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Genetic Incorporation of Ne-Formyllysine, a New Histone

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Genetic Incorporation of Ne-Formyllysine, a New Histone DOI:10.1002/cbic.201500170 Communications Genetic Incorporation of Ne-Formyllysine, aNew Histone Post-translationalModification Tianyuan Wang,[a] Qing Zhou,*[b] FahuiLi,[b] Yang Yu,[c] Xuebin Yin,*[a, d] and JiangyunWang*[b] Lysine formylation is anewly discoveredpost-translational lysine,and Ne-methyllysine) have been successfully genetically modification (PTM) in histones and other nuclear proteins;it encoded.[5] Lysine formylation is anewly discovered PTM in his- has awell-recognized but poorlydefined role in chromatin tones and other nuclear proteins,and it is believed to be asso- conformation modulation and gene expression. To date, there ciated with oxidative stress under pathological conditions.[6] is no general method to site-specifically incorporate Ne-formyl- The formyl moiety can come from 3’-formylphosphate residues lysine at adefined site of aprotein. Here we report the highly arising from 5’-oxidation of deoxyribose in DNA, caused by the efficient genetic incorporation of the unnatural amino acid Ne- enediyne neocarzinostatin. Onlyone methyl group shorter formyllysine into proteins produced in Escherichiacoli and than AcK,[7] Ne-formyllysine (ForK) is not only structurally simi- mammalian cells, by using an orthogonal Ne-formyllysinetRNA- lar,but also appears at the same sites of histones. This raises synthetase/tRNACUA pair. This techniquecan be appliedto the questionastowhetherForK and AcK have similarroles in study the role of lysine formylation in epigenetic regulation. chromatin structure modulation and gene expression. Howev- er,the lack of methods to site-specifically incorporateForK into defined sites of proteinslimits our ability to probe the function Post-translationalmodification (PTM) of histones is vital for the regulation of diversebiological process- es, including DNA replication, DNA repair,and main- tenanceofgenomic stability.[1] Aberrant histone modification leads to many diseases in human.[2] To reveal the role of aspecific PTM in histones, it is es- sentialtoproduce homogeneous recombinantpro- tein that contains the site-specific PTM. Semisynthet- ic and enzyme-catalyzed methods have been devel- oped to realize site-specific histone PTM.[3] Com- pared to these methods, the genetic code expansion technique has the uniqueadvantage that unnatural amino acids (UAAs) can be directly incorporated at Figure 1. Site-specific ForK incorporation. A) Chemicalstructure of ForK.B)SDS-PAGE analysis of myoglobin-TAG4-ForK expressioninthe presence (+UAA) or absence ( UAA) [4] À specific sites of aprotein of interest. For example, of 1mm ForK. C) SDS-PAGE analysis of the expressionofhistone H3-TAG23 ForK in the three lysine PTMs (Ne-acetyllysine (AcK), Ne-crotonyl- presence(+UAA) or absence ( UAA) of 1mm ForK. À [a] T. Wang, Prof. X. Yin SchoolofEarth and Space Science of ForK in vitro and in vivo. University of Science and Technology of China (USTC) Here we report the highly efficient geneticincorporation of 96 Jinzhai Road, Hefei, Anhui230026 (China) the UAA ForK (Figure 1A)into Escherichia coli and mammalian E-mail:[email protected] cells by using an orthogonal Ne-formyllysine tRNA synthetase/ [b] Dr.Q.Zhou, Dr.F.Li, Prof. J. Wang Pyl LaboratoryofRNA Biology tRNACUA pair.Wefound that ForK-bearing proteins are not rec- Institute of Biophysics, Chinese Academy of Sciences ognized by anti-AcK antibody,thus indicating that the in vivo 15 Datun Road,ChaoyangDistrict,Beijing 100101 (China) proteins bearing ForK might interactwith other proteins differ- E-mail:[email protected] ently from those bearingAcK. [email protected] To selectively incorporate ForK at defined sites in proteins, [c] Dr.Y.Yu Institute of Industrial Biotechnology, Chinese Academy of Sciences we performedthree rounds of positive and two rounds of neg- 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin 300308 (China) ative selection with a Methanosarcina barkeri pyrrolysyl-tRNA- [d] Prof. X. Yin synthetase (MbPylRS)library,inorder to evolve an orthogonal Suzhou Key Lab for Selenium and Human Health tRNA/aminoacyl-tRNAsynthetase pair that selectivelycharges Suzhou Institute for Advanced Study,USTC ForK in response to the amber (TAG) codon, as previously de- 166 Ren’ai Road, DushuLake Higher Education Area, Industrial Park [4i, 8] Suzhou, Jiangsu 215123 (China) scribed. The selected ForK-specific PylRS (“ForKRS”) had five Supportinginformation for this article is available on the WWW under mutations: L266M, L270I, Y271F,L274A, and C313F.ForKRS was http://dx.doi.org/10.1002/cbic.2.201500170. digestedand two copiesofForKRS gene were ligated sequen- ChemBioChem 2015, 16,1440 –1442 1440 2015 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Communications tially into SalI/BglII and Pst1/Nde1 restriction sites of the pEVOLvector[9] to generate pEVOL1.Then, Methanosarcina bar- keri pyrrolysyl-tRNACUA(pylT) was ligated into the ApaL1/Xho1 sites of pEVOL1 to generate pEVOL-ForKRS, whichwas used to test the efficiency and selectivity of ForKRS, as previously re- ported.[4c] To determine whetherForK can be incorporated into apro- tein with high efficiencyand fidelity,anamber stopcodon was substituted for Ser4 in sperm whale myoglobin to construct the myoglobin-TAG4 expression vector.Protein expression was carriedout in E. coli in the presence of the selected synthetase Pyl (ForKRS), MbtRNACUA(pylT) and1mm ForK (or in the absence of ForK as anegative control). Analysisofthe purifiedprotein by SDS-PAGE showedthat full-length myoglobin was expressed only in the presence of ForK (Figure 1B), thus indicating that ForKRS recognizesForK specifically.ESI-MS analysis of myoglo- bin-TAG4-ForK gave an observed averagemass of 18422.0 Da (Figure 2), which was in agreement with the calculated mass (18423 Da). This indicates that ForK had been incorporated at the defined site of myoglobin. ForK was also incorporated into the position 23 of histone H3 (Figure 1C); this has been identi- fied as alysine formylation and acetylation site.[6a] The yield of 1 “H3-TAG23-ForK” was2mg LÀ ,which is comparable to yields reported forthe incorporation of other UAAs with the pylRS/ Pyl [5] MbtRNACUA pair. To examine whether ForK is recognized by an anti-AcKanti- body,anamber stop codon was substituted for Lys3 and Tyr151 in superfolder green fluorescent protein (sfGFP) to con- struct the sfGFP-TAG3 and sfGFP-TAG151 expression vectors, respectively.AcK or ForK was incorporated into position151 of sfGFP,and analyzed by western blotting. sfGFP-TAG3-AcK was detectedefficiently by an anti-AcKantibody;sfGFP-TAG3-ForK was not (Figure 3). This implies that lysine formylation PTM is Figure 3. Western blot analysis of wild-type and mutant sfGFP: lane 1) wild- type sfGFP; lane2)sfGFP-TAG3-AcK;lane 3) sfGFP-TAG3-ForK. The samples were probed by using anti-his tag and anti-Ne-acetyllysine antibodies. asignificantly different to lysine acetylation. As the pyrrollysine tRNA synthetase/tRNA pair is orthogonal in both bacterial and mammalian cells,[10] we tested whether Pyl the ForKRS and MbtRNACUA pair could perform site-specific incorporation of ForK in mammalian cells. We cloned ForKRS into the pCMV-NBK-1 vector[10] to generatepCMV-ForKRS, in Pyl which transcription of MbtRNACUA is under the controlofthe Figure 2. ESI-MS analysis of myoglobin.A)Wild-type (calcd:18354 Da; humanU6promoter and the expression of ForKRS is driven by found:18355.0 Da). B) Myoglobin-TAG4-ForK(calcd:18423.0 Da;found: the CMV promoter.Plasmids pSwan-EGFP37TAG[4i] and pCMV- 18422.0 Da). ForKRS wereco-transfected into 293T human embryonic kidneycells. The cellswere grown in the absence or presence of 1mm ForK for 36 h. EGFP fluorescencewas observed only ChemBioChem 2015, 16,1440 –1442 www.chembiochem.org 1441 2015 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Communications tain any studies with human or animal subjects performed by any of the authors. Keywords: amino acids · formyllysine · gene technology · histones · post-translationalmodifications [1] a) B. D. Strahl, C. D. Allis, Nature 2000, 403,41–45;b)X.-J. Yang, E. Seto, Mol. Cell 2008, 31,449–461;c)A.L.Burlingame, X. Zhang,R.J.Chalkley, Methods 2005, 36,383–394;d)F.Chu, D. A. Nusinow, R. J. Chalkley,K. Plath, B. Panning, A. L. Burlingame, Mol. Cell.Proteomics 2006, 5,194 – 203. [2] a) G. Egger,G.Liang, A. Aparicio,P.A.Jones, Nature 2004, 429,457 – Figure 4. Genetic incorporation of ForK at position 37 of EGFP in 293T cells 463;b)A.Portela, M. Esteller, Nat. Biotechnol. 2010, 28,1057 –1068. Pyl [3] M. Shogren-Knaak, H. Ishii, J.-M. Sun,M.J.Pazin, J. R. Davie,C.L.Peter- by using the ForKRS/MbtRNACUA pair.The EGFPfluorescence is observed only in the presence of 1mm ForK. son, Science 2006, 311,844 –847. [4] a) J. W. Chin, Annu. Rev.Biochem. 2014, 83,379–408;b)X.Liu, J. Li, J. Dong, C. Hu, W. Gong, J. Wang, Angew.Chem. Int. Ed. 2012, 51,10261 – for cells grown in the presence of ForK (Figure 4), thus indicat- 10265; Angew.Chem. 2012, 124,10407–10411; c) F. Li, H. Zhang, Y. Sun, Pyl Y. Pan, J. Zhou,J.Wang, Angew.Chem. Int. Ed. 2013, 52,9700 –9704; ing that when using the ForKRS/MbtRNACUA pair,ForK was site- Angew.Chem. 2013, 125,9882–9886;d)C.Hu, S. I. Chan,E.B.Sawyer, Y. specifically introduced at position 37 of EGFP throughamber Yu,J.Wang, Chem.Soc. Rev. 2014, 43,6498–6510;e)M.J.Schmidt, D. codon suppression in mammalian cells. Summerer, Angew.Chem.Int. Ed. 2013, 52,4690–4693; Angew.Chem. 2013, 125,4788–4791;f)M.J.Schmidt,J.Borbas, M. Drescher,D.Sum- merer, J. Am. Chem.Soc. 2014, 136,1238 –1241;g)E.Arbely,J.Torres- Conclusion Kolbus, A. Deiters, J. W. Chin, J. Am. Chem. Soc. 2012, 134,11912 – 11915;h)D.P.Nguyen, M. Mahesh,S.J.Elsässer,S.M.Hancock, C. Utta- We have demonstratedthe highly efficient geneticincorpora- mapinant, J. W. Chin, J. Am. Chem. Soc. 2014, 136,2240 –2243;i)Z.Yu, tion of ForK into E. coli and human cells. The ForK-incorporated Y.
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