Heterochromatin Protein 1 Is Extensively Decorated with Histone Code-like Post-translational Modifications

Abstract

Heterochromatin protein 1 (HP1) family members (α, β, and γ) bind histone H3 methylated at Lys-9, leading to gene silencing and heterochromatin formation. Several previous reports have suggested that HP1s are post-translationally modified, yet sites of modification have not yet been exhaustively determined. Here we perform the first comprehensive proteomic analysis of all HP1 isoforms using tandem mass spectrometry. Our data reveal that all HP1 isoforms are highly modified in a manner analogous to histones including phosphorylation, acetylation, methylation, and formylation, including several sites having multiple different types of modifications. Additionally, many of these modifications are found in both the chromo- and chromoshadow domains, suggesting that they may have an important role in modulating HP1 interactions or functions. These studies are the first to systematically map the abundant sites of covalent modifications on HP1 isoforms and provide the foundation for future investigations to test whether these modifications are essential in heterochromatin maintenance or other nuclear processes. Heterochromatin protein 1 (HP1) family members (α, β, and γ) bind histone H3 methylated at Lys-9, leading to gene silencing and heterochromatin formation. Several previous reports have suggested that HP1s are post-translationally modified, yet sites of modification have not yet been exhaustively determined. Here we perform the first comprehensive proteomic analysis of all HP1 isoforms using tandem mass spectrometry. Our data reveal that all HP1 isoforms are highly modified in a manner analogous to histones including phosphorylation, acetylation, methylation, and formylation, including several sites having multiple different types of modifications. Additionally, many of these modifications are found in both the chromo- and chromoshadow domains, suggesting that they may have an important role in modulating HP1 interactions or functions. These studies are the first to systematically map the abundant sites of covalent modifications on HP1 isoforms and provide the foundation for future investigations to test whether these modifications are essential in heterochromatin maintenance or other nuclear processes. The mammalian HP1α, β, and γ proteins are a family of chromatin-associated proteins that are homologs of the Drosophila heterochromatin protein 1(HP1) 1The abbreviations used are:HP1heterochromatin protein 1CDchromodomainCSDC-terminal chromoshadow domainPTMpost-translational modificationHEKhuman embryonic kidneyMSmass spectrometryLC-MS/MSliquid chromatography tandem mass spectrometry., originally identified as a protein required for position effect variegation. Position effect variegation is the silencing phenomenon that occurs when a block of euchromatin is placed adjacent to a region of heterochromatin (1Elgin S.C. Grewal S.I. Heterochromatin: silence is golden.Curr. Biol. 2003; 13: R895-R898Abstract Full Text Full Text PDF PubMed Google Scholar). HP1α, β, and γ proteins all contain a N-terminal chromodomain (CD) and a C-terminal chromoshadow domain (CSD) that are linked together by a flexible hinge region (2Fanti L. Pimpinelli S. HP1: a functionally multifaceted protein.Curr. Opin. Genet. Dev. 2008; 18: 169-174Crossref PubMed Scopus (100) Google Scholar). The CD in HP1 is one of many specialized domains found in a number of other chromatin-associated proteins that recognizes histone post-translational modifications (PTMs). In particular, x-ray crystallographic studies have demonstrated that the HP1 chromodomain binds directly to the histone H3 protein trimethylated on Lys-9 (3Jacobs S.A. Khorasanizadeh S. Structure of HP1 chromodomain bound to a lysine 9-methylated histone H3 tail.Science. 2002; 295: 2080-2083Crossref PubMed Scopus (651) Google Scholar). heterochromatin protein 1 chromodomain C-terminal chromoshadow domain post-translational modification human embryonic kidney mass spectrometry liquid chromatography tandem mass spectrometry. The C-terminal CSD domain is structurally similar to the CD and is thought to be responsible for dimerization and interactions with other proteins. The mammalian HP1α, β, and γ proteins have been shown to form both homo- and heterodimers with each other (4Nielsen A.L. Oulad-Abdelghani M. Ortiz J.A. Remboutsika E. Chambon P. Losson R. Heterochromatin formation in mammalian cells: interaction between histones and HP1 proteins.Mol. Cell. 2001; 7: 729-739Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). Although the HP1α, β, and γ proteins have similar structures and dimerize with each other, studies suggest they have non-overlapping functions. As the name implies, the HP1 proteins are generally associated with constitutive heterochromatic loci; however, HP1γ localizes with both hetero- and euchromatin (5Minc E. Courvalin J.C. Buendia B. HP1γ associates with euchromatin and heterochromatin in mammalian nuclei and chromosomes.Cytogenet. Cell Genet. 2000; 90: 279-284Crossref PubMed Scopus (127) Google Scholar). Moreover, although microscopy analysis has demonstrated that HP1α and HP1β localize together at many heterochromatic loci, they do not completely overlap, further suggesting that they can function independently (6Dialynas G.K. Terjung S. Brown J.P. Aucott R.L. Baron-Luhr B. Singh P.B. Georgatos S.D. Plasticity of HP1 proteins in mammalian cells.J. Cell Sci. 2007; 120: 3415-3424Crossref PubMed Scopus (63) Google Scholar). Furthermore, HP1γ was found to be associated with the DNA of actively transcribed genes by chromatin immunoprecipitation (7Vakoc C.R. Mandat S.A. Olenchock B.A. Blobel G.A. Histone H3 lysine 9 methylation and HP1gamma are associated with transcription elongation through mammalian chromatin.Mol. Cell. 2005; 19: 381-391Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar). All three of the mammalian HP1 proteins can complex with the co-repressor protein, KAP-1/Tif1β. Interestingly, this protein is also a post-translational code-reading protein that contains a bromodomain (acetyl-lysine binding) and a plant homeodomain (methyl-lysine binding). While the majority of KAP-1/Tif1β co-localizes with HP1α and HP1β, there are some non-overlapping foci, suggesting the bromodomain and plant homeodomain of KAP-1/Tif1β is reading a sub-code found within some heterochromatic loci (8Cammas F. Janoshazi A. Lerouge T. Losson R. Dynamic and selective interactions of the transcriptional corepressor TIF1 beta with the heterochromatin protein HP1 isotypes during cell differentiation.Differentiation. 2007; 75: 627-637Crossref PubMed Scopus (27) Google Scholar, 9Bártová E. Pacherník J. k J. Kozubík A. Kozubek S. Differentiation-specific association of HP1alpha and HP1beta with chromocentres is correlated with clustering of TIF1beta at these sites.Histochem. Cell Biol. 2007; 127: 375-388Crossref PubMed Scopus (29) Google Scholar). Although it is not clear how the HP1 proteins complex with KAP-1/Tif1β, the CSD is required for this interaction (10Lechner M.S. Schultz D.C. Negorev D. Maul G.G. Rauscher 3rd, F.J. The mammalian heterochromatin protein 1 binds diverse nuclear proteins through a common motif that targets the chromoshadow domain.Biochem. Biophys. Res. Commun. 2005; 331: 929-937Crossref PubMed Scopus (162) Google Scholar). Interestingly, it has been reported that KAP-1/Tif1β is a protein kinase that can phosphorylate all three of the mammalian HP1s and autophosphorylate itself (11Nielsen A.L. Ortiz J.A. You J. Oulad-Abdelghani M. Khechumian R. Gansmuller A. Chambon P. Losson R. Interaction with members of the heterochromatin protein 1 (HP1) family and histone deacetylation are differentially involved in transcriptional silencing by members of the TIF1 family.EMBO J. 1999; 18: 6385-6395Crossref PubMed Scopus (296) Google Scholar). The HP1 proteins have also been shown to interact with a number of other proteins/complexes that are involved with chromatin metabolism such as; the CAF-1 complex, a histone deposition chaperone; the origin recognition complex, a protein complex required for DNA replication that binds to origins of replication; and the BRG1 complex, an ATP-dependent chromatin remodeling machine (12Pak D.T. Pflumm M. Chesnokov I. Huang D.W. Kellum R. Marr J. Romanowski P. Botchan M.R. Association of the origin recognition complex with heterochromatin and HP1 in higher eukaryotes.Cell. 1997; 91: 311-323Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 13Nielsen A.L. Sanchez C. Ichinose H. Cerviño M. Lerouge T. Chambon P. Losson R. Selective interaction between the chromatin-remodeling factor BRG1 and the heterochromatin-associated protein HP1alpha.EMBO J. 2002; 21: 5797-5806Crossref PubMed Scopus (74) Google Scholar, 14Murzina N. Verreault A. Laue E. Stillman B. Heterochromatin dynamics in mouse cells: interaction between chromatin assembly factor 1 and HP1 proteins.Mol. Cell. 1999; 4: 529-540Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Like the target histones, the HP1 proteins themselves have been suggested to contain numerous PTMs such as phosphorylation, acetylation, or methylation. For example, it is well known that there are phosphorylation sites within a Drosophila HP1 protein mediated by several kinases, and that hyperphosphorylation of this HP1 is associated with heterochromatin formation and affects chromosomal localization (15Eissenberg J.C. Ge Y.W. Hartnett T. Increased phosphorylation of HP1, a heterochromatin-associated protein of Drosophila, is correlated with heterochromatin assembly.J. Biol. Chem. 1994; 269: 21315-21321Abstract Full Text PDF PubMed Google Scholar). Meanwhile, mutations at these HP1 phosphorylation sites attenuate or abolish silencing activity (16Zhao T. Heyduk T. Eissenberg J.C. Phosphorylation site mutations in heterochromatin protein 1 (HP1) reduce or eliminate silencing activity.J. Biol. Chem. 2001; 276: 9512-9518Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Interestingly, a hypophosphorylated form of Drosophila HP1 has also been shown to associate with the subunits of the origin recognition complex, while the hyperphosphorylated form is more tightly associated with interphase chromatin indicating that phosphorylation may regulate HP1 function (17Huang D.W. Fanti L. Pak D.T. Botchan M.R. Pimpinelli S. Kellum R. Distinct cytoplasmic and nuclear fractions of Drosophila heterochromatin protein 1: their phosphorylation levels and associations with origin recognition complex proteins.J. Cell Biol. 1998; 142: 307-318Crossref PubMed Scopus (103) Google Scholar). HP1γ phosphorylation at Ser-83 is found to be an exclusive euchromatin mark interacting with the regulatory protein Ku70 and this modified HP1γ isoform also demonstrates impaired silencing properties and is targeted to areas of transcriptional elongation (18Lomberk G. Bensi D. Fernandez-Zapico M.E. Urrutia R. Evidence for the existence of an within the histone Cell Biol. PubMed Scopus Google Scholar). HP1β phosphorylation at has been shown to be important during of the DNA as of the kinase kinase a in HP1 at sites and further N. J.A. chromatin that the DNA 2008; PubMed Scopus Google Scholar). The HP1 proteins have also been shown to other covalent modifications such as acetylation, methylation, and by with (18Lomberk G. Bensi D. Fernandez-Zapico M.E. Urrutia R. Evidence for the existence of an within the histone Cell Biol. PubMed Scopus Google Scholar). all this a of HP1 modification sites have been as comprehensive mass analysis has identified all HP1 we to systematically map the PTMs on the three mammalian HP1 homologs (α, β, and γ) using liquid chromatography to a mass Our that all HP1s are highly modified on many sites with many types of modifications. Several of the sites we found are sites within the chromo- and chromoshadow Interestingly, many HP1 found to be modified with different a of data be for future studies to the of the HP1α, β, and γ proteins as well as their to different of HP1α, HP1β, and HP1γ by a human of the the with an N-terminal was by the S. J. I. D. A. and transcriptional by family Cell. Biol. 2002; PubMed Scopus Google Scholar). The in modified by the proteins All used in the histone and 1 1 and 1 For protein nuclei by in 1 and transcription factor has an associated activity by the region of Sci. S. A. PubMed Scopus Google Scholar). the nuclei by in immunoprecipitation and The was by at for and the proteins by the with at with The three with and the proteins by with at for The proteins by and with was not in HP1s to both and with multiple For HP1 was with of and with for 1 at by with for at in the this HP1 was for at using at a of at a of or at a of Additionally, HP1 was on a and in and as with or as A. M. M. of proteins Chem. PubMed Scopus Google Scholar). The to A. M. M. of proteins Chem. PubMed Scopus Google Scholar). All using J. M. and for and in Chem. 2003; 75: PubMed Scopus Google to in a and in of for was not in liquid chromatography tandem mass spectrometry was on a mass to an and an on with an that with of by using a to in at a of for The was in using a of to a by in the for placed on an for to The with an target of and of a mass of using an of and a of for All with a of the or through the both the for a of and of using the to or a the HP1 protein the protein and the human protein modification of for and lysine modifications of or and or and phosphorylation used in the The was used to and protein at as by the A. E. R. to the of by and Chem. 2002; PubMed Scopus Google or also the HP1 to the of modified all data modified proteins embryonic using an and of the that protein HP1α and HP1β have higher mass with HP1γ we HP1 to on an mass we and for HP1α, HP1β, and isoform immunoprecipitation the other HP1 isoforms as well not this is in with previous data that HP1 isoforms interact with one the chromoshadow domain (2Fanti L. Pimpinelli S. HP1: a functionally multifaceted protein.Curr. Opin. Genet. Dev. 2008; 18: 169-174Crossref PubMed Scopus (100) Google Scholar). of the data using the a of PTMs on all three HP1 isoforms and be that we also the PTMs on HP1 isoforms that are with the HP1 we that on the PTMs are with modification of of HP1 modifications. was on all isoforms in with PTMs are shown in PTMs identified phosphorylation and with previous we found the HP1 proteins to be at (15Eissenberg J.C. Ge Y.W. Hartnett T. Increased phosphorylation of HP1, a heterochromatin-associated protein of Drosophila, is correlated with heterochromatin assembly.J. Biol. Chem. 1994; 269: 21315-21321Abstract Full Text PDF PubMed Google Scholar, T. Heyduk T. Eissenberg J.C. Phosphorylation site mutations in heterochromatin protein 1 (HP1) reduce or eliminate silencing activity.J. Biol. Chem. 2001; 276: 9512-9518Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, N. H. T. H. of heterochromatin protein 1 (HP1) as a phosphorylation target by kinase and the effect of phosphorylation on the transcriptional function of 2000; PubMed Scopus Google Scholar, E. Courvalin J.C. Buendia B. and phosphorylation of HP1 proteins during the cell in mammalian 1999; PubMed Scopus Google Scholar, T. Eissenberg J.C. Phosphorylation of heterochromatin protein 1 by kinase is required for heterochromatin in Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the tandem mass of a HP1γ that was found to be and was as the phosphorylation site by the at and also by the at the was not on it also be that of the we have in this tandem mass for a phosphorylation as well at and at as in mass phosphorylation in has been found on HP1γ that is to euchromatin (18Lomberk G. Bensi D. Fernandez-Zapico M.E. Urrutia R. Evidence for the existence of an within the histone Cell Biol. PubMed Scopus Google Scholar). found phosphorylation sites on other HP1 isoforms as The of the HP1α is shown in Interestingly, all phosphorylation sites we found to the hinge of phosphorylation sites the of HP1α and with multiple phosphorylation sites in different also (18Lomberk G. Bensi D. Fernandez-Zapico M.E. Urrutia R. Evidence for the existence of an within the histone Cell Biol. PubMed Scopus Google used to suggest that HP1 proteins can be modified with other PTMs of the including acetylation, methylation, and this we also data PTMs and that HP1s modified on with several types of modifications. the of a at The was found to have a on the the of the mass to be an a methylation mark In HP1α was found to be highly with the other HP1 isoforms in with the (18Lomberk G. Bensi D. Fernandez-Zapico M.E. Urrutia R. Evidence for the existence of an within the histone Cell Biol. PubMed Scopus Google Scholar). methylation was also found on the isoforms as shown in for a at HP1γ that a modification is on was found to be the identified of lysine methylation on the three phosphorylation, the lysine and methylation sites to the chromoshadow or a of lysine on all of the HP1 proteins. the of the a of the was that the lysine not an the The mass of this with The of HP1α, is shown in that is the modified lysine As we not or in of the we that these modifications be in The majority of the on the chromoshadow and on the sites or methylation we and on HP1 proteins. the of the and mass of the and to the of HP1γ at as the modified Interestingly, was also found to be of proteins has as an in covalent modification on histones and other nuclear proteins A. M. of lysine is a post-translational modification of nuclear proteins at involved in of chromatin Res. 2008; PubMed Scopus Google Scholar, T. M. of lysine in histone proteins as a modification DNA Sci. S. A. 2007; PubMed Scopus Google and these future on the in the of in silencing through or on HP1γ is of the at a that the modification is on the of the at with the a and the to for and with indicating the HP1 is an important nuclear protein first shown to be required for variegation and is in heterochromatic of the The CD of HP1 has been shown to bind histone methylation, (3Jacobs S.A. Khorasanizadeh S. Structure of HP1 chromodomain bound to a lysine 9-methylated histone H3 tail.Science. 2002; 295: 2080-2083Crossref PubMed Scopus (651) Google as well as histone methylation S. T. N. R. HP1 binds to histone phosphorylation HP1 Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google a silencing have suggested that the role of HP1 in heterochromatin formation may be by post-translational modifications. studies have on the function of HP1 phosphorylation a of and Eissenberg (15Eissenberg J.C. Ge Y.W. Hartnett T. Increased phosphorylation of HP1, a heterochromatin-associated protein of Drosophila, is correlated with heterochromatin assembly.J. Biol. Chem. 1994; 269: 21315-21321Abstract Full Text PDF PubMed Google that in Drosophila HP1 is that phosphorylation levels during multiple HP1 isoforms Additionally, HP1 phosphorylation is first during the nuclear is the when heterochromatin first a role for HP1 phosphorylation in heterochromatin Our proteomic data are with HP1 at multiple sites in and we have identified HP1 as the N-terminal of of the HP1 that HP1 be a target by several such as kinase and kinase or protein kinase C. has been as a kinase involved in HP1γ phosphorylation that was identified through a and shown to bind HP1γ the chromoshadow domain N. H. T. 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HP1γ was found to be at Ser-83 and this mark was found on a of HP1γ that was in euchromatin and impaired silencing Our mass that Ser-83 is a modification site on we found that this site was at a and was more that Ser-83 may be to a of be a mark that is on the more abundant HP1γ heterochromatin was found to be on HP1β during DNA by kinase and phosphorylation was found to be chromatin phosphorylation of DNA we not this site as an in phosphorylation to role in DNA and in In with (18Lomberk G. Bensi D. Fernandez-Zapico M.E. Urrutia R. Evidence for the existence of an within the histone Cell Biol. PubMed Scopus Google we found that HP1β the of phosphorylation of all the HP1 we that mass of human have also data on the phosphorylation of HP1α on the multiple and on HP1γ at and in of B. F. B. C. P. M. in and phosphorylation dynamics in 127: Full Text Full Text PDF PubMed Scopus Google Scholar, H. N. A. proteomic of using tandem mass Sci. S. A. 2007; PubMed Scopus Google Scholar). Urrutia and (18Lomberk G. Bensi D. Fernandez-Zapico M.E. Urrutia R. Evidence for the existence of an within the histone Cell Biol. PubMed Scopus Google by that HP1 isoforms to be or In we also identified several sites of and methylation on all HP1 not sites of or on HP1 to their in to be at levels Additionally, these or HP1 may also be as Interestingly, in with (18Lomberk G. Bensi D. Fernandez-Zapico M.E. Urrutia R. Evidence for the existence of an within the histone Cell Biol. PubMed Scopus Google we found that the number of sites is on the HP1α and methylation sites more the protein including the hinge domains, and chromoshadow The of these modifications to be All HP1 isoforms are known to complex with the histone through a CSD interaction M. M. L. P. G. T. analysis of a role in heterochromatin and Cell. Biol. 2000; PubMed Scopus Google it is to that the methylation sites on HP1 in that region may be a of that all HP1 isoforms directly interact with the co-repressor KAP-1/Tif1β, and KAP-1/Tif1β binds a domain protein with histone activity D.C. Negorev D. Maul G.G. Rauscher 3rd, F.J. a histone lysine that to silencing of genes by Dev. 2002; PubMed Scopus Google Scholar). a effect of the interaction may be the methylation of R. A. A. HP1 to chromatin methylated at is by Cell. Biol. 2007; PubMed Scopus Google that while HP1 binds to methylated H3 it to bind to methylated to HP1 for and the that HP1 may have other sites on chromatin that are with the These also be methylation of HP1 itself is it can bind Our data also that HP1 methylation and on the to similar on histones as histone or or These different modifications at the may as to HP1 function in the that may regulate histone H3 as in the of with gene and with transcriptional and and modification in histone and 2003; PubMed Scopus Google Scholar, The of covalent histone 2000; PubMed Scopus Google Scholar). demonstrated that HP1 proteins are also highly on multiple The of proteins can in by the of G. L. J.A. histone modifications in Res. 2008; 21: PubMed Scopus Google or J.A. A.L. of on for of sites by tandem mass Cell. 2007; Full Text Full Text PDF PubMed Scopus Google and by using of and and and by mass Chem. 1998; PubMed Scopus Google Scholar). we not of these in of protein has been reported on histones, and other nuclear proteins A. M. of lysine is a post-translational modification of nuclear proteins at involved in of chromatin Res. 2008; PubMed Scopus Google Scholar). T. M. of lysine in histone proteins as a modification DNA Sci. S. A. 2007; PubMed Scopus Google demonstrated that the the of in DNA can a modification on the of lysine on While this be a of on HP1 HP1 has been shown to be involved in DNA be associated with the DNA N. J.A. chromatin that the DNA 2008; PubMed Scopus Google Scholar, M.R. and are transcriptional that localization properties to Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google with to HP1 HP1 proteins themselves do not bind DNA HP1 is well known to bind histone and in the several many histone have been with of the known the of higher of methylation Lys-9 on histone H3 a of Dynamic of histone lysine methylation by Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). that as histones are by they of that with including histones and it has been that Drosophila HP1α with the histone through chromoshadow domain and the activity of B. S. L. Heterochromatin protein histone H3 lysine by the Drosophila Cell. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). of HP1α also to in is to that many of the sites we in the on including the that directly binds of this region may HP1 further to The majority of the sites on lysine that are also methylated as for histone A. M. of lysine is a post-translational modification of nuclear proteins at involved in of chromatin Res. 2008; PubMed Scopus Google Scholar). These that HP1 lysine may with chromatin on the and of this we for the of and on HP1, to has not been to we that the to be can also be as the that these may HP1 function through for with