Structural and Biophysical Analysis of the DNA Binding Properties of Myelin Transcription Factor 1

Abstract

Zinc binding domains, or zinc fingers (ZnFs), form one of the most numerous and most diverse superclasses of protein structural motifs in eukaryotes. Although our understanding of the functions of several classes of these domains is relatively well developed, we know much less about the molecular mechanisms of action of many others. Myelin transcription factor 1 (MyT1) type ZnFs are found in organisms as diverse as nematodes and mammals and are found in a range of sequence contexts. MyT1, one of the early transcription factors expressed in the developing central nervous system, contains seven MyT1 ZnFs that are very highly conserved both within the protein and between species. We have used a range of biophysical techniques, including NMR spectroscopy and data-driven macromolecular docking, to investigate the structural basis for the interaction between MyT1 ZnFs and DNA. Our data indicate that MyT1 ZnFs recognize the major groove of DNA in a way that appears to differ from other known zinc binding domains. Zinc binding domains, or zinc fingers (ZnFs), form one of the most numerous and most diverse superclasses of protein structural motifs in eukaryotes. Although our understanding of the functions of several classes of these domains is relatively well developed, we know much less about the molecular mechanisms of action of many others. Myelin transcription factor 1 (MyT1) type ZnFs are found in organisms as diverse as nematodes and mammals and are found in a range of sequence contexts. MyT1, one of the early transcription factors expressed in the developing central nervous system, contains seven MyT1 ZnFs that are very highly conserved both within the protein and between species. We have used a range of biophysical techniques, including NMR spectroscopy and data-driven macromolecular docking, to investigate the structural basis for the interaction between MyT1 ZnFs and DNA. Our data indicate that MyT1 ZnFs recognize the major groove of DNA in a way that appears to differ from other known zinc binding domains. It is predicted that there are at least 15,000 zinc fingers (ZnFs) 6The abbreviations used are: ZnFzinc fingerMyT1myelin transcription factor 1PLPproteolipid proteinNZF1neuronal zinc finger 1β-RAREβ-retinoic acid receptor geneSPRsurface plasmon resonanceITCisothermal titration calorimetryF5Finger 5MES4-morpholineethanesulfonic acidNOEnuclear Overhauser effectNOESYnuclear Overhauser effect spectroscopyAIRambiguous interaction restraintr.m.s.d.root mean square deviationChIPchromatin immunoprecipitation. in over 1,000 different proteins in humans (1Rubin G.M. Yandell M.D. Wortman J.R. Gabor Miklos G.L. Nelson C.R. Hariharan I.K. Fortini M.E. Li P.W. Apweiler R. Fleischmann W. Cherry J.M. Henikoff S. Skupski M.P. Misra S. Ashburner M. Birney E. Boguski M.S. Brody T. Brokstein P. Celniker S.E. Chervitz S.A. Coates D. Cravchik A. Gabrielian A. Galle R.F. Gelbart W.M. George R.A. Goldstein L.S. Gong F. Guan P. Harris N.L. Hay B.A. Hoskins R.A. Li J. Li Z. Hynes R.O. Jones S.J. Kuehl P.M. Lemaitre B. Littleton J.T. Morrison D.K. Mungall C. O'Farrell P.H. Pickeral O.K. Shue C. Vosshall L.B. Zhang J. Zhao Q. Zheng X.H. Lewis S. Science. 2000; 287: 2204-2215Crossref PubMed Scopus (1373) Google Scholar). These domains fall into more than twenty subclasses, based on their fold and zinc-ligation topology, and different members can mediate interactions with DNA, RNA, proteins, and other molecules (2Gamsjaeger R. Liew C.K. Loughlin F.E. Crossley M. Mackay J.P. Trends Biochem. Sci. 2007; 32: 63-70Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). The structures and functions of several classes of ZnFs, including classical and GATA-type domains, have been analyzed in detail; in contrast, some classes of ZnFs are not well understood. zinc finger myelin transcription factor 1 proteolipid protein neuronal zinc finger 1 β-retinoic acid receptor gene surface plasmon resonance isothermal titration calorimetry Finger 5 4-morpholineethanesulfonic acid nuclear Overhauser effect nuclear Overhauser effect spectroscopy ambiguous interaction restraint root mean square deviation chromatin immunoprecipitation. Myelin transcription factor 1 (MyT1, or neural zinc finger 2 (NZF2)) is a transcription factor that contains seven copies of a ZnF with a C2HC consensus sequence. The seven ZnFs are arranged in a 1 + 2 + 4 topology (see Fig. 1), although a second isoform exists that lacks finger 1 (F1) (3Nielsen J.A. Berndt J.A. Hudson L.D. Armstrong R.C. Mol. Cell Neurosci. 2004; 25: 111-123Crossref PubMed Scopus (78) Google Scholar). MyT1 was first isolated on the basis of its interaction with DNA elements in the promoter of the proteolipid protein (PLP) gene (4Kim J.G. Hudson L.D. Mol. Cell. Biol. 1992; 12: 5632-5639Crossref PubMed Scopus (112) Google Scholar). MyT1 acts during central nervous system development to induce differentiation of early neuronal precursors and to commit glial cell precursors to the glial cell lineage (5Bellefroid E.J. Bourguignon C. Hollemann T. Ma Q. Anderson D.J. Kintner C. Pieler T. Cell. 1996; 87: 1191-1202Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). After birth, MyT1 activates the PLP gene in Schwann cells, leading to the production of the myelin sheath around axons in the central nervous system (6Matsushita F. Kameyama T. Marunouchi T. Mech. Dev. 2002; 118: 209-213Crossref PubMed Scopus (21) Google Scholar, 7Kim J.G. Armstrong R.C. v Agoston D. Robinsky A. Wiese C. Nagle J. Hudson L.D. J. Neurosci. Res. 1997; 50: 272-290Crossref PubMed Scopus (101) Google Scholar). Orthologs of MyT1 are found in organisms ranging from nematodes to mammals, and functional data indicate that MyT1 is essential for neural development in Xenopus laevis (5Bellefroid E.J. Bourguignon C. Hollemann T. Ma Q. Anderson D.J. Kintner C. Pieler T. Cell. 1996; 87: 1191-1202Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Humans contain two other likely paralogs of MyT1: MyT1-like (MyT1L/NZF1) and Suppressor of Tumorigenicity 18 (ST18/NZF3). MyT1L appears to be involved in neural development (7Kim J.G. Armstrong R.C. v Agoston D. Robinsky A. Wiese C. Nagle J. Hudson L.D. J. Neurosci. Res. 1997; 50: 272-290Crossref PubMed Scopus (101) Google Scholar), whereas ST18 was reported to be a breast cancer tumor suppressor (8Jandrig B. Seitz S. Hinzmann B. Arnold W. Micheel B. Koelble K. Siebert R. Schwartz A. Ruecker K. Schlag P.M. Scherneck S. Rosenthal A. Oncogene. 2004; 23: 9295-9302Crossref PubMed Scopus (66) Google Scholar). At least three additional vertebrate proteins that contain one MyT1-type ZnF have been identified (L3MBTL, L3MBTL3, and L3MBTL4), and one of these, L3MBTL, is a possible tumor suppressor (9Boccuni P. MacGrogan D. Scandura J.M. Nimer S.D. J. Biol. Chem. 2003; 278: 15412-15420Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Several biochemical studies have demonstrated that MyT1-type ZnFs from MyT1, MyT1L, and ST18 can recognize DNA in a sequence-specific manner (5Bellefroid E.J. Bourguignon C. Hollemann T. Ma Q. Anderson D.J. Kintner C. Pieler T. Cell. 1996; 87: 1191-1202Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 10Jiang Y. Yu V.C. Buchholz F. O'Connell S. Rhodes S.J. Candeloro C. Xia Y.R. Lusis A.J. Rosenfeld M.G. J. Biol. Chem. 1996; 271: 10723-10730Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 11Yee K.S. Yu V.C. J. Biol. Chem. 1998; 273: 5366-5374Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The consensus sequence, AAGTT, is the core element of the cis-regulatory element of the β-retinoic acid receptor gene (β-RARE) and is also found in the human PLP promoter. Until now, the molecular details of DNA recognition by MyT1-type zinc fingers have not been addressed. In this work, we used surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) to delineate the relative importance of each ZnF in MyT1 for DNA binding. Finger 5 (F5) has the highest affinity for the β-RARE site. We went on to use a combination of NMR and mutagenesis to probe the structural basis for the MyT1-DNA interaction. A data-driven model of the interaction, calculated using HADDOCK (12van Dijk A.D. Bonvin A.M. Bioinformatics (Oxford, England). 2006; 22: 2340-2347Crossref PubMed Scopus (123) Google Scholar, 13van Dijk M. van Dijk A.D. Hsu V. Boelens R. Bonvin A.M. Nucleic Acids Res. 2006; 34: 3317-3325Crossref PubMed Scopus (152) Google Scholar, 14Dominguez C. Boelens R. Bonvin A.M. J. Am. Chem. Soc. 2003; 125: 1731-1737Crossref PubMed Scopus (2189) Google Scholar), reveals that a whole MyT1-type ZnF can sit snugly in the major groove of double-stranded DNA, forming a number of specific electrostatic and hydrophobic contacts. This mode of recognition is distinct from that observed for other classes of DNA-binding ZnFs. Subcloning, Expression, and Purification of MyT1 Constructs–The original plasmid encoding mouse 6-ZnF myelin transcription factor 1 (mMyT1) was a generous gift of Dr. Lynn Hudson (National Institutes of Health). Ten different constructs of MyT1 from the original plasmid and using (see Fig. constructs as with at was using the in a M. Y. K. G.M. A.M. R. J. 1998; PubMed Scopus Google Scholar). using affinity and in 1 by and by at and P. Biochem. Scopus Google Scholar). at β-RARE DNA and β-RARE DNA and and DNA for from in to for 5 to over the of and using calculated by at on a system at of in DNA was on MyT1 was in and binding was binding for different or constructs in a of each or and the The system was with 1 each studies and using the In the or was to DNA in the of 2 or 5 of DNA and DNA the of β-RARE DNA was into or on a at each of of at for of from of DNA into and analyzed using as R. J.M. Crossley M. Mackay J.P. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). NMR or was into NMR 1 or and in to and at 4 on a and NMR with and data used to by of the F. W.M. J. PubMed Scopus Google Scholar), and from and of with β-RARE DNA in with in the of 1 at and for both and the with and and for the calculated as a of and using to a reported W.M. J. PubMed Scopus Google Scholar, C. Dev. 1996; PubMed Scopus Google Scholar). of the DNA from NMR data using and analyzed with D. and D. of at structures of calculated in P. C. K. J. Mol. Biol. 1997; 273: PubMed Scopus Google from and from both and the of identified by of the structures calculated in the of using J.P. M. W. M. 2003; PubMed Scopus Google in G.M. P. J. M. T. G.L. D. Biol. 1998; PubMed Scopus Google Scholar), using the with the zinc and the of the J.G. S. Sci. PubMed Scopus Google Scholar). by and In the of the structures in a of using and for details J.P. Bonvin A.M. M. 2003; 50: PubMed Scopus Google and J. J. Chem. Scopus Google Scholar). The with the of analyzed using R. M. K. J. Mol. 1996; PubMed Scopus Google and R.A. J.A. R. J.M. J. 1996; PubMed Scopus Google Scholar). HADDOCK was to the DNA using the HADDOCK (12van Dijk A.D. Bonvin A.M. Bioinformatics (Oxford, England). 2006; 22: 2340-2347Crossref PubMed Scopus (123) Google Scholar, 13van Dijk M. van Dijk A.D. Hsu V. Boelens R. Bonvin A.M. Nucleic Acids Res. 2006; 34: 3317-3325Crossref PubMed Scopus (152) Google Scholar, 14Dominguez C. Boelens R. Bonvin A.M. J. Am. Chem. Soc. 2003; 125: 1731-1737Crossref PubMed Scopus (2189) Google Scholar). The structures for the a model of the DNA with the Nucleic T. Nucleic and the structures of on NMR titration of the and seven of the from the and for both the protein and the DNA based on data and by the A was used to the ambiguous interaction the DNA and within the consensus sequence as based on the observed from the of and to from of and of whereas for and 18 the whole was of the of these Bonvin A.M. M. A. van R. Boelens R. J. Mol. Biol. 2006; PubMed Scopus Google Scholar). The and as between of and and of and DNA of the protein as the surface of and A of 18 from and used as in to and for the DNA. the structures and the based on the used for the by The using a of based on the The and the of HADDOCK with the structures of the Bonvin A.M. M. A. van R. Boelens R. J. Mol. Biol. 2006; PubMed Scopus Google Scholar). The structures as in a of The structures over of this analyzed using HADDOCK and used to a model of the The DNA-binding of MyT1 ZnF the DNA-binding of MyT1 ZnFs, we first expressed and to different of the MyT1 ZnFs A and and their to a the β-RARE sequence Fig. NMR and data that this is a well in the not The of the was by and K. NMR of and Nucleic Scholar). We first the affinity of ZnFs for the β-RARE DNA. in Fig. in the binding observed between The data for finger 5 (F5) well to a binding a of (see Fig. of the from to the binding affinity to a electrostatic to the interaction. In the binding of was at and and that affinity not be of the relative of each ZnF for DNA, we the the of of protein in Fig. for data on and This a of the relative for the protein used is of the of the was in these in Fig. the different ZnFs have different DNA-binding in and to with whereas and relatively of binding using this and in with the the of finger constructs less data that to β-RARE with a and affinity of that these data the the of the and the receptor this the was data are with in the range the data are in has been that can be for interactions are as as or less J. Am. Chem. Soc. 2003; 125: PubMed Scopus Google Scholar). The binding for the interaction well with that by A finger both and also in a two of more Fig. binding of the β-RARE probe is by a of the MyT1 ZnFs and and also the other fingers can a to binding at a site. of to the of DNA binding by MyT1-type ZnFs in more NMR data that ZnFs 2 and in as that in on the binding for the interaction not be by of these are with of the a that was by We on the interaction of with DNA. the importance of each DNA for the interaction, β-RARE DNA was and was in the of a of a This of was based on with each in to to and these β-RARE the by and other with this The and less for binding than the other that the central sequence is likely to the for The was for the finger three additional of the central demonstrated to to binding. the in relative at the for with of the different used by the of protein and DNA and the binding for the different of we used and NMR to the of structures calculated in P. C. K. J. Mol. Biol. 1997; 273: PubMed Scopus Google using restraint data that and the zinc The was in J.P. M. W. M. 2003; PubMed Scopus Google Scholar, J.P. M. J. PubMed Scopus Google Scholar), with the of to the zinc D. Y. A. J. Mol. Biol. 1992; PubMed Scopus Google Scholar). The structures from a of used to the of These structures and restraint or 5 The is well with for of lacks elements of and several with a central zinc The with and a hydrophobic Fig. electrostatic surface of a distinct groove on one and a of and on the A electrostatic also appears to on one surface of and of the surface to the of as by NMR spectroscopy not with a for these electrostatic interactions in of and NMR range range ambiguous from from in most in additional in in in a the by NMR MyT1-type ZnFs recognize DNA, we analyzed a titration of with Fig. a of the of during the The of a of with the that observed in the the of more than one of DNA not a specific interaction between and DNA. The interaction is to on the as for the of a with a A of is in Fig. and in Fig. that both and have a are a of These on one of the that this surface the DNA-binding surface of The is a of and hydrophobic and and Fig. also using finger constructs and not for the by in whereas in the of observed for in both ZnFs. with a for the finger constructs to the of their interaction with DNA. of of the also to be for in the DNA and the observed for the central of the with the A of the the was the of our NMR be identified from or a combination of and J. J.M. J. PubMed Scopus (57) Google Scholar, M. V. V. J. 1992; PubMed Scopus Google Scholar). from a number of DNA and as well as of that are likely to form the not be in of the These most likely to into the likely DNA-binding mode of we used our and mutagenesis data to a model of the using the data-driven HADDOCK (12van Dijk A.D. Bonvin A.M. Bioinformatics (Oxford, England). 2006; 22: 2340-2347Crossref PubMed Scopus (123) Google Scholar, 13van Dijk M. van Dijk A.D. Hsu V. Boelens R. Bonvin A.M. Nucleic Acids Res. 2006; 34: 3317-3325Crossref PubMed Scopus (152) Google Scholar, 14Dominguez C. Boelens R. Bonvin A.M. J. Am. Chem. Soc. 2003; 125: 1731-1737Crossref PubMed Scopus (2189) Google Scholar). HADDOCK a of that on of the J.P. M. W. M. 2003; PubMed Scopus Google and G.M. P. J. M. T. G.L. D. Biol. 1998; PubMed Scopus Google is to use biochemical biophysical interaction data to and to to of a that the data D. M. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, M.E. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. E. Li Y. Xia B. C. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). In the protein and DNA as that both in and surface from protein and to and DNA and that the DNA is and the is from DNA on the basis of the DNA data and a docking, and Bonvin A.M. M. A. van R. Boelens R. J. Mol. Biol. 2006; PubMed Scopus Google Scholar), the structures to a model of the These structures with of over of the restraint observed in the structures The DNA-binding surface of in the between the two of zinc and this into the major groove and over the of the consensus sequence. Ten and hydrophobic are observed in of the structures and the interactions can be in Fig. The of both and a in DNA the two with and as well as hydrophobic with and whereas the with and as well as van to and to are by the of whereas and form with in the the of the of is with other well with the that the of a with of both and interactions between and hydrophobic in a The of the DNA the was analyzed using the Nucleic Acids Res. 2003; PubMed Scopus Google Scholar). the DNA double-stranded the although some from observed in the Fig. The of the major groove of DNA is at a between and the of and In to our we of and form the of specific with the DNA as well as and NMR of and that the more and the DNA-binding of to NMR and in in DNA binding we although the DNA binding of and is different the acid sequence of the two domains is conserved at the DNA binding In to the leading to this was to This is one of the in the that is not in is The in a of DNA binding with that the electrostatic between and a in the The DNA of MyT1 Zinc this we have to the molecular basis for DNA recognition by MyT1-type ZnFs. Our data the on the DNA by a MyT1 ZnF and the molecular basis for the DNA-binding of these domains. The model that we have from a combination of NMR and binding data that MyT1 ZnFs form structures that can sit within the major groove of DNA. The of a of and specific with DNA in the of the MyT1 consensus sequence, and these interactions most likely to as well as binding The of of the and van interactions of the a in the recognition of the DNA 5 and during DNA binding has been observed with other proteins Bonvin A.M. R. Boelens R. R. J. 2002; PubMed Scopus Google Scholar). Our model reveals that there is between MyT1 ZnFs and the DNA major groove The of the in the major groove data on the also interaction with the major groove Y. Yu V.C. Buchholz F. O'Connell S. Rhodes S.J. Candeloro C. Xia Y.R. Lusis A.J. Rosenfeld M.G. J. Biol. Chem. 1996; 271: 10723-10730Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, J.M. 2004; PubMed Scopus Google Scholar). HADDOCK we observed of structures in the protein interactions with the DNA and the of in the major groove by In these the of the into the major groove was a of calculated details G.M. P. J. M. T. G.L. D. Biol. 1998; PubMed Scopus Google a with our model In observed in of these structures this Although many zinc fingers recognize the major groove of DNA, the MyT1 this of the in the with the and receptor ZnFs. These domains to and in many other domains, including and other proteins, of the between the and the DNA major MyT1-type ZnFs are found in proteins than classical ZnFs, and in this are more to as and zinc finger domains, are also data that the binding of or MyT1 zinc finger constructs to DNA is than that observed for sequence-specific DNA-binding proteins is to its the MyT1-DNA interaction is sequence our that the binding of to a of data from A. Res. 2006; PubMed Scopus Google that interactions are and are likely to be is to use the and is to that these are not The interaction between MyT1 and DNA is relatively a of data that is highly the NMR titration was to the DNA sequence and the each of the protein A is that is that interactions are to be the not be in the cell at the there is of data in the that this as domains and J. M. S.D. J. 2004; 23: PubMed Scopus Google Scholar), the transcription factors and K. Liew C.K. J.M. Crossley M. Mackay J.P. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus (21) Google Scholar), or and their recognition domains and and S. W. J. D.J. 2006; PubMed Scopus Google Scholar, F. Zhao R. 2006; PubMed Scopus Google Scholar, M. M. Y. W. F. S. PubMed Scopus Google have in the that interactions are known to be the that can be from these data is that binding in not the a for be by or the of in binding are a relative different domains or a of for the of their is to and DNA has our understanding of the zinc fingers of MyT1 to recognize DNA. although well to DNA, can each with to the affinity of MyT1 for DNA. Our data indicate that the finger is likely to the of a that a is both and to recognition of the β-RARE site. the between the of and and the of the DNA is likely that of the finger the sequence in that by from that observed for binding to This is with our model of the the of the of the β-RARE as well as with the binding of and the The DNA recognition identified for MyT1 (4Kim J.G. Hudson L.D. Mol. Cell. Biol. 1992; 12: 5632-5639Crossref PubMed Scopus (112) Google in the human PLP promoter contains a of sequence and is by three studies also that of the are for DNA binding Y. Yu V.C. Buchholz F. O'Connell S. Rhodes S.J. Candeloro C. Xia Y.R. Lusis A.J. Rosenfeld M.G. J. Biol. Chem. 1996; 271: 10723-10730Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). This is of the of the and is conserved in the the that mouse and MyT1 a with the human protein (5Bellefroid E.J. Bourguignon C. Hollemann T. Ma Q. Anderson D.J. Kintner C. Pieler T. Cell. 1996; 87: 1191-1202Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar), in the DNA-binding identified in this a not be at the in these a very was found of the by three It is possible that this the for and Yu K.S. Yu V.C. J. Biol. Chem. 1998; 273: 5366-5374Abstract Full Text Full Text PDF PubMed Scopus (54) Google have found that the of the MyT1 to a DNA probe two the reported using of MyT1 zinc finger domains, and we have observed in other that the of to can to Loughlin and J. P. that there are within the promoter of the human or the as to MyT1 three domains and the It is possible that distinct chromatin by binding to highly DNA This possible is in of the that MyT1 can E. J.A. J.G. Hudson L.D. J. PubMed Scopus Google and indicate a in the binding of MyT1 to and the of in the of chromatin of this type have been for many including proteins that can contain to zinc we have very of In we have used a range of biophysical to delineate the of DNA recognition by the MyT1 of zinc Our indicate that MyT1 ZnFs recognize of double-stranded DNA in a sequence-specific manner by the whole into the major groove of the DNA. These data our understanding of We Bonvin and van Dijk for with the and Lynn Hudson for the original