to the nucleus in the mammary gland or in the BAT. Indeed, in addition to its previously established localization on lipid droplets, a substantial portion of Cidea protein was detected in the nuclear fraction, together with C/EBPβ and the nuclear matrix protein Lamin B, in both lactating mammary glands and BAT (Fig. 5a and Supplementary Fig. 5a). Notably, Fsp27 was also detected in the nuclear fraction of BAT and of differentiated 3T3-L1 adipocytes (Fig. 5b). Cidea and Fsp27 were also detected in the nuclear fraction


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Figure 4 Cidea interacts with C/EBPβ and activates the Xdh promoter. (a) Sequence analysis of the promoter region 0 of mouse Xdh (encoding XOR) reveals the conserved C/EBP binding element (CBE). Red line represents crossspecies similarity within promoter region of Xdh. (b) Expression of luciferase reporter constructs in response to Cidea overexpression in undifferentiated HC11 cells. (c,d) Endogenous XOR mRNA levels (c) and TAG secretion (d) Ad-Cidea in differentiated HC11 cells infected with adenoviruses as in Figure 2i,j. Ad-C/EBPβ-KD expresses a C/EBPβ-knockdown sequence. (e) Immunoblot (IB) showing coprecipitation of C/EBPβ upon immunoprecipitation (IP) of Cidea in nuclear extract from wild-type or Cidea−/− mammary glands. Anti-IgG was used as a negative control. (f,g) Coimmunoprecipitation of constructs coexpressed in 293T cells (as indicated above gels). Flag-tagged Cidea constructs in f,g included full-length (residues 1–217) and truncated sequences (residues 1–117, 118–217, 1–163 or 164–217), and deletion mutants (∆118–163, ∆164–200). Red asterisks represent the correct size proteins. (h) GST pull-down assays of C/EBPβ and Cidea 33–110 or Cidea110–163. (i,j) Endogenous XOR mRNA levels (i) and TAG secretion (j) in differentiated HC11 cells infected with adenovirus (Ad) expressing indicated constructs. Error bars show means ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; NS, no statistical significance. Ad


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when they were ectopically expressed (Supplementary Fig. 5b). When overexpressed, GFP-tagged Cidea and Fsp27 were detected both in the nucleus and on the surfaces of lipid droplets (Fig. 5 and Supplementary Fig. 5c,d). In addition, fluorescence recovery after photobleaching (FRAP) analysis showed that the rate of Cidea-GFP import into the nucleus was faster than that of GFP alone, with a halfmaximal recovery time after fluorescent bleaching (t1/2) of approximately 30 s for Cidea-GFP, compared with 75 s for GFP alone (Fig. 5c and Supplementary Fig. 5e). Furthermore, leptomycin B (LMB), a CRM-1 inhibitor26,27, had no effect on the nuclear accumulation of Cidea-GFP in HC11 cells (Supplementary Fig. 5f ). These data suggest that nuclear accumulation of Cidea-GFP is an active transport process and that it might be CRM-1 independent. When overexpressed, GFP-tagged Cidea1–164 or Cidea with a deletion of the lipid droplet–association domain (∆164–200) were present in the nucleus, whereas Cidea1–117, Cidea118–217, Cidea164–217 or Cidea with a deletion of the C/EBPβ interaction interface (∆118–163) showed substantially weaker signals in the nucleus (Fig. 5d and Supplementary Fig. 5g). These data indicate that specific residues in the N-terminal region and its interaction with C/EBPβ are both required for Cidea nuclear localization. Sequence alignment of the N-terminal region of Cidea from different species revealed several conserved lysine and arginine residues (including Lys23 and Lys24; Fig. 5e and Supplementary Fig. 5h) that may have a role in determining Cidea nuclear localization. Indeed, amino acid substitution of both Lys23 and Lys24 with alanine (K23A K24A) substantially reduced

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the nuclear localization of Cidea (Fig. 5d,f and Supplementary Fig. 5i). Notably, substitution of Lys23 with arginine (K23R) led to markedly more nuclear Cidea (Fig. 5d,f and Supplementary Fig. 5i). Consistent with its inability to localize to the nucleus, Cidea harboring the K23A K24A mutations did not induce XOR expression (Fig. 5g). Therefore, Lys23, Lys24 and the C/EBPβ-interacting domain of Cidea are required for its nuclear localization. Cidea functions as a transcriptional cofactor for C/EBPb To further explore the molecular mechanism by which Cidea induces XOR expression, we measured the amount of Cidea and C/EBPβ associated with the Xdh promoter by chromatin immunoprecipitation (ChIP). ChIP analysis revealed that Cidea was associated with the Xdh promoter, and this association was reduced when C/EBPβ was knocked down or when the C/EBPβ-interacting domain of Cidea was deleted (Fig. 6a and Supplementary Fig. 6a,b). Notably, the association of C/EBPβ with the Xdh promoter was substantially greater when Cidea was overexpressed but markedly less in Cidea-KD cells (Fig. 6b). Enhanced association of C/EBPβ with the Xdh promoter was not

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Figure 5 Cidea and Fsp27 are localized to the nucleus. (a,b) Western blotting of proteins in fractions of lactating mammary gland (a) and differentiated 3T3-L1 adipocytes (b). β-tubulin, cytosol marker; Lamin B, nuclear marker; calnexin, microsome marker; Plin2, lipid droplet marker; Cox4, mitochondria marker. (c) FRAP analysis to examine the time of nuclear import of Cidea-GFP and a GFP control in HC11 cells. Data are from 12 cells. (d) Immunofluorescence staining showing the nuclear localization of GFP-tagged Cidea (1–217, full length) and Cidea mutants expressed in HC11 cells. Bright circles are lipid droplets that have Cidea associated with their surfaces. Cells were pretreated with 20 µg ml−1 digitonin before fixation and stained with DAPI (blue). Scale bars, 5 µm. (e) Schematic of Cidea showing position of K23A K24A mutation (red). (f) Biochemical fractionation of 293T cells expressing wild-type (WT) Cidea and Cidea mutants. CM, nonnuclear (cytosol plus crude membranes) fraction; N, nuclear fraction. (g) Endogenous XOR mRNA levels in differentiated HC11 cells infected with adenovirus (Ad) expressing GFP (control) or Cidea constructs. Error bars show means ± s.e.m. *P < 0.05, ***P < 0.001; NS, no statistical significance.

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observed upon expression of Cidea118–163, which lacks the Cidea-C/EBPβ inter action domain (Supplementary Fig. 6c). Furthermore, a Cidea truncation mutant lacking the lipid droplet-associated domain (Cidea164–200) associated markedly more strongly with the Xdh promoter than did a full-length Cidea protein (Supplementary Fig. 6b). In contrast, the Cidea K23A K24A mutant had considerably lower binding affinMerge ity for the Xdh promoter (Supplementary Fig. 6d). Because C/EBPβ-mediated activation of downstream target genes involves dissociation of the HDAC1-containing tranMerge scription corepressor complex from the promoter28,29, we examined the association of HDAC1 with the Xdh promoter in the presence and absence of Cidea. The association of HDAC1 with the Xdh promoter was approximately threefold higher in Cidea-KD cells (Fig. 6c, right), whereas the overexpression of Cidea decreased the association of HDAC1 with the Xdh promoter (Fig. 6c, left). The association of p300, a transcriptional coactivator30, with the promoter was not significantly affected by the expression level of Cidea (Fig. 6c and Supplementary Fig. 6e). Furthermore, the overexpression of Cidea resulted in markedly more acetylation of histone 3 at Lys9 (H3K9ac), a marker of active chromatin31, at the Xdh promoter (Fig. 6d and Supplementary Fig. 6f). These data indicate that by interacting with C/EBPβ, Cidea strengthens the association of C/EBPβ with the Xdh promoter and increases histone acetylation. Cidea also dissociates HDAC1 from the promoter. We next evaluated the expression of several other genes that are known downstream targets of C/EBPβ and found that Socs3, Socs1, Tgfbr and Tgfb1 (genes in the inflammation pathway)32–36; and prolactin receptor (Prlr)37,38 and Id2 (refs. 39,40); and Igf1 (refs. 41,42) (genes relating to MFG production) were all expressed at lower levels in Cidea−/− mammary glands (Fig. 6e). However, expression


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of Csn (casein) was similar between wild-type and Cidea−/− mammary glands (Fig. 6e). Consistent with this, the overexpression of Cidea in mammary epithelial cells increased the expression of Id2, Igf1, Prlr, Socs3 and Socs1 but not Csn (Supplementary Fig. 7a,b). ChIP analysis revealed that Cidea was associated with the promoters of Id2 and Igf1 but not with that of Csn (Fig. 6f). In addition, Cidea promoted the association of C/EBPβ with, and the dissociation of HDAC1 from, the promoters of Id2 and Igf1 but not that of Csn (Fig. 6g and Supplementary Fig. 7c–e). Thus, Cidea seems to induce the expression of a specific subset of C/EBPβ target genes. Because Fsp27 localizes to the nucleus and interacts with C/EBPβ, we assessed the expression of a similar subset of C/EBPβ target genes in the white adipose tissue (WAT) of wild-type and Fsp27-deficient mice and found that the expression levels of Socs3, Socs1, Tgfb1, Tgfbr, Id2 and Xdh were markedly reduced (Supplementary Fig. 8a). In addition, knocking down Fsp27 in 3T3-L1 adipocytes also decreased expression of these genes (Supplementary Fig. 8b). These data suggest that Fsp27 may act as a coactivator of C/EBPβ in the WAT to control the expression of a subset of C/EBPβ downstream target genes. Notably, the expression levels of Socs3, Socs1, Tgfb1, Tgfbr, Id2 and Xdh were similar in the BAT of wild type and Cidea−/− mice (Supplementary Fig. 8c). In contrast to a previous report14, we have detected moderate levels of Fsp27 proteins in BAT using antibody to


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Figure 6 Cidea binds to promoters of a subset of C/EBPβ target genes and enhances the DNA binding affinity of C/EBPβ. (a) ChIP analysis of the Xdh promoter, using antibody to HA in HC11 cells infected with adenoviruses expressing HA-tagged Cidea (Ad-Cidea), a C/EBPβknockdown sequence, and controls described in Figure 2i,j. Experiments were repeated three times. (b,c) ChIP analysis of the Xdh promoter with antibodies to C/EBPβ, HDAC and p300. (d) ChIP analysis of the Xdh promoter with antibodies to H3K9ac and H3 and determination of the H3K9ac/H3 ratio after transfection of Ad-GFP or Ad-Cidea. (e) Expression of other C/EBPβ target genes in the mammary gland from wild-type and Cidea−/− mice. (f) ChIP analysis of Cidea binding to the promoters of Id2, Igf1 and Csn. (g) ChIP analysis of Cidea’s effects on the association of C/EBPβ and HDAC1 with promoters of Id2, Igf1 and Csn. (h) Schematic illustration of the role of Cidea in regulating milk lipid secretion. Error bars show means ± s.e.m. *P < 0.05, **P < 0.01; NS, no statistical significance.

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residues 1–90 of Fsp27 (ref. 15). Thus, similar levels of gene expression between wild-type and Cidea−/− BAT are probably attributable to the compensating effect of Fsp27 (Supplementary Fig. 8c). DISCUSSION In this study, we observed that Cidea is expressed at very high levels in the mammary glands of pregnant and lactating mice. Cidea deficiency results in markedly reduced milk lipid secretion and premature pup death. The lipid secretion defect is due to Cidea’s role in inducing XOR expression because Cidea-deficient mammary glands and Cidea-KD MECs have less XOR expression and lipid secretion, whereas the overexpression of Cidea markedly increases the expression of endogenous XOR, activates the promoter driving this expression and increases lipid secretion in vivo. In addition, reintroduction of XOR into Cidea-KD MECs restores lipid secretion. C/EBPβ is required for Cidea-induced XOR expression and lipid secretion, mediating this via its interaction with Cidea. A specific and direct interaction between Cidea and C/EBPβ was revealed by their in vivo reciprocal coimmunoprecipitation in lactating mammary cells, in cells coexpressing Cidea and C/EBPβ, and in a GST pull-down assay. In addition, a deletion analysis identified a specific region of Cidea (amino acid residues 118–163) that mediates its interaction with C/EBPβ. Moreover, Cidea lacking this region is defective at inducing XOR expression and lipid secretion.

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Articles To our surprise, biochemical fractionation, immunofluorescence staining, ChIP and deletion and mutation analyses revealed that Cidea protein localizes to the nucleus in lactating mammary glands, BAT and cultured MECs. We observed that two basic residues in the N-terminal region of Cidea (Lys23 and Lys24) and the middle region of Cidea (residues 118–163, responsible for its interaction with C/EBPβ) are both required for strong nuclear association of Cidea and full activation of XOR expression. The N-terminal lysine residues may be responsible for the shuttling of Cidea from the cytosol to the nucleus, whereas its interaction with C/EBPβ may further enhance its nuclear retention. Mechanistically, we demonstrated that Cidea associates with the promoter regions of Xdh (encoding XOR) and several other C/EBPβ downstream target genes in a C/EBPβ-dependent manner. Moreover, Cidea increases histone acetylation and the DNA binding affinity of C/EBPβ by forming a complex on the Xdh promoter, from which it promotes the release of HDAC1. Notably, Cidea regulates the expression of only a subset of C/EBPβ downstream target genes in the lipid secretion, MFG production and inflammatory pathways. The functional specificity of Cidea-regulated gene expression is probably due to the recruitment of Cidea to specific promoters. This recruitment depends not only on the presence of C/EBPβ but also on the temporal dynamics of transcription factors and their coregulators for each target gene, and it is therefore promoter context dependent, as in the case of p53 (refs. 43,44) and NF-κB (ref. 45). We consider it plausible that the differentially regulated target genes of C/EBPβ contain intrinsically different combinations of regulatory cis-acting elements that dictate distinct loading patterns with the transcription factors and cofactors available in given tissues or cell types. The interaction between C/EBPβ and Cidea, the nuclear localization of Cidea, and its association with and activation of a subset of C/EBPβ target genes indicate that Cidea serves as a transcriptional coactivator with C/EBPβ. Fsp27, another CIDE-family protein, may also act as a coactivator of C/EBPβ, given that it is localized to the nucleus, interacts with C/EBPβ and regulates similar subsets of C/EBPβ downstream target genes in white adipocytes. Recently, Cidea has been shown to interact with LXR and regulate its activity46. It is therefore important to note that Cidea may act as a coregulator with many other unidentified transcription factors in lipid metabolism. Cidea and Fsp27 are known to be crucial in promoting formation of large lipid droplets in brown and white adipocytes15,20. We have shown here that Cidea-deficient MECs accumulate smaller lipid droplets, whereas Cidea overexpression results in the formation of large lipid droplets. Therefore, Cidea seems to have dual roles in mammary epithelial cells: inducing lipid secretion and promoting large lipid droplet formation. Evidence suggests that these two pathways are controlled by independent mechanisms. First, the lipid droplet localization domain of Cidea is required for its ability to promote formation of large lipid droplets, but it is dispensable for Cidea-induced XOR expression and lipid secretion. Second, introduction of XOR into Cidea-KD cells restores lipid secretion without affecting the size of their lipid droplets. The markedly reduced milk lipid levels in Cideadeficient female mice are probably a combined effect of smaller lipid droplets and reduced MFG secretion. Overall, we have defined a previously unknown regulatory pathway controlled by Cidea that relates to milk lipid secretion and offspring survival (Fig. 6h). When induced in pregnant and lactating mammary glands, Cidea is transported into the nucleus, where it interacts with C/EBPβ. This interaction enhances the association of C/EBPβ with the promoters of a subset of downstream target genes, leading to the dissociation of HDAC1 from these promoters. This ultimately results in higher expression of target genes (including Xdh), which is vital to proper milk

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lipid secretion for nursing newborn pups5,41. Therefore, in addition to its role in controlling lipid droplet size, Cidea may act as an important transcriptional coactivator with C/EBPβ to control the expression of a subset of its downstream targets. Thus, Cidea may represent an important candidate gene for screening for lactation insufficiency, which occurs in approximately 5% of all lactating human females47. Methods Methods and any associated references are available in the online version of the paper at http://www.nature.com/naturemedicine/. Note: Supplementary information is available on the Nature Medicine website. Acknowledgments We thank L. Yu, L. Huang, Y. Hong and Y. Zhou for helpful discussions, and B. Groner (Georg-Speyer-Haus Institute for Biomedical Research) for providing HC11 cells. This work was supported by grants from the National Basic Research Program (2007CB914404, 2011CB910800) from the Ministry of Science and Technology of China and the National Natural Science Foundation of China (30925017 and 31030038 to P.L., 90913024 to Y.S.), and from the National University of Singapore and the Singapore National Research Foundation under CRP award no. 2007-04 to M.R.W. AUTHOR CONTRIBUTIONS W.W. devised the hypothesis, designed and performed the experiments, analyzed the data and wrote the first draft of manuscript. N.L., S.Z., J.Z., H.Q. and Y.C. performed the experiments. G.S., M.R.W. performed lipidomics analysis. J.Y. performed EM analysis. Y.X. and Y.S. helped with experimental design and data analysis. P. L. is responsible for the formulation of the hypothesis, experimental design, data coordination, analysis and interpretation. P.L. is also responsible for the writing, revision and finalization of the manuscript as well as for the decision to submit the manuscript for publication. All authors read and approved the final manuscript. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Published online at http://www.nature.com/naturemedicine/. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html.

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14. Nishino, N. et al. FSP27 contributes to efficient energy storage in murine white adipocytes by promoting the formation of unilocular lipid droplets. J. Clin. Invest. 118, 2808–2821 (2008). 15. Toh, S.Y. et al. Up-regulation of mitochondrial activity and acquirement of brown adipose tissue-like property in the white adipose tissue of fsp27 deficient mice. PLoS ONE 3, e2890 (2008). 16. Ye, J. et al. Cideb, an ER- and lipid droplet-associated protein, mediates VLDL lipidation and maturation by interacting with apolipoprotein B. Cell Metab. 9, 177–190 (2009). 17. Li, J.Z. et al. Cideb regulates diet-induced obesity, liver steatosis, and insulin sensitivity by controlling lipogenesis and fatty acid oxidation. Diabetes 56, 2523–2532 (2007). 18. Pettersson, A.T. et al. Characterization of the human CIDEA promoter in fat cells. Int. J. Obes. (Lond.) 32, 1380–1387 (2008). 19. Puri, V. et al. Cidea is associated with lipid droplets and insulin sensitivity in humans. Proc. Natl. Acad. Sci. USA 105, 7833–7838 (2008). 20. Zhou, Z. et al. Cidea-deficient mice have lean phenotype and are resistant to obesity. Nat. Genet. 35, 49–56 (2003). 21. Dahlman, I. et al. The CIDEA gene V115F polymorphism is associated with obesity in Swedish subjects. Diabetes 54, 3032–3034 (2005). 22. Neville, M.C. & Picciano, M.F. Regulation of milk lipid secretion and composition. Annu. Rev. Nutr. 17, 159–183 (1997). 23. Shekar, P.C. et al. κ-casein-deficient mice fail to lactate. Proc. Natl. Acad. Sci. USA 103, 8000–8005 (2006). 24. Jeong, J. et al. The PRY/SPRY/B30.2 domain of butyrophilin 1A1 (BTN1A1) binds to xanthine oxidoreductase: implications for the function of BTN1A1 in the mammary gland and other tissues. J. Biol. Chem. 284, 22444–22456 (2009). 25. McManaman, J.L., Palmer, C.A., Anderson, S., Schwertfeger, K. & Neville, M.C. Regulation of milk lipid formation and secretion in the mouse mammary gland. Adv. Exp. Med. Biol. 554, 263–279 (2004). 26. Kudo, N. et al. Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1. Exp. Cell Res. 242, 540–547 (1998). 27. Xu, L., Kang, Y., Col, S. & Massague, J. Smad2 nucleocytoplasmic shuttling by nucleoporins CAN/Nup214 and Nup153 feeds TGFβ signaling complexes in the cytoplasm and nucleus. Mol. Cell 10, 271–282 (2002). 28. Wiper-Bergeron, N., Wu, D., Pope, L., Schild-Poulter, C. & Hache, R.J. Stimulation of preadipocyte differentiation by steroid through targeting of an HDAC1 complex. EMBO J. 22, 2135–2145 (2003). 29. Paz-Priel, I., Houng, S., Dooher, J. & Friedman, A.D. C/EBPα and C/EBPα oncoproteins regulate nfkb1 and displace histone deacetylases from NF-κB p50 homodimers to induce NF-κB target genes. Blood 117, 4085–4094 (2011). 30. Lee, S., Miller, M., Shuman, J.D. & Johnson, P.F. CCAAT/Enhancer-binding protein β DNA binding is auto-inhibited by multiple elements that also mediate association with p300/CREB-binding protein (CBP). J. Biol. Chem. 285, 21399–21410 (2010).

31. Kouzarides, T. Chromatin modifications and their function. Cell 128, 693–705 (2007). 32. Borland, G., Bird, R.J., Palmer, T.M. & Yarwood, S.J. Activation of protein kinase Cα by EPAC1 is required for the ERK- and CCAAT/enhancer-binding protein β-dependent induction of the SOCS-3 gene by cyclic AMP in COS1 cells. J. Biol. Chem. 284, 17391–17403 (2009). 33. Cui, T.X. et al. C/EBPβ mediates growth hormone-regulated expression of multiple target genes. Mol. Endocrinol. 25, 681–693 (2011). 34. Sahay, B. et al. CD14 signaling restrains chronic inflammation through induction of p38-MAPK/SOCS-dependent tolerance. PLoS Pathog. 5, e1000687 (2009). 35. Yarwood, S.J., Borland, G., Sands, W.A. & Palmer, T.M. Identification of CCAAT/ enhancer-binding proteins as exchange protein activated by cAMP-activated transcription factors that mediate the induction of the SOCS-3 gene. J. Biol. Chem. 283, 6843–6853 (2008). 36. McCarthy, T.L., Pham, T.H., Knoll, B.I. & Centrella, M. Prostaglandin E2 increases transforming growth factor-β type III receptor expression through CCAAT enhancerbinding protein δ in osteoblasts. Mol. Endocrinol. 21, 2713–2724 (2007). 37. Dong, J., Tsai-Morris, C.H. & Dufau, M.L. A novel estradiol/estrogen receptor alphadependent transcriptional mechanism controls expression of the human prolactin receptor. J. Biol. Chem. 281, 18825–18836 (2006). 38. Goldhar, A.S., Duan, R., Ginsburg, E. & Vonderhaar, B.K. Progesterone induces expression of the prolactin receptor gene through cooperative action of Sp1 and C/EBP. Mol. Cell. Endocrinol. 335, 148–157 (2011). 39. Karaya, K. et al. Regulation of Id2 expression by CCAAT/enhancer binding protein β. Nucleic Acids Res. 33, 1924–1934 (2005). 40. Mori, S., Nishikawa, S.I. & Yokota, Y. Lactation defect in mice lacking the helixloop-helix inhibitor Id2. EMBO J. 19, 5772–5781 (2000). 41. Kleinberg, D.L., Feldman, M. & Ruan, W. IGF-I: an essential factor in terminal end bud formation and ductal morphogenesis. J. Mammary Gland Biol. Neoplasia 5, 7–17 (2000). 42. Wessells, J., Yakar, S. & Johnson, P.F. Critical prosurvival roles for C/EBPβ and insulin-like growth factor I in macrophage tumor cells. Mol. Cell. Biol. 24, 3238–3250 (2004). 43. Drost, J. et al. BRD7 is a candidate tumour suppressor gene required for p53 function. Nat. Cell Biol. 12, 380–389 (2010). 44. Gomes, N.P. & Espinosa, J.M. Gene-specific repression of the p53 target gene PUMA via intragenic CTCF-Cohesin binding. Genes Dev. 24, 1022–1034 (2010). 45. Amir-Zilberstein, L. et al. Differential regulation of NF-κB by elongation factors is determined by core promoter type. Mol. Cell. Biol. 27, 5246–5259 (2007). 46. Kulyté, A. et al. CIDEA interacts with liver X receptors in white fat cells. FEBS Lett. 585, 744–748 (2011). 47. Neifert, M.R. Prevention of breastfeeding tragedies. Pediatr. Clin. North Am. 48, 273–297 (2001).

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Milk collection and analysis. Milk was collected from lactating female mice as described10. Briefly, female mice were separated from their newborns at day 2 of lactation for 3 h and injected with 10 units of oxytocin (Sigma). At 10 min after the injection, milk was manually collected from the fourth pair of mammary glands for further analysis. Milk protein concentration was determined using a Bradford assay kit (Thermo Scientific). A total of 5 µg of total milk protein was separated by 15% SDS-PAGE and stained with Coomassie blue dye.

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Isolation of mammary epithelial cells and adipocytes. Methods for the isolation of mammary epithelial cells and white adipocytes were essentially as described10 with minor modifications. Briefly, mammary glands containing surrounding adipose tissue were minced and digested in a collagenase solution (1 mg ml−1 collagenase) containing 1 mg ml−1 fatty acid–free BSA in HBSS for 1 h at 37 °C. The tissue mixture was centrifuged at 100g for 5 min. The floating fraction was collected as the adipocyte fraction. The pellet containing epithelial cells was further incubated with collagenase solution for an additional 1 h. Epithelial cells were collected from the pellet after the reaction mixture was centrifuged at 233g for 4 min, washed twice and harvested for further use. Biochemical fractionation of subcellular organelles from mammary glands, differentiated 3T3-L1 adipocytes and 293T cells. The procedures for the isolation of subcellular organelles were essentially as described16 with minor modifications. Briefly, mammary glands from wild-type females on day 2 of lactation were minced and rapidly homogenized in an ice-cold lysis buffer (10 mM TrisHCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.25 M sucrose) with a tight-fitting dounce. The homogenates were laid on top of 3 ml of 2 M sucrose in Beckman polycarbonate centrifuge tubes and centrifuged in a precooled Beckman SW41 swinging bucket rotor in a Beckman XP-100 ultracentrifuge at 16,000g at 4 °C for 30 min. The resulting crude nuclear pellet was harvested and washed twice with a lysis buffer containing 1% vol/vol NP-40 to remove adherent membrane structures. The resulting pellet was saved as the nuclear fraction. Mitochondria, microsomes, cytosol and lipid droplets were isolated as described16. The procedure for preparing non-nuclear (cytosol plus crude membranes) and nuclear fractions from 293T cells was essentially as described48. Mouse handling, HC11 cell culture, plasmid construction and adenovirus preparation. Cidea- and Cidec (Fsp27)–deficient mice were generated and maintained as described20. The procedure for HC11 cell culture was essentially as described49. A full-length mouse cDNA encoding XOR was cloned into the vector pCMV5 with an N-terminal hemagglutinin (HA) tag. A full-length mouse cDNA encoding C/EBPβ was purchased from Addgene (plasmid no. 12557). Cidea and its various truncations were subcloned into pCMV6 (Origene) with a C-terminal Flag tag or pEGFP (Clontech) with a C-terminal GFP tag. Then, shRNAs against Cidea and C/EBPβ were subcloned into pSilencer 2.0-U6 (Ambion) and subsequently incorporated into an adenoviral vector. The fidelity of each construct was confirmed by sequencing analysis. Recombinant adenoviruses expressing mouse full-length Cidea, truncated Cidea, XOR, GFP, Cidea shRNA, C/EBPβ shRNA and scrambled shRNA were constructed using the AdEasy-1 system (Stratagene). The nucleotide sequences for the Cidea and C/EBPβ shRNAs are available in Supplementary Table 1.

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Luciferase reporter assay. Reporter assays were performed in undifferentiated HC11 cells as described12, and luciferase activity was measured using the dualluciferase reporter assay system (Promega). Chromatin immunoprecipitation. ChIP analysis was performed as described12. Antibodies against C/EBPβ (Santa Cruz), HDAC1 (Santa Cruz), p300 (Cell Signaling), HA (Santa Cruz), H3K9ac (Cell Signaling), histone H3 (Cell Signaling) and rabbit IgG (Santa Cruz) were used for the ChIP assays. We quantified promoter occupancy by measuring the percentage of input chromatin that immunoprecipitated by real-time PCR analysis. The nucleotide sequences of the primers used for ChIP analysis are available in Supplementary Table 2. Western blotting, coimmunoprecipitation and GST pull-down assay. Western blotting and coimmunoprecipitation were performed as described50. Primary antibodies against C/EBPβ, β-actin, XOR, casein and Lamin B were obtained from Santa Cruz. Antibodies against β-tubulin (Sigma), Plin2 (Fitzgerald), and Plin (Research & Diagnostic) were also used. Nuclear fractions isolated from HC11 cells or lactating mammary glands were used for in vivo coimmuno precipitation experiments as described50. GST pull-down assays were performed essentially as described51. Full-length C/EBPβ protein was synthesized in vitro by the cell-free expression of recombinant protein using the Escherichia coli lysate system (Qiagen). GST-Cidea33–110 and GST-Cidea110–163 were isolated from bacteria using a standard protocol51. Quantitative real-time PCR analyses. Gene expression was assessed using real-time PCR with SYBR-Green PCR master mix and the 7500 real-time PCR system (ABI). The list of primers for quantitative PCR is available in Supplementary Table 3. Immunofluorescence and fluorescence recovery after photobleaching. HC11 cells were transfected with GFP-tagged Cidea and its truncation plasmids for 24 h and incubated with a digitonin-containing buffer (10 mM PIPES pH 7.3, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA and 20 µg ml−1 digitonin) for 5 min and then fixed with 4% wt/vol paraformaldehyde. Fluorescence images were obtained with a Zeiss LSM7100Meta confocal microscope and pro cessed with Adobe Photoshop for presentation. The FRAP assay was performed essentially as described48. Statistical analyses. Data are presented as the mean ± s.e.m. Statistical significance was determined using two-tailed unpaired t-test with a significance level of 0.05. Additional methods. Detailed methodology is described in the Supplementary Methods. 48. Trotman, L.C. et al. Ubiquitination regulates PTEN nuclear import and tumor suppression. Cell 128, 141–156 (2007). 49. Ball, R.K., Friis, R.R., Schoenenberger, C.A., Doppler, W. & Groner, B. Prolactin regulation of beta-casein gene expression and of a cytosolic 120-kd protein in a cloned mouse mammary epithelial cell line. EMBO J. 7, 2089–2095 (1988). 50. Qi, J. et al. Downregulation of AMP-activated protein kinase by Cidea-mediated ubiquitination and degradation in brown adipose tissue. EMBO J. 27, 1537–1548 (2008). 51. Li, Q. et al. Axin determines cell fate by controlling the p53 activation threshold after DNA damage. Nat. Cell Biol. 11, 1128–1134 (2009).

doi:10.1038/nm.2614