Phosphorylated Intrinsically Disordered Region of FACT Masks Its Nucleosomal DNA Binding Elements

Abstract

FACT is a heterodimer of SPT16 and SSRP1, which each contain several conserved regions in the primary structure. The interaction of FACT with nucleosomes induces chromatin remodeling through the combinatorial action of its distinct functional protein regions. However, there is little mechanistic insight into how these regions cooperatively contribute to FACT functions, particularly regarding the recognition of nucleosomal DNA. Here, we report the identification of novel phosphorylation sites of Drosophila melanogaster FACT (dFACT) expressed in Sf9 cells. These sites are densely concentrated in the acidic intrinsically disordered (ID) region of the SSRP1 subunit and control nucleosomal DNA binding by dFACT. This region and the adjacent segment of the HMG domain form weak electrostatic intramolecular interactions, which is reinforced by the phosphorylation, thereby blocking DNA binding competitively. Importantly, this control mechanism appears to support rapid chromatin transactions during early embryogenesis through the dephosphorylation of some sites in the maternally transmitted dSSRP1. FACT is a heterodimer of SPT16 and SSRP1, which each contain several conserved regions in the primary structure. The interaction of FACT with nucleosomes induces chromatin remodeling through the combinatorial action of its distinct functional protein regions. However, there is little mechanistic insight into how these regions cooperatively contribute to FACT functions, particularly regarding the recognition of nucleosomal DNA. Here, we report the identification of novel phosphorylation sites of Drosophila melanogaster FACT (dFACT) expressed in Sf9 cells. These sites are densely concentrated in the acidic intrinsically disordered (ID) region of the SSRP1 subunit and control nucleosomal DNA binding by dFACT. This region and the adjacent segment of the HMG domain form weak electrostatic intramolecular interactions, which is reinforced by the phosphorylation, thereby blocking DNA binding competitively. Importantly, this control mechanism appears to support rapid chromatin transactions during early embryogenesis through the dephosphorylation of some sites in the maternally transmitted dSSRP1. FACT (facilitates chromatin transcription), 2The abbreviations used are: FACTfacilitates chromatin transcriptionAPasealkaline phosphataseIDintrinsically disorderedCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidEMSAelectrophoretic mobility shift assayHMGhigh-mobility groupAFMatomic force microscopyWTwild type. an evolutionarily conserved protein in eukaryotes, is a heterodimer consisting of structure-specific recognition protein-1 (SSRP1) and SPT16 with a larger molecular mass than SSRP1 (1.Orphanides G. Wu W.H. Lane W.S. Hampsey M. Reinberg D. 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Orphanides G. Studitsky V.M. Reinberg D. Science. 2003; 301: 1090-1093Crossref PubMed Scopus (647) Google Scholar). The FACT subunits also display a range of physical and genetic interactions with other factors (2.Reinberg D. Sims 3rd, R.J. J. Biol. Chem. 2006; 281: 23297-23301Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 3.Heo K. Kim H. Choi S.H. Choi J. Kim K. Gu J. Lieber M.R. Yang A.S. An W. Mol. Cell. 2008; 30: 86-97Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 6.Tan B.C. Chien C.T. Hirose S. Lee S.C. EMBO J. 2006; 25: 3975-3985Crossref PubMed Scopus (132) Google Scholar, 11.Shimojima T. Okada M. Nakayama T. Ueda H. Okawa K. Iwamatsu A. Handa H. Hirose S. Genes Dev. 2003; 17: 1605-1616Crossref PubMed Scopus (97) Google Scholar, 13.VanDemark A.P. Blanksma M. Ferris E. Heroux A. Hill C.P. Formosa T. Mol. Cell. 2006; 22: 363-374Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), suggesting that FACT directs several different functions by interacting with multiple complexes. facilitates chromatin transcription alkaline phosphatase intrinsically disordered 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid electrophoretic mobility shift assay high-mobility group atomic force microscopy wild type. At the molecular level, FACT initially binds nucleosomes and/or nucleosomal DNA, and then destabilizes the interactions between the H2A/H2B dimers and the H3/H4 tetramer within nucleosomes (2.Reinberg D. Sims 3rd, R.J. J. Biol. Chem. 2006; 281: 23297-23301Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Therefore, most studies have so far focused on interactions between FACT and histones. For example, it has been previously reported that the C-terminal region of human SPT16 (hSPT16) directly binds to H2A/H2B dimers (12.Belotserkovskaya R. Oh S. Bondarenko V.A. Orphanides G. Studitsky V.M. Reinberg D. Science. 2003; 301: 1090-1093Crossref PubMed Scopus (647) Google Scholar). A recent study has revealed that the N-terminal amino peptidase-like domain of Schizosaccharomyces pombe SPT16 associates with the H3/H4 histones, suggesting that this SPT16 may contribute to binding, eviction, and/or deposition of all histones (14.Stuwe T. Hothorn M. Lejeune E. Rybin V. Bortfeld M. Scheffzek K. Ladurner A.G. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 8884-8889Crossref PubMed Scopus (102) Google Scholar). However, it remains unclear how the FACT protein interacts with the nucleosomal DNA at the initial step of chromatin remodeling, although several studies have reported that the high-mobility group (HMG) box domain of SSRP1 binds to DNA nonspecifically or by recognizing specific structures of DNA (15.Yarnell A.T. Oh S. Reinberg D. Lippard S.J. J. Biol. 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Genetics. 2000; 155: 1593-1606Crossref PubMed Google Scholar) and animal (19.Cao S. Bendall H. Hicks G.G. Nashabi A. Sakano H. Shinkai Y. Gariglio M. Oltz E.M. Ruley H.E. Mol. Cell. Biol. 2003; 23: 5301-5307Crossref PubMed Scopus (60) Google Scholar) viability. This protein contains two structural domains, a structure-specific recognition (SSRC) motif (amino acids 186–436 in Drosophila) and an HMG-box domain (amino acids 555–624 in Drosophila). In yeast, the bipartite SSRP1 analog consists of Pob3 and Nhp6 (20.Brewster N.K. Johnston G.C. Singer R.A. Mol. Cell. Biol. 2001; 21: 3491-3502Crossref PubMed Scopus (103) Google Scholar). Further sequence analysis revealed that the other regions could be distinguished as four evolutionarily conserved regions: an N-terminal region (amino acids 1–186 in Drosophila), an acidic ID region (amino acids 437–518 in Drosophila) with limited homology to nucleolin, an HMG-flanking ID segment (amino acids in Drosophila), and a ID region at the (amino acids in Drosophila). SSRP1 initially on the nucleosomal DNA in the chromatin remodeling through the combinatorial action of its distinct functional protein such as the HMG is to the molecular mechanism by which SSRP1 interacts with the nucleosomal DNA. several studies have reported the phosphorylation of SSRP1 Y. Keller D.M. Scott J.D. Lu H. J. Biol. Chem. 2005; 280: 11869-11875Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, P. R. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, D.M. Lu H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, D.M. Y. M. H. R. G. Y. Lu H. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), it remains unclear how the phosphorylation of SSRP1 DNA The ID regions of FACT are particularly For the of SSRP1 and SPT16 of in and (Fig. 1A). In it has been that the ID region of SPT16 is essential for the transcriptional by FACT (12.Belotserkovskaya R. Oh S. Bondarenko V.A. Orphanides G. Studitsky V.M. Reinberg D. Science. 2003; 301: 1090-1093Crossref PubMed Scopus (647) Google Scholar). These disordered regions in some into binding to Mol. Cell Biol. 2005; PubMed Scopus Google Scholar, J.D. P. Oh J. R. W. J. Mol. 2001; PubMed Scopus Google Scholar, J. 2006; PubMed Scopus Google Scholar). we have the ID regions of Drosophila melanogaster FACT (dFACT) in by atomic force microscopy A. Y. T. K. Hirose S. K. T. 2008; PubMed Scopus Google Scholar). However, the and functional of the FACT ID regions at the molecular the expressed in Sf9 we how the phosphorylation of melanogaster induces the of DNA that the phosphorylation sites the nucleosomal DNA binding are concentrated in the acidic ID region of dSSRP1. This acidic ID region an intramolecular interaction with the HMG domain and the ID phosphorylation of the acidic ID region this thereby blocking the DNA binding to the HMG The of these is by that some phosphorylation sites in the maternally transmitted to rapid chromatin transactions during early used the as in study T. Okada M. Nakayama T. Ueda H. Okawa K. Iwamatsu A. Handa H. Hirose S. Genes Dev. 2003; 17: 1605-1616Crossref PubMed Scopus (97) Google Scholar). the DNA the and into the and sites of the for and the DNA into the and sites of the in the and then the DNA into the and sites the control of the in the of in and or and 1A). The also into the and sites the control of the in the to and the of the the the and the within the protein and factor sites between amino acid and also the by these used to The complex into Sf9 cells. The Sf9 from and at in II The at The expressed at for in Sf9 which at a of with the the The by and in and a of the by two of at for the a the with A and the with A and The then to a which with a of in A. the FACT complex A and then on a by with a of in A. The and of from E. have been previously A. Y. T. K. Hirose S. K. T. 2008; PubMed Scopus Google Scholar, Y. Hirose S. K. S. J. 2005; PubMed Scopus Google Scholar). The of the and to in the cells. we expressed the protein with two sites between amino acid and and a factor between amino acid and in Sf9 and then with and factor to these the protein with two sites with for at The protein to a in A and the the The protein on a by with a of in A. this protein with factor for at during A The protein to a in A The protein with a of in A. of the A used the as in the study T. Okada M. Nakayama T. Ueda H. Okawa K. Iwamatsu A. Handa H. Hirose S. Genes Dev. 2003; 17: 1605-1616Crossref PubMed Scopus (97) Google Scholar). and from a at and from the segment of from the in and The and in and a and the as the The then with as for to the with of alkaline phosphatase in and at for The and the from and with These by in and then on with used the as in the study T. Okada M. Nakayama T. Ueda H. Okawa K. Iwamatsu A. Handa H. Hirose S. 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Res. 2005; PubMed Scopus Google Scholar). with the FACT in and at for The the at on a in and then by acid the of The by in A with an of the for at in and also with an of for at in the In the with an of for at The with and of and then for at the The at by in and then by and E. by the protein in as the to the The protein in a and in The protein also in the and The for the protein in the and with a J. A.G. Scholar). A of for with different of to the The of the protein to to and the The on a at for the at with the S. Zhu G. J. A. J. PubMed Scopus Google Scholar). by in the R. J. Scopus Google Scholar), which the as an of of for all The shift in in and are the in for the and and the for the and to and in the on a and melanogaster in the cell and from a of Sf9 we the as a complex between and The to and at (Fig. and This could be to phosphorylation of as phosphorylation of by the DNA binding of FACT Y. Keller D.M. Scott J.D. Lu H. J. Biol. Chem. 2005; 280: 11869-11875Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). this the protein with alkaline phosphatase and then the from the complex with to The the mobility of to suggesting that some of the of (Fig. The with the nucleosomal as with the form (Fig. and and and and Therefore, we that the phosphorylation of its binding to nucleosomal DNA. we the binding of the between the and The complex with the of as revealed by the of nucleosomes These that the interaction is to an in DNA In the interacts with nucleosomes that contain DNA, in with the that HMG-box to DNA. For example, the interaction with the which than that with the (Fig. and and However, this is not with the that the DNA not the binding of the complex S. Formosa T. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). This may be to the that contain the HMG domain within the of the domain is on a in the FACT The study has that the as phosphorylation sites in the phosphorylation of a in binding between and DNA Y. Keller D.M. Scott J.D. Lu H. J. Biol. Chem. 2005; 280: 11869-11875Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). This to for the functional phosphorylation sites in for the nucleosomal DNA An sequence of revealed phosphorylation sites (Fig. and of as or the phosphorylation sites in and to in (Fig. which consists of the the acidic the and the HMG These phosphorylation from to of sites in than that for the (Fig. For example, the of the phosphorylation in of the than of the and the (Fig. for (Fig. in the sites (Fig. to be in Sf9 cells. The phosphorylation sites by the mobility as the protein expressed in E. (Fig. in which the sites to different and as to that of (Fig. the phosphorylation sites we and The of and of and to the than that of of to the of sites The of the to the The also by a (Fig. the of These that the region of amino acids of contains at specific phosphorylation which are by the phosphorylation within the region of amino acids the nucleosomal DNA binding of we the of as in The to to the in with (Fig. and and The binding of the and to the in the of at the phosphorylation sites (Fig. the we also these sites are to the nucleosomal DNA binding In with the with the the from the complex in the of on the than the protein (Fig. also that the and to nucleosomes with than the protein (Fig. and we that the C-terminal region of (amino acids contains other phosphorylation sites (Fig. and its phosphorylation in the of nucleosomal DNA binding For example, the consisting of to the nucleosomes, in with the and and the the nucleosomal DNA binding the of the C-terminal region and and Therefore, it is that the C-terminal region of has a on the interaction between and nucleosomes, not an These that the of for nucleosomal DNA is by phosphorylation or dephosphorylation at the sites within the acidic ID The phosphorylation induces the acidic region of to the DNA However, this region interactions with DNA. In the (Fig. which of this acidic not with (Fig. and the protein (Fig. the segment and its HMG to directly with DNA (Fig. also that the segment essential to the for the DNA binding The acidic region and the segment are as by the A. Y. T. K. Hirose S. K. T. 2008; PubMed Scopus Google Scholar) and not it is that the acidic ID region intramolecular interactions with the HMG domain and the ID thereby on the DNA binding of dSSRP1. this we the interaction between the acidic region and the HMG domain within by The protein by two sites with and factor as also that the with two sites the of phosphorylation as that of the protein not The and to to the and the of these to for the and which are of the acidic the segment and the HMG with to the of the ID segment on the interaction between the HMG domain and the acidic ID region The HMG with the ID segment and multiple with the of the phosphorylation although the HMG protein could not and with the from the binding this that the ID segment to the intramolecular interaction with the acidic the interaction between the acidic region and the HMG domain the ID we the the protein and the which contains a of the ID segment and the HMG domain (Fig. In the shift at in the ID segment the binding to the protein (Fig. from to and This is with acids in the HMG domain also shift by the (Fig. from to These that the protein interacts with the ID segment and the HMG The the that the HMG domain and ID segment are in binding between the and this the complex between the and with to a of (Fig. that the protein with the is the of an of (Fig. the protein to be with the in the of a of (Fig. These that the acidic ID region interacts with the ID segment or the HMG domain with than and the of in the of the acidic ID However, the phosphorylation the of the acidic ID thereby blocking the interaction between and the HMG The and of (Fig. suggesting that the phosphorylation not structures in the acidic be that phosphorylation two and phosphorylation sites the of the acidic ID region by Therefore, it is that these the interactions of the unstructured acidic region with the HMG domain and the ID segment by electrostatic the of the phosphorylation, we the phosphorylation of in the of the and the early The from the of the with the mobility as that from Sf9 in (Fig. The protein mobility as with that of the the (Fig. of the of by the These that the in the is to the as the protein in Sf9 cells. the other the in the early as with the (Fig. and the dephosphorylation of in the (Fig. the in the of the the the in the of the (Fig. In the early the from the and the (Fig. and these that is as the form in the and is into the form through dephosphorylation The a mechanistic of FACT the phosphorylation of the ID region the binding between and the nucleosomal DNA to the form for (Fig. In the the acidic ID region weak intramolecular interactions with the HMG domain and the ID the HMG domain and the ID segment to DNA, the for DNA the interactions of the acidic region with the the other the phosphorylation the of within the acidic ID this in the of the intramolecular interactions, which the of the DNA binding of the ID thereby blocking the DNA binding (Fig. This mechanism on the of the ID region with the phosphorylation and it be distinct from the that in some binding of ID regions. 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