Dr Spencer Lai (賴鶴明博士): 破解減肥密碼BAF60c基因

破解減肥密碼BAF60c基因

華裔正主導研究減肥靈藥將問世

綠色部分就是在肝臟細胞中的BAF60c基因。

王凱峰有信心，他的BAF60c基因技術在數年內就可以製成減肥靈藥。

斷絕BAF60c功能的老鼠，體重明顯下降。

科學期刊「分子細胞」（Molecular Cell）6日在網站上刊登柏克萊加大營養學和毒理學部門的研究報告，阻止醣類轉化成脂肪的基因研究已宣告完成。相關研究將進入製藥階段，如果成功，可為脂肪肝、糖尿病和肥胖症的患者帶來低價的特效藥。

從2007年起主導這項研究的華裔科學家王凱峰表示，新發現的BAF60c基因是細胞中把碳水化合物轉為脂肪的專職基因，以藥物阻擋它的作用將不會產生過多的後遺症。他預期最快在兩到三年內，以這種基因技術製成的減肥藥就可以產生。

美國權威科學期刊「細胞」（Cell）在2009年9月就曾刊登王凱峰團隊的研究。當時找出的DNA-PK基因是科學界首次找出脂肪和碳水化合物的關聯。王凱峰解釋，人體進食碳水化合物後血糖會提高，引發胰島素分泌。科學界很早就知道胰島素和轉變過程有關，但之前的知識不足以製藥。

新發現的BAF60c基因位於DNA-PK同一圖譜的更深層，因此阻擋BAF60c的功能，不會產生改變DNA-PK所帶來的後遺症。在BAF60c發現之前，所有實驗都只能在老鼠身上進行。現在終於可以開始在靈長類動物身上實驗。

這項長達五年的研究共耗資50萬元，由國家保健學會（National Institute of Health）出資。王凱峰表示，因為研究階段都是由政府出錢，所以未來製成藥品的價格不會很高。他希望製成一天1到2元，不需處方在藥房就可以買到的成藥。

王凱峰稱，BAF60c發現之後該計畫的科學研究階段已告完成，他肯定未來的動物實驗將不會發現導致重新開始科研的問題。另外，由於BAF60c位在人類基因圖譜的深層，減肥藥的效果不會因為每個人不同的基因而有異，適合所有人使用。

他說：「這可以稱為減肥的靈藥（Magic Pill）。因為正常的人體只會將攝取過多的醣類轉換為脂肪儲存，所以如果每天攝取的碳水化合物和身體消耗的能量達成平衡，就算用這種藥也不會無止盡地減重。」

王凱峰今年29歲，高中之後從香港到美國留學，沙加緬度州大生物化學畢業後，在柏大參與相關研究。也因為DNA-PK的發現在2010年順利取得博士學位。他在BAF60c研究完成之後已經轉職到灣區一藥廠，繼續從事與新陳代謝有關的研究。

Available online 6 December 2012

In Press, Corrected Proof — Note to users

Article

Phosphorylation and Recruitment of BAF60c in Chromatin Remodeling for Lipogenesis in Response to Insulin

Yuhui Wang ¹^,⁵,

Roger H.F. Wong ²^,⁵,

Tianyi Tang ³,

Carolyn S. Hudak ¹,

Di Yang ¹,

Robin E. Duncan ¹^,⁴,

Hei Sook Sul ¹^,²^,³^,^,

¹ Department of Nutritional Science and Toxicology, University of California, Berkeley, Berkeley, CA 94720, USA

² Comparative Biochemistry Program, University of California, Berkeley, Berkeley, CA 94720, USA

³ Endocrinology Program, University of California, Berkeley, Berkeley, CA 94720, USA

Corresponding author

Received 21 May 2012

Revised 19 August 2012

Accepted 25 October 2012

Available online 6 December 2012

Published online: December 6, 2012

http://dx.doi.org/10.1016/j.molcel.2012.10.028, How to Cite or Link Using DOI

Summary

Fatty acid and triglyceride synthesis is induced in response to feeding and insulin. This lipogenic induction involves coordinate transcriptional activation of lipogenic enzymes, including fatty acid synthase and glycerol-3-phosphate acyltransferase. We recently reported the importance of USF-1 phosphorylation and subsequent acetylation in insulin-induced lipogenic gene activation. Here, we show that Brg1/Brm-associated factor (BAF) 60c is a specific chromatin remodeling component for lipogenic gene transcription in liver. In response to insulin, BAF60c is phosphorylated at S247 by atypical PKCζ/λ, which causes translocation of BAF60c to the nucleus and allows a direct interaction of BAF60c with USF-1 that is phosphorylated by DNA-PK and acetylated by P/CAF. Thus, BAF60c is recruited to form the lipoBAF complex to remodel chromatin structure and to activate lipogenic genes. Consequently, BAF60c promotes lipogenesis in vivo and increases triglyceride levels, demonstrating its role in metabolic adaption to activate the lipogenic program in response to feeding and insulin.

Highlights

► BAF60c is required for feeding/insulin-mediated lipogenic gene activation ► BAF60c interacts with USF-1 to be recruited to lipogenic genes, such as FAS ► BAF60c is phosphorylated at S247 by aPKC to interact with P-S262, Act-K237 USF-1 ► BAF60c forms lipoBAF complex on lipogenic promoters to remodel chromatin structure

Figure 1. Identification of BAF Subunits as USF-1 Interacting Proteins(A) Immunoblotting of TAP-purified USF-1-associated proteins with indicated antibodies (left). RT-PCR for BAF subunits (right).(B) Immunoblotting of TAP eluates from 293F cells overexpressing BAF60c-HA and USF-1-Flag-TAP (a). CoIP of cells overexpressing BAF60c-HA and USF-1-Flag (b). Immunoblotting of liver lysates from fasted and fed mice after IP with USF-1 antibody (c).(C) GST-USF-1 protein was incubated with in vitro translated 35S-labeled BAF60c before GST pull-down.(D) RT-qPCR. Means ± SEM are shown. ∗∗p < 0.01 (left). Immunoblotting of liver lysates from fasted and fed mice (right).(E) ChIP assay of livers of −444 FAS-CAT transgenic mice.(F) ChIP assay (a, left) and quantification for enrichment of BAF60c by qPCR (a, right). Re-ChIP with USF-1 antibody followed by BAF60c antibody (b).(G) ChIP on FAS in HepG2 cells (left) and re-ChIP (right).(H) FAS promoter activity in 293 cells upon BAF60c overexpression.(I) FAS promoter activity in BAF60c knockdown cells treated with insulin. Means ± SEM are shown. ∗∗p < 0.01.(J) Immunoblotting of lysates from HepG2 cells infected with shUSF-1 lentivirus (left). ChIP assay (middle). Expression of lipogenic genes (right).See also Figures S1 and S2.

Figure 2. Insulin/Feeding Induces S247 Phosphorylation and Nuclear Localization of BAF60c(A) Immunoblotting of cell lysates from cells transfected with wild-type BAF60c (WT) or S247A BAF60c mutant (S247A) with S247 phosphorylation-specific BAF60c antibody.(B) Lysates of livers and HepG2 cells.(C) Immunoblotting for nuclear and cytosolic fractions of livers.(D) Immunoblotting for total cell lysates and nuclear and cytosolic fractions of HepG2 cells.(E) Confocal fluorescence microscopy for BAF60c localization in HepG2 cells (left) and BAF60c-GFP transfected 293 cells (right).(F) Immunofluorescence of endogenous BAF60a, BAF60b, and BAF60c in HepG2 cells (top) and endogenous BAF60c and phosphorylated BAF60c (P247S) in livers (bottom).(G) Immunoblotting for phosphorylation at S247 in nuclear and cytosolic fractions of HepG2 cells upon insulin treatment (left) and BAF60c in total lysates and cytosolic and nuclear fractions of 293 cells transfected with wild-type BAF60c or BAF60c S247A mutant after insulin treatment (right).See also Figure S3.

Figure 3. S247 Phosphorylation of BAF60c Enhances the Interaction with USF-1(A) Immunoblotting of lysates from 293 cells transfected with Flag-tagged USF-1 and HA-tagged BAF60c wild-type (WT), S247A, and S247D mutant BAF60c (left) and after IP with USF-1 antibody (right).(B) Lysates of cells transfected with Flag-tagged USF-1 and BAF60c were incubated with either wild-type 247 peptide (peptide S247) or peptide containing S247A mutation (peptide S247A) before IP and immunoblotting (left). Lysates from cells transfected with only USF-1 were used (right).(C) GST-USF-1 fusion protein was used for pull-down of BAF60c with glutathione beads (left). GST-USF-1 was incubated with in vitro translated BAF60c in the presence of S247D or S247A peptides.(D) Luciferase activity. Means ± SEM are shown. ∗∗p < 0.01.(E) ChIP for BAF60c binding by qPCR. Means ± SEM are shown.

Figure 4. BAF60c Is Phosphorylated at S247 by aPKC(A) In vitro-translated BAF60c-HA was in vitro phosphorylated with aPKCλ (left) and ζ (right) in the presence or absence of inhibitory peptide (100 mM) before immunoblotting.(B) In vitro translated wild-type or S247A mutant BAF60c were in vitro phosphorylated before immunoblotting.(C) BAF60c-HA-transfected cells were incubated with or without aPKC inhibitory peptide.(D) Lysates from 293 cells transfected with BAF60c, with or without PKCζ (left), and PKCζ-transfected cells treated with GF10920 at 5 mM or control (DMSO) for 30 min (right).(E) Immunoblotting of 293 cells cotransfected with BAF60c along with wild-type or dominant negative PKCζ.(F) Lysates from HepG2 cells infected with adenovirus for wild-type PKCζ or dominant negative PKCζ (PKCζ-DN) overexpression (left) and after IP with USF-1 antibody (right) before immunoblotting.(G) aPKC protein levels in cells transfected with aPKCζ and aPKCλ siRNA (aPKC siRNA) (top). Immunoblotting of lysates from 293 cells cotransfected with control or aPKC siRNA along with BAF60c or S247A mutant (bottom).(H) FAS promoter activity. Means ± SEM are shown. ∗∗p < 0.01.(I) Immunoblotting of liver lysates from mice after administration of PKCζ-DN (left). Expression of lipogenic genes (right). Means ± SEM are shown. ∗p < 0.05, ∗∗p < 0.01, n = 3–4.

Figure 5. Posttranslational Modification of Both BAF60c and USF-1 Mediates Their Interaction(A) IP of USF-1 with Flag-antibody of 293 cells cotransfected with Flag-tagged USF-1 (WT) or its acetylation mutants (K237R and K237Q) with HA-tagged BAF60 or its phosphorylation mutants (S247A and S247D) and immunoblotting with anti-HA antibody for BAF60c.(B) Input of in vitro translated K237A and K237R mutant USF-1 and wild-type and S247A mutant BAF60c. USF-1 and BAF60c were used for coIP followed by immunoblotting.(C) ChIP analysis for BAF60c bound to the FAS-Luc promoter in cells transfected with USF-1 or its acetylation mutants along with BAF60c or its mutant by PCR (left) or qPCR (right).(D) Luciferase activity in cells transfected with −444 FAS-Luc, USF-1, and BAF60c or its mutants. Means ± SEM are shown. ∗∗p < 0.01.(E) Immunoblotting of lysates from insulin-treated HepG2 cells after IP with anti-USF-1 antibody.(F) Immunoblotting of total liver lysates from 6-week-old SCID mice.(G) Immunoblotting of liver nuclear extracts after IP with USF-1 antibody.(H) ChIP for FAS and mGPAT promoters after IP with anti-BAF60c and anti-USF antibodies in livers from SCID mice (left) and quantification by qPCR (right). Means ± SEM are shown.

Figure 6. BAF60c Enhances Chromatin Modification of Lipogenic Genes in Response to Insulin(A) Nuclear extracts from HepG2 cells treated with insulin for 10 min were subjected to MNase assay with 80 U/ml. qPCR with primers spanning 500 bp of the FAS promoter region.(B) MNase assay in HepG2 cells treated with insulin. Fold change over non-MNase treated cells. Means ± SEM are shown. ∗p < 0.05.(C) ChIP assay in HepG2 cells (left) and in mouse livers (right).(D) BAF60c protein levels in HepG2 cells infected with control or BAF60c adenovirus (a, top). Infected cells treated with insulin for 10 min for MNase assay (a, bottom). ChIP for the FAS promoter region in HepG2 cells after 10 min of insulin treatment using anti-H1, anti-p-H3-S10, anti-Ac-H3-K14, and anti-Pol II antibodies and qPCR for the FAS promoter (b). Nuclear run-on assay for FAS transcription in cells infected with BAF60c after insulin treatment and RT-qPCR for FAS run-on assay (c). RT-qPCR for lipogenic gene expression in cells overexpressing BAF60c (d). Means ± SEM are shown. ∗p < 0.05, ∗∗p < 0.01.(E) BAF60c protein levels in shBAF60c adenovirus infected cells (a, top). MNase assays by qPCR (a, bottom). ChIP for FAS promoter in HepG2 cells after BAF60c knockdown (b). Run-on assay using RT-qPCR for FAS transcription (c). Lipogenic gene expression by RT-qPCR in BAF60c knockdown cells (d). Means ± SEM are shown. ∗p < 0.05, ∗∗p < 0.01.

Figure 7. BAF60c Promotes Lipogenesis In Vivo(A) BAF60c protein levels in livers of mice 14 days after tail vein administration of BAF60c adenovirus (top left). mRNA levels (top middle) and nascent RNA levels (top right). Percent of newly synthesized fatty acids in liver (bottom left) and hepatic triglyceride levels (bottom right). Means ± SEM are shown. ∗p < 0.05, ∗∗p < 0.01, n = 3–5.(B) BAF60c protein levels in livers from mice 14 days after tail vein administration of shBAF60c adenovirus (top left). Expression of lipogenic genes (top right), de novo lipogenesis (bottom left) and triglyceride levels (bottom right) in the liver. Means ± SEM are shown. ∗p < 0.05, ∗∗p < 0.01, n = 3–5.(C) Expression levels of BAF60c and Akt 10 days after tail vein injection of adenoviral shBAF60c and adenoviral Akt in livers of mice (a, left). Expression levels of lipogenic genes in livers (b, right), n = 4. Expression levels of BAF60c and Akt in HepG2 cells infected with adenoviral shBAF60c and adenoviral Akt (b, left). Expression levels of lipogenic genes 72 hr after infection (a, right). Means ± SEM are shown.(D) Insulin signaling pathway for posttranslational modifications of BAF60c and USF-1 in chromatin remodeling and activation of lipogenic genes.See also Table S1.

Corresponding author

4Present address: Department of Kinesiology, University of Waterloo, Waterloo, ON N2L 3G1, Canada

5These authors contributed equally to this work

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