The C-terminal Domain Is the Primary Determinant of Histone H1 Binding to Chromatin in Vivo

We have used a combination of kinetic measurements and targeted mutations to show that the C-terminal domain is required for high-affinity binding of histone H1 to chromatin, and phosphorylations can disrupt binding by affecting the secondary structure of the C terminus. By measuring the fluorescence recovery after photo-bleaching profiles of green fluorescent protein-histone H1 proteins in living cells, we find that the deletion of the N terminus only modestly reduces binding affinity. Deletion of the C terminus, however, almost completely eliminates histone H1.1 binding. Specific mutations of the C-terminal domain identified Thr-152 and Ser-183 as novel regulatory switches that control the binding of histone H1.1 in vivo. It is remarkable that the single amino acid substitution of Thr-152 with glutamic acid was almost as effective as the truncation of the C terminus to amino acid 151 in destabilizing histone H1.1 binding in vivo. We found that modifications to the C terminus can affect histone H1 binding dramatically but have little or no influence on the charge distribution or the overall net charge of this domain. A comparison of individual point mutations and deletion mutants, when reviewed collectively, cannot be reconciled with simple charge-dependent mechanisms of C-terminal domain function of linker histones. We have used a combination of kinetic measurements and targeted mutations to show that the C-terminal domain is required for high-affinity binding of histone H1 to chromatin, and phosphorylations can disrupt binding by affecting the secondary structure of the C terminus. By measuring the fluorescence recovery after photo-bleaching profiles of green fluorescent protein-histone H1 proteins in living cells, we find that the deletion of the N terminus only modestly reduces binding affinity. Deletion of the C terminus, however, almost completely eliminates histone H1.1 binding. Specific mutations of the C-terminal domain identified Thr-152 and Ser-183 as novel regulatory switches that control the binding of histone H1.1 in vivo. It is remarkable that the single amino acid substitution of Thr-152 with glutamic acid was almost as effective as the truncation of the C terminus to amino acid 151 in destabilizing histone H1.1 binding in vivo. We found that modifications to the C terminus can affect histone H1 binding dramatically but have little or no influence on the charge distribution or the overall net charge of this domain. A comparison of individual point mutations and deletion mutants, when reviewed collectively, cannot be reconciled with simple charge-dependent mechanisms of C-terminal domain function of linker histones. Histone H1 is the fifth histone subtype and is not one of the histones that form the histone octamer of the nucleosome. Rather, histone H1 binds to the surface of the nucleosome and interacts with nucleosomal DNA at the entry and exit points (1Vignali M. Workman J.L. Nat. Struct. Biol. 1998; 5: 1025-1028Crossref PubMed Scopus (67) Google Scholar, 2Deleted in proofGoogle Scholar). In doing so, histone H1 is critical in determining the higher-order folding states of chromatin. Because of this property, histone H1 has traditionally been considered a general repressor of transcription (3Thomas J.O. Curr. Opin. Cell Biol. 1999; 11: 312-317Crossref PubMed Scopus (182) Google Scholar). Consistent with this hypothesis, histone H1 was found to be modestly depleted in transcriptionally active genes (4Kamakaka R.T. Thomas J.O. EMBO J. 1990; 9: 3997-4006Crossref PubMed Scopus (135) Google Scholar, 5Bresnick E.H. Bustin M. Marsaud V. Richard-Foy H. Hager G.L. Nucleic Acids Res. 1992; 20: 273-278Crossref PubMed Scopus (189) Google Scholar, 6Garrard W.T. Bioessays. 1991; 13: 87-88Crossref PubMed Scopus (45) Google Scholar). More recently, genetic studies have revealed contributions of H1 histones to the establishment of epigenetic silencing (7Jedrusik M.A. Schulze E. Development. 2001; 128: 1069-1080PubMed Google Scholar, 8Gabrilovich D.I. Cheng P. Fan Y. Yu B. Nikitina E. Sirotkin A. Shurin M. Oyama T. Adachi Y. Nadaf S. Carbone D.P. Skoultchi A.I. J. Leukoc. Biol. 2002; 72: 285-296PubMed Google Scholar, 9Jedrusik M.A. Schulze E. Mol. Cell. Biol. 2003; 23: 3681-3691Crossref PubMed Scopus (17) Google Scholar, 10Alami R. Fan Y. Pack S. Sonbuchner T.M. Besse A. Lin Q. Greally J.M. Skoultchi A.I. Bouhassira E.E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5920-5925Crossref PubMed Scopus (96) Google Scholar). In addition to a structural role, histone H1 also functions in gene-specific regulation. A large number of studies have demonstrated that H1 histones or specific variants are directly involved in the regulation of specific genes (3Thomas J.O. Curr. Opin. Cell Biol. 1999; 11: 312-317Crossref PubMed Scopus (182) Google Scholar, 11Folco H.D. Freitag M. Ramon A. Temporini E.D. Alvarez M.E. Garcia I. Scazzocchio C. Selker E.U. Rosa A.L. Eukaryot. Cell. 2003; 2: 341-350Crossref PubMed Scopus (36) Google Scholar, 12Koop R. Di Croce L. Beato M. EMBO J. 2003; 22: 588-599Crossref PubMed Scopus (67) Google Scholar, 13Takami Y. Nishi R. Nakayama T. Biochem. Biophys. Res. Commun. 2000; 268: 501-508Crossref PubMed Scopus (40) Google Scholar, 14Crane-Robinson C. Bioessays. 1999; 21: 367-371Crossref PubMed Scopus (36) Google Scholar), consistent with the observation of differential gene expression when the sole histone H1 gene was knocked out in Tetrahymena thermophila (15Shen X. Gorovsky M.A. Cell. 1996; 86: 475-483Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). The structure of H1 histones is typically considered to consist of three separate domains (16Allan J. Hartman P.G. Crane-Robinson C. Aviles F.X. Nature. 1980; 288: 675-679Crossref PubMed Scopus (536) Google Scholar). A short stretch of amino acids on the N terminus and a much larger stretch that comprises the C terminus show significant variability between individual subtypes as well as between species. The amino and carboxyl termini have diverged considerably throughout the evolution of metazoans (17Kasinsky H.E. Lewis J.D. Dacks J.B. Ausio J. FASEB J. 2001; 15: 34-42Crossref PubMed Scopus (183) Google Scholar). If we restrict the analysis to mammals, the C termini diverge between individual histone H1 variants, but the sequences of the individual C termini are well conserved between species. When histone H1 sequences are examined in a broader range of species, the centrally located region of the protein, the globular domain, is the most highly conserved region among H1 histone family members (18Ponte I. Vila R. Suau P. Mol. Biol. Evol. 2003; 20: 371-380Crossref PubMed Scopus (33) Google Scholar). The structure of the central region of the protein has been solved by x-ray crystallography (19Ramakrishnan V. Finch J.T. Graziano V. Lee P.L. Sweet R.M. Nature. 1993; 362: 219-223Crossref PubMed Scopus (665) Google Scholar) and is sufficient for binding to the nucleosome in vitro (20Vermaak D. Steinbach O.C. Dimitrov S. Rupp R.A. Wolffe A.P. Curr. Biol. 1998; 8: 533-536Abstract Full Text Full Text PDF PubMed Google Scholar). However, studies with reconstituted systems showed that the C-terminal domain (CTD) 1The abbreviations used are: CTD, C-terminal domain; GFP, green fluorescent protein; FRAP, fluorescence recovery after photobleaching. is required to condense chromatin into higher order structures (16Allan J. Hartman P.G. Crane-Robinson C. Aviles F.X. Nature. 1980; 288: 675-679Crossref PubMed Scopus (536) Google Scholar, 23Allan J. Mitchell T. Harborne N. Bohm L. Crane-Robinson C. J. Mol. Biol. 1986; 187: 591-601Crossref PubMed Scopus (269) Google Scholar). This is also consistent with the presence of only C-terminal-like domains in some protists. Because of the high density of positively charged amino acids within the CTD, it is commonly believed that condensation is mediated through charge-neutralization of the negatively charged linker DNA. In a recent study, Lu and Hansen (22Lu X. Hansen J.C. J. Biol. Chem. 2004; 279: 8701-8707Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) found that the ability of histone H1° to stabilize chromatin folding was not evenly distributed; rather, it was localized to two specific subdomains in the CTD. Because the density of positively charged lysine and arginine amino acids is very similar throughout the ∼100 amino acids of the C terminus, binding does not correlate in a simple manner with the abundance of positively charged amino acids within the domain. Lu and Hansen (22Lu X. Hansen J.C. J. Biol. Chem. 2004; 279: 8701-8707Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) proposed that histone H1 initially binds with low specificity in a charge-dependent manner. Upon binding to the DNA, the C terminus then acquires secondary structure. This feature of protein folding has been described as “intrinsic disorder.” This mechanism of histone H1 binding contrasts with the binding properties that would be expected if histone H1 were to function according to the “charge patch” hypothesis. The charge patch hypothesis proposes that the clustered positively charged lysines in the C-terminal domain bind DNA and facilitate condensation through neutralization of phosphates on the DNA (24Dou Y. Gorovsky M.A. Mol. Cell. 2000; 6: 225-231Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 25Dou Y. Bowen J. Liu Y. Gorovsky M.A. J. Cell Biol. 2002; 158: 1161-1170Crossref PubMed Scopus (80) Google Scholar). Although this mechanism of binding may apply to the evolutionarily divergent H1 of T. thermophila, recent structural studies of histone H1s from mammals indicate that regions within the C-terminal domain adopt an α-helical structure when associated with DNA (26Vila R. Ponte I. Collado M. Arrondo J.L. Suau P. J. Biol. Chem. 2001; 276: 30898-30903Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 27Vila R. Ponte I. Jimenez M.A. Rico M. Suau P. Protein Sci. 2000; 9: 627-636Crossref PubMed Scopus (41) Google Scholar, 28Vila R. Ponte I. Collado M. Arrondo J.L. Jimenez M.A. Rico M. Suau P. J. Biol. Chem. 2001; 276: 46429-46435Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Molecular modeling techniques also predict the adoption of secondary structure in the C terminus of histone H1 (21Bharath M.M. Chandra N.R. Rao M.R. Nucleic Acids Res. 2003; 31: 4264-4274Crossref PubMed Scopus (65) Google Scholar). More specifically, the modeling studies reveal that the C terminus may adopt an high mobility group-box-like structure, and that the C terminus SPKK motifs are sites of DNA binding and function in the compaction of the DNA (21Bharath M.M. Chandra N.R. Rao M.R. Nucleic Acids Res. 2003; 31: 4264-4274Crossref PubMed Scopus (65) Google Scholar). We and others have previously used fluorescence recovery after photobleaching (FRAP) to quantify the binding of histone H1 proteins in living cells (29Lever M.A. Th'ng J.P. Sun X. Hendzel M.J. Nature. 2000; 408: 873-876Crossref PubMed Scopus (355) Google Scholar, 30Misteli T. Gunjan A. Hock R. Bustin M. Brown D.T. Nature. 2000; 408: 877-881Crossref PubMed Scopus (526) Google Scholar, 31Contreras A. Hale T.K. Stenoien D.L. Rosen J.M. M.A. Mol. Cell. Biol. 2003; 23: PubMed Scopus Google Scholar). studies revealed that histone H1 binds to the chromatin of living In this study, we quantify the specific contributions of the and C-terminal domains of histone H1.1 as well as the motifs in the to the in chromatin binding of histone We find that the C-terminal domain of histone H1 a in the of histone H1 binding in vivo. Cell cells were in in the presence of The cells were according to the were for of the by for in the presence of and cells that the fluorescent histone H1.1 were and used in recovery after photobleaching was a as previously (29Lever M.A. Th'ng J.P. Sun X. Hendzel M.J. Nature. 2000; 408: 873-876Crossref PubMed Scopus (355) Google Scholar). the of at from three separate The are not but are typically for of histone H1.1 was described by (29Lever M.A. Th'ng J.P. Sun X. Hendzel M.J. Nature. 2000; 408: 873-876Crossref PubMed Scopus (355) Google Scholar). of and sites on histone H1.1 was by as described in the in Molecular for were by The sequences of the to mutations and and and and and and and The were into the and then into or of to the mutations were by Molecular and the in among Histone H1 conserved of of the histone H1 variants a central domain, a large carboxyl terminus in and and sites within the domain. The C-terminal domain, of the of the H1 protein, for most of the between histone H1 variants (18Ponte I. Vila R. Suau P. Mol. Biol. Evol. 2003; 20: 371-380Crossref PubMed Scopus (33) Google Scholar), and this domain was to be for high in binding to chromatin (29Lever M.A. Th'ng J.P. Sun X. Hendzel M.J. Nature. 2000; 408: 873-876Crossref PubMed Scopus (355) Google Scholar, 30Misteli T. Gunjan A. Hock R. Bustin M. Brown D.T. Nature. 2000; 408: 877-881Crossref PubMed Scopus (526) Google Scholar). the amino acid of the C-terminal domains of individual histone H1 subtypes of and subtype has to in histones and in the C-terminal and one at the N The specific lysines to bind and the DNA (21Bharath M.M. Chandra N.R. Rao M.R. Nucleic Acids Res. 2003; 31: 4264-4274Crossref PubMed Scopus (65) Google Scholar) are in the of in histone H1 binding in we to histone H1.1 histone and histone Biochem. Cell Biol. 2001; PubMed Scopus Google This H1 subtype has only two C-terminal reduces the of determining the of on histone H1 binding to chromatin binding in vivo. studies of histone binding to chromatin in vitro M.M. S. Chandra N.R. Rao M.R. 2002; PubMed Scopus Google Scholar) and a number of recent structural and modeling studies predict that the C-terminal domain of histone H1 is when to DNA and chromatin (26Vila R. Ponte I. Collado M. Arrondo J.L. Suau P. J. Biol. Chem. 2001; 276: 30898-30903Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 27Vila R. Ponte I. Jimenez M.A. Rico M. Suau P. Protein Sci. 2000; 9: 627-636Crossref PubMed Scopus (41) Google Scholar). The lysines by modeling (21Bharath M.M. Chandra N.R. Rao M.R. Nucleic Acids Res. 2003; 31: 4264-4274Crossref PubMed Scopus (65) Google Scholar) to with the DNA within the are as in In this the Thr-152 with a conserved in lysines are to binding DNA. The conserved at Ser-183 in histone H1.1 is amino acids from the two lysines as DNA in the If histone H1 a C terminus, the of or sites within the C terminus of histone H1.1 may have a much on histone H1 binding in vivo. The in histone H1.1 on chromatin after with the general is consistent with a of chromatin binding in living cells (29Lever M.A. Th'ng J.P. Sun X. Hendzel M.J. Nature. 2000; 408: 873-876Crossref PubMed Scopus (355) Google Scholar). to histone H1 structure and we to the specific contributions of individual regions of histone H1 protein to chromatin binding in living C-terminal the of Histone H1 recovery after photobleaching was used to the binding of green fluorescent protein histone H1 to chromatin in living We by measuring the fluorescence recovery of histones or C-terminal by to and by specific the recovery of histone H1.1 protein in The two of histone H1.1 with the to the chromatin. However, when the was on the C terminus, the recovery was when the was at the N terminus. The of binding by the C-terminal was almost as as that when the domain was Deletion of the N terminus the binding of the histone the C-terminal of the with a that when the was at the N terminus. When was to the recovery was by also When the C terminus was the H1.1 deletion protein not bind well and at on studies were with the histone H1.1 into the at the N terminus to of mutations to or glutamic acid on histone H1.1 measurements of the mobility of histone H1.1 Thr-152 and Ser-183 to glutamic acid or The the recovery of fluorescence after photobleaching for The histone H1.1 is as a for of C-terminal of Histone the of the C terminus to histone H1.1 binding in we a of to the contributions of the two C-terminal this C-terminal region of histone two sites that are by J.M. Finch J.T. Thomas J.O. EMBO J. 1991; PubMed Scopus Google Scholar). motifs are of the commonly SPKK is to bind DNA in vitro M. EMBO J. 8: PubMed Scopus Google Scholar). The that motifs be to a critical in DNA binding and histone condensation of the chromatin can be from of the structure of the C-terminal domain in with DNA and the C-terminal domain within the (21Bharath M.M. Chandra N.R. Rao M.R. Nucleic Acids Res. 2003; 31: 4264-4274Crossref PubMed Scopus (65) Google Scholar, M.M. S. Chandra N.R. Rao M.R. 2002; PubMed Scopus Google Scholar, M.M. Chandra N.R. Rao M.R. 2002; PubMed Scopus (33) Google Scholar). We examined the contributions of the individual regions of the C-terminal deletion of histone Deletion of amino acids in a protein that has binding and the region to be in the that this region of the to the binding to chromatin. C-terminal to lysine 151 in a protein that to chromatin with much and in of the required for the protein When recovery were the of of the deletion a recovery that as a In the deletion and the protein into two kinetic in This may the kinetic of two binding involved in the of the globular domain with the surface of the nucleosome. of Thr-152 and the deletion showed that the regions from Thr-152 and Ser-183 to the of the C-terminal domain the binding of histone H1.1 in not reveal the that the sites may in chromatin binding. the specific contributions of of of we H1.1 proteins in the Thr-152 was to or the Ser-183 was to to at the of on cells histone H1.1 The show that has a significant on binding of the histone H1.1 protein to chromatin. The recovery profiles of the deletion are in this to the of the in binding to the in the deletion of Ser-183 to glutamic acid binding properties very similar to of the that was at this This that this was not in DNA binding according to the modeling of (21Bharath M.M. Chandra N.R. Rao M.R. Nucleic Acids Res. 2003; 31: 4264-4274Crossref PubMed Scopus (65) Google Scholar), is located in a region in a disrupt DNA binding of the lysines that are C-terminal to The of a glutamic acid in has a much on the of histone H1.1 binding in does the It is that the of Thr-152 with glutamic acid histone H1.1 binding truncation of the C terminus at lysines and an The of of Thr-152 and Ser-183 on Histone H1.1 the of amino acids in the binding of histone H1.1 in we glutamic acid and the recovery profiles of Thr-152 and Ser-183 to glutamic acid or the single point mutations to glutamic acid are also in this It is remarkable that the of amino acids to glutamic acid a histone H1.1 protein with a recovery almost to that of the single glutamic acid substitution at amino acid The of Thr-152 and Ser-183 to would and this would the mobility of histone if functions to disrupt binding by charge When the recovery of histone H1 mutations was we a of histone H1.1 binding in to the histone The histone H1.1 has a binding that is between the Ser-183 glutamic acid and the histone H1.1 of Thr-152 and Ser-183 the of of Histone the addition of to the highly C terminus a influence on the binding of histone H1.1 to chromatin in living cells, we Thr-152 or Ser-183 with lysines and the binding of histone H1.1 that the of a lysine at the of histone H1.1 binding in vivo. This is consistent with the that overall net charge of regions is in histone H1.1 binding. However, when are the protein with single the recent of proteins for in FRAP, studies functions to the structure of histone H1 in chromatin been to in vitro reconstituted histone H1.1 and variants, we have the of the and domains to the binding of histone H1.1 to chromatin in living The of is that the C-terminal domain of histone H1 is for high-affinity binding of histone H1 to chromatin in and that high-affinity binding can be directly by at Thr-152 and a to a recent in vitro of the functions of the histone H1 C terminus. a of reconstituted Lu and Hansen (22Lu X. Hansen J.C. J. Biol. Chem. 2004; 279: 8701-8707Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) have that specific regions of the histone H1 C terminus function in folding reconstituted nucleosomal in studies that deletion of of the C-terminal domain the binding of histone H1 for chromatin. studies the hypothesis that the C terminus functions as a that on the DNA and of the influence of secondary structure. Lu and Hansen (22Lu X. Hansen J.C. J. Biol. Chem. 2004; 279: 8701-8707Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) that chromatin binding was not evenly the CTD. a the of the C terminus not affect histone H1 binding in However, the amino acid on of this are for the of the chromatin when we examined the binding of histone H1.1 in living cells, we that the single substitution of Thr-152 with glutamic acid the of histone H1 binding to a deletion of the C terminus at a region that of the positively charged amino acids found in the C-terminal domain. studies also the two of the C termini that motifs as regions of two histone H1 It is to however, that studies variants with only two studies are consistent with DNA binding to motifs within the C terminus, we cannot that the motifs are directly involved in DNA binding in vivo. studies with histone H1 variants be required to the specific of motifs commonly found in H1 C We can however, that the sites within the C terminus of histone H1.1 can directly the of histone H1 binding in vivo. We find that of the two C-terminal sites to glutamic acid in a significant of histone H1.1 binding in vivo. Although this is in general with the of A. Hale T.K. Stenoien D.L. Rosen J.M. M.A. Mol. Cell. Biol. 2003; 23: PubMed Scopus Google Scholar), showed that the of the and sites to the binding to chromatin, to that mutations of sites chromatin binding. This may be by of a C-terminal with that we have to the of histone H1 binding to chromatin studies have that the is critical to the binding of linker histones and that this domain a secondary structure for function as an the studies the was localized at the N terminus, with chromatin binding was studies showed that the domain have two the that is to the central globular domain a α-helical structure when associated with DNA. The of the N terminus of histone the is is and does not bind to the chromatin R. Ponte I. Collado M. Arrondo J.L. Jimenez M.A. Rico M. Suau P. J. Biol. Chem. 2001; 276: 46429-46435Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, R. Ponte I. Jimenez M.A. Rico M. Suau P. Protein Sci. 2002; 11: PubMed Scopus Google Scholar). the N terminus may be as a to the from regions directly involved in chromatin binding. Although are consistent with a for the sites in we find that the two C-terminal sites in histone H1.1 in the to affect binding. The contributions of subdomains of the to linker histone binding was demonstrated when mutations of the individual Thr-152 and Ser-183 showed on Rather, of Thr-152 to glutamic acid the binding of histone H1.1 to the as mutations of sites to glutamic would not be expected if the C-terminal domain were to with DNA on the of In the addition of two from the of two would not be sufficient to the net of the lysine and arginine found within the CTD. Rather, the of the Thr-152 in the of histone H1.1 binding in is consistent with the of this specific conserved in the secondary structure of histone H1 within the and consistent with the recent in vitro of Lu and Hansen (22Lu X. Hansen J.C. J. Biol. Chem. 2004; 279: 8701-8707Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). In this may be to histone H1 from chromatin much of the H1 are Bioessays. 1992; PubMed Scopus Google Scholar). In we have that the C terminus is critical for high-affinity binding of histone H1.1 to chromatin. In the of the CTD, the globular and N terminus does not bind well to chromatin. studies have traditionally the of the globular domain of the H1 histones. The of the globular domain with DNA and have been in vitro (1Vignali M. Workman J.L. Nat. Struct. Biol. 1998; 5: 1025-1028Crossref PubMed Scopus (67) Google Scholar, J.O. Curr. Opin. Cell Biol. 1999; 11: 312-317Crossref PubMed Scopus (182) Google Scholar). The of the C terminus in the of histone H1 binding in that the among individual histone H1 variants in in chromatin binding in living Consistent with this hypothesis, we in the of binding of individual histone H1 variants when are used to H1 binding in vivo. P. H. Th'ng and M. J. in In of and recent on the of the C terminus in chromatin binding and folding in vitro (1Vignali M. Workman J.L. Nat. Struct. Biol. 1998; 5: 1025-1028Crossref PubMed Scopus (67) Google Scholar, J.O. Curr. Opin. Cell Biol. 1999; 11: 312-317Crossref PubMed Scopus (182) Google Scholar), the C terminus is as critical to the function of H1 histones in vivo.

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