H1 Family Histones in the Nucleus

H1 histones bind to DNA as they enter and exit the nucleosome. H1 histones have a tripartite structure consisting of a short N-terminal domain, a highly conserved central globular domain, and a lysine-and arginine-rich C-terminal domain. The C-terminal domain comprises approximately half of the total amino acid content of the protein, is essential for the formation of compact chromatin structures, and contains the majority of the amino acid variations that define the individual histone H1 family members. This region contains several cell cycle-regulated phosphorylation sites and is thought to function through a charge-neutralization process, neutralizing the DNA phosphate backbone to allow chromatin compaction. In this study, we use fluorescence microscopy and fluorescence recovery after photobleaching to define the behavior of the individual histone H1 subtypes in vivo. We find that there are dramatic differences in the binding affinity of the individual histone H1 subtypes in vivo and differences in their preference for euchromatin and heterochromatin. Further, we show that subtype-specific properties originate with the C terminus and that the differences in histone H1 binding are not consistent with the relatively small changes in the net charge of the C-terminal domains. H1 histones bind to DNA as they enter and exit the nucleosome. H1 histones have a tripartite structure consisting of a short N-terminal domain, a highly conserved central globular domain, and a lysine-and arginine-rich C-terminal domain. The C-terminal domain comprises approximately half of the total amino acid content of the protein, is essential for the formation of compact chromatin structures, and contains the majority of the amino acid variations that define the individual histone H1 family members. This region contains several cell cycle-regulated phosphorylation sites and is thought to function through a charge-neutralization process, neutralizing the DNA phosphate backbone to allow chromatin compaction. In this study, we use fluorescence microscopy and fluorescence recovery after photobleaching to define the behavior of the individual histone H1 subtypes in vivo. We find that there are dramatic differences in the binding affinity of the individual histone H1 subtypes in vivo and differences in their preference for euchromatin and heterochromatin. Further, we show that subtype-specific properties originate with the C terminus and that the differences in histone H1 binding are not consistent with the relatively small changes in the net charge of the C-terminal domains. The H1 or “linker” histones are a family of very lysine-rich proteins that associate with the stretch of DNA that enters and exits the nucleosome. In the absence of histone H1, the nucleosome comprises ∼146 bp of DNA that wraps 1.75 turns around the outer surface of the histone octamer (1Whitlock Jr., J.P. Simpson R.T. Biochemistry. 1976; 15: 3307-3314Crossref PubMed Scopus (125) Google Scholar, 2Allan J. Hartman P.G. Crane-Robinson C. Aviles F.X. Nature. 1980; 288: 675-679Crossref PubMed Scopus (526) Google Scholar, 3Furrer P. Bednar J. Dubochet J. Hamiche A. Prunell A. J. Struct. Biol. 1995; 114: 177-183Crossref PubMed Scopus (52) Google Scholar). In the presence of histone H1, the resulting chromatosome contains two complete turns of 168-bp DNA. Histone H1, which is in an ∼1:1 ratio with the number of nucleosomes in the cell, bends and alters the path of the DNA entering and exiting the nucleosome such that the nucleosome adopts a lollipop-like conformation (4Bednar J. Horowitz R.A. Grigoryev S.A. Carruthers L.M. Hansen J.C. Koster A.J. Woodcock C.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14173-14178Crossref PubMed Scopus (452) Google Scholar, 5Sivolob A. Prunell A. J. Mol. Biol. 2003; 331: 1025-1040Crossref PubMed Scopus (27) Google Scholar). This change in the trajectory of the linker DNA defines the first step in folding the polynucleosome chain into interphase chromosomes. The discovery of the H1 histones and the identification of its seven variants in the mid 1970s suggested possible roles in development (6Lennox R.W. J. Biol. Chem. 1984; 259: 669-672Abstract Full Text PDF PubMed Google Scholar). An additional variant, referred to as H1oo, was recently isolated from the oocytes of mice (7Tanaka M. Hennebold J.D. Macfarlane J. Adashi E.Y. Development (Camb.). 2001; 128: 655-664Crossref PubMed Google Scholar). These studies revealed associations between histone H1 subtypes in cell growth and differentiation (8Lennox R.W. Cohen L.H. J. Biol. Chem. 1983; 258: 262-268Abstract Full Text PDF PubMed Google Scholar, 9Lennox R.W. Cohen L.H. Dev. 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Differentiation. 1977; 7: 167-180Crossref PubMed Scopus (69) Google Scholar, 14Moorman A.F. de Boer P.A. Charles R. Lamers W.H. Differentiation. 1987; 35: 100-107Crossref PubMed Scopus (22) Google Scholar, 15Lennox R.W. Dev. Biol. 1986; 118: 319-323Crossref PubMed Scopus (4) Google Scholar). In simplistic terms, variants that have been associated with cellular differentiation are the histone H5 and histone H1.0 subtypes, and they are closer in amino acid sequences and most divergent from the other somatic histone H1 variants. Histone H5 is restricted to amphibian and reptile species, where it is found in high abundance in the nucleated but transcriptionally inert erythrocytes (16Parseghian M.H. Hamkalo B.A. Biochem. Cell Biol. 2001; 79: 289-304Crossref PubMed Scopus (106) Google Scholar, 17Doenecke D. Albig W. Bode C. Drabent B. Franke K. Gavenis K. Witt O. Histochem. Cell Biol. 1997; 107: 1-10Crossref PubMed Scopus (96) Google Scholar). The nuclei of these mature erythrocytes are almost entirely heterochromatic, allowing them to maintain a very small total volume. In this case, histone H5 expression correlates with the cessation of the RNA polymerase II transcriptional program (18Andreeva N.B. Vishnevskaia T. Gazarian K.G. Mol. Biol. 1978; 12: 123-134Google Scholar, 19Bergman M.G. Wawra E. Winge M. J. Cell Sci. 1988; 91: 201-209Crossref PubMed Google Scholar, 20Doenecke D. Tonjes R. Kress H. Adv. Enzyme Regul. 1988; 27: 107-120Crossref PubMed Scopus (1) Google Scholar). It is thought that histone H5 functions to transcriptionally inactivate the genome as part of the terminal differentiation program of the erythrocyte (19Bergman M.G. Wawra E. Winge M. J. Cell Sci. 1988; 91: 201-209Crossref PubMed Google Scholar, 21Sun J.M. Wiaderkiewicz R. Ruiz-Carrillo A. Science. 1989; 245: 68-71Crossref PubMed Scopus (58) Google Scholar). Histone H1.0 is the mammalian homolog and is closer in structure to H5 than the other H1 variants (22Schulze E. Schulze B. J. Mol. Evol. 1995; 41: 833-840Crossref PubMed Scopus (27) Google Scholar). Early studies showed that its abundance is increased in cells that are quiescent or terminally differentiated (14Moorman A.F. de Boer P.A. Charles R. Lamers W.H. Differentiation. 1987; 35: 100-107Crossref PubMed Scopus (22) Google Scholar). Consistent with this is a recent study showing that overexpression of histone H1.0 can slow cell cycle progression and repress gene expression (23Brown D.T. Alexander B.T. Sittman D.B. Nucleic Acids Res. 1996; 24: 486-493Crossref PubMed Scopus (106) Google Scholar). Logan et al. (24Logan K.A. Dahmus M.E. Bradbury E.M. J. Biol. Chem. 1988; 263: 9658-9662Abstract Full Text PDF PubMed Google Scholar) showed that histone H1.0 and RNA polymerase II shared a common antigenic epitope and suggested that H1.0 may compete for similar regions of the chromatin with RNA polymerase II. There are six histone H1 variants in the somatic cells in mammals, and they have a tripartite structure consisting of a central globular domain flanked by tail domains. The structure of the globular domain has been solved by X-ray crystallography (25Ramakrishnan V. Finch J.T. Graziano V. Lee P.L. Sweet R.M. Nature. 1993; 362: 219-223Crossref PubMed Scopus (661) Google Scholar), and this domain has routinely been used for studies on reconstituted nucleosomes and chromatin (26Chan D.C. Biard-Roche J. Gorka C. Girardet J.L. Lawrence J.J. Piette L.I. J. Biomol. Struct. Dyn. 1984; 2: 319-332Crossref PubMed Scopus (4) Google Scholar, 27Hayes J.J. Pruss D. Wolffe A.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7817-7821Crossref PubMed Scopus (75) Google Scholar, 28Vermaak 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, 29Thomas J.O. Curr. Opin. Cell Biol. 1999; 11: 312-317Crossref PubMed Scopus (181) Google Scholar). Although there are variations in the amino acid sequences, structural studies showed that the globular domain of histone H1.0 adopts a secondary structure that is similar to those of the other variants. The major differences between the variants lie in the flanking domains. Early studies done to define the functions of these domains using reconstituted chromatin showed that the N terminus was not required for inducing higher order structures (30Allan J. Mitchell T. Harborne N. Bohm L. Crane-Robinson C. J. Mol. Biol. 1986; 187: 591-601Crossref PubMed Scopus (263) Google Scholar). In contrast, the C terminus was required, in addition to the globular domain, for H1-dependent chromatin folding but was not sufficient for the full nuclease protection afforded the nucleosome upon H1 binding (30Allan J. Mitchell T. Harborne N. Bohm L. Crane-Robinson C. J. Mol. Biol. 1986; 187: 591-601Crossref PubMed Scopus (263) Google Scholar). Several recent studies have also revealed the prominent role that the C-terminal domain (CTD) 1The abbreviations used are: CTD, C-terminal domain; FRAP, fluorescence recovery after photobleaching; eGFP, enhanced green fluorescent protein. plays in determining the binding properties of histone H1s in vivo (31De S. Brown D.T. Lu Z.H. Leno G.H. Wellman S.E. Sittman D.B. Gene (Amst.). 2002; 292: 173-181Crossref PubMed Scopus (38) Google Scholar, 32Lever M.A. Th'ng J.P. Sun X. Hendzel M.J. Nature. 2000; 408: 873-876Crossref PubMed Scopus (354) Google Scholar, 33Hendzel M.J. Lever M.A. Crawford E. Th'ng J.P. J. Biol. Chem. 2004; 279: 20028-20034Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 34Lu X. Hansen J.C. J. Biol. Chem. 2004; 279: 8701-8707Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) However, studies comparing the binding properties of the variants have been limited by the low sensitivity of the in vitro methods for assaying H1 binding. We have previously shown that fluorescence recovery after photobleaching (FRAP) has the sensitivity to detect subtle differences in binding affinities of histone H1 in live cells (33Hendzel M.J. Lever M.A. Crawford E. Th'ng J.P. J. Biol. Chem. 2004; 279: 20028-20034Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar), and we have used it here to examine the role of the CTD in affecting the binding affinities of individual variants. The C-terminal domain makes up half of the linker histone molecule, and the distinct features (e.g. the number of lysine residues and the (S/T)PXK phosphorylation motifs) of this domain in each variant are highly conserved between species. However, these features vary between the individual variants (35Ponte I. Vidal-Taboada J.M. Suau P. Mol. Biol. Evol. 1998; 15: 702-708Crossref PubMed Scopus (68) Google Scholar). Therefore, it is likely that the the mammalian subtypes of histone H1 in differences in the C-terminal domain. to the histone proteins that the nucleosome H1 histones have relatively low binding affinity to chromatin and have short This has been in vitro and in vivo A.P. Hansen J.C. 2001; Full Text Full Text PDF PubMed Google Scholar). H1 proteins in or cell it is possible to this the nuclei of live cells G. Crawford E. Th'ng J. de G. Hendzel M.J. 2004; PubMed Scopus Google Scholar). and using histone showed that a phosphorylation regions the C-terminal domain can the of the histone (33Hendzel M.J. Lever M.A. Crawford E. Th'ng J.P. J. Biol. Chem. 2004; 279: 20028-20034Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). This is consistent with those using histone H1.0 on reconstituted chromatin X. Hansen J.C. J. Biol. Chem. 2004; 279: 8701-8707Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), that the from these in vitro can to the of the of the C terminus of histone revealed that this domain plays a role in linker histone the using reconstituted This an for the of the C-terminal sequences of each variant of the C terminus each species. the that the binding and the function of H1 histones in we the binding properties of the six somatic mammalian subtypes in cells and the of the C-terminal domain. We showing that the individual histone variants vary with to their in chromatin and that they have distinct in the This study, a for the differences between individual histone H1 subtypes and on these differences can cellular of Histone H1 of histone H1 variants by Lever et al. M.A. Th'ng J.P. Sun X. Hendzel M.J. Nature. 2000; 408: 873-876Crossref PubMed Scopus (354) Google Scholar), using by from sequences from The and the sequences for into The for histone H1.0 number the and histone number and histone number the and histone number the and histone number the and histone number the and histone by terminal amino acid was used as the was with DNA polymerase using DNA from cells as and the into with and and to restricted of the C-terminal the used for histone CTD was and the for histone was and the was those used for of the individual variants. The for into and These used as to the to the between Histone H1 domain between histone H1 the N-terminal half the N-terminal and globular domains using the for each of the variants and the was to the of the highly conserved globular domain. The used to the C-terminal domain has the and contains a stretch of that is to the use for the globular domain. The used to the CTD the used to the histone variants. The N-terminal and C-terminal from the individual and and for using the and for the individual histone variants that to The and into as by the to the after with individual histone H1 using and cells by in in cells number in a small of by a with on a region the of the cell was and recovery was and to G. Crawford E. Th'ng J. de G. Hendzel M.J. 2004; PubMed Scopus Google Scholar) between Histone H1 proteins of histone H1 subtypes as as a that is into as cells enter or a of the C-terminal domain the differences and where structure or function have been define the roles by the in each of the we proteins of histone H1 subtypes as as The was to the N terminus of each histone H1 variant of that to the C terminus binding of histone H1 to as the CTD contains the variations between the individual Histone and have the C-terminal histones and have the C-terminal domains. of the C-terminal domain where functions have been are with in Although these domains are very each variant contains a of and that net on the C The CTD of histone has the with and histones amino and and amino have the number of amino is histone the it also from the subtypes in two of the conserved phosphorylation histone H1.0 is the of the linker with a CTD of amino but contains the of amino with and It also has than histone of Histone H1 in has to a for the chromatin binding properties of histone H1 and for to We have previously used this to an of histone binding to chromatin through a amino acid This consistent with a recent in vitro study on the of the C-terminal tail of histone H1.0 and histone is with the of histone that is on on this we that the small but differences in the six somatic cell H1 histones in distinct binding properties in vivo. of comparing the recovery of fluorescence for each of the six major somatic H1 The H1 in show binding properties in vivo. and which are by the C-terminal binding and to histone H1.0 has in amino acid and the highly divergent C-terminal domain is the of the H1 and histone H1.0 to several to in the It is with the H1 histones that have C-terminal which These and as as to after the photobleaching we the recovery of cells and the individual histone H1 subtypes for differences in it can that the histone H1 subtypes a of recovery The that the histone H1 variants to chromatin with affinities in vivo. the recovery for histone H1 variants in histone and histone to chromatin with the and this affinity increased with CTD of histones and We the of the differences in recovery required for and this the H1 variants into major binding and each binding properties distinct from other histone H1 variants. Histone H1.0 and histone showed differences that not from each other but from the histone H1 variants. and that similar to each other but from H1.0 and This that the of the CTD of histone have a major on the binding to Although histone H1.0 has the CTD, the binding studies that other variations in the of the may to its increased binding such as the higher of lysine and also the and of the The of C-terminal on in the that the C-terminal domain defines the in vivo binding properties of the histone H1 subtypes, we domain between histone the histone H1 with the binding and histones and the subtypes with the binding that the consisting of the N terminus and globular domains of histone to the CTD of histone to than the which of the N terminus and globular domains of histone to the CTD of histone The of the C-terminal domain of or with that of the of required for the to to that for histone the C-terminal domain of histone was with the C-terminal domain of histone or histone the of required for the to photobleaching was the proteins are also of the C in Histone of the differences between the binding histone H1 and the binding histone and is the of the C This to a in the net charge comparing with for there is change in the number of sites previously as DNA binding domains. Therefore, we this of the C terminus a in the binding affinity of the protein. the recovery of and histone The C-terminal of histone in a in the required for the to in the of Histone H1 the histone H1 subtypes have been to have in the these differences not C-terminal histones used in a study of H1 we these with H1 histone cell have distinct and regions in their nuclei and used to or not these H1 subtypes in they the The of each H1 histone with is to cells they are as of Although subtypes in we distinct differences in the of the individual H1 The most is histone which was found to in quiescent and differentiated cells and has the number of changes with the other somatic subtypes a of a where H1.0 is with in a cell The DNA by is that regions of with the green fluorescent histones as The green fluorescent histone H1.0 to in the euchromatin regions of the a domain is in histone the to of histone The other subtypes of linker histones also showed distinct through the The green fluorescence showed that histones are found in euchromatin histones and are in regions of the the for cells for each H1 histone of cells showing in fluorescence in distinct chromatin of chromatin are the chromatin the the regions of of regions regions where is as chromatin or domains and regions where is but distinct chromatin structures are to in a The between and individual variants is higher than the between individual variants each (16Parseghian M.H. Hamkalo B.A. Biochem. Cell Biol. 2001; 79: 289-304Crossref PubMed Scopus (106) Google Scholar, 17Doenecke D. Albig W. Bode C. Drabent B. Franke K. Gavenis K. Witt O. Histochem. Cell Biol. 1997; 107: 1-10Crossref PubMed Scopus (96) Google Scholar). It a study comparing the in vivo binding of histone H1 subtypes and the homolog of histone to a between the two T. A. R. M. Brown D.T. Nature. 2000; 408: PubMed Scopus Google Scholar). This has been in where differences between histone H1 subtypes has been shown for binding and the to into structures H. N. W. H. B. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). This differences between the histone H1 subtypes, in the of development of a for differences between histone H1 variants is to and the that subtypes are not for may to an on the of to to and histone H1.0 was as it has recently been that there is a in cell In this study, we have the in vivo binding properties of histone H1 variants to or not these variants in their binding in study this using histone H1 subtypes T. A. R. M. Brown D.T. Nature. 2000; 408: PubMed Scopus Google Scholar). of this study was the use of C-terminal We have recently that C-terminal of to histone H1 the binding of this in vivo (33Hendzel M.J. Lever M.A. Crawford E. Th'ng J.P. J. Biol. Chem. 2004; 279: 20028-20034Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). This is by the differences between histone H1 variants that are in these which use of N-terminal We found that to the N terminus has on chromatin most likely studies showed that the of the N-terminal domain is and not bind chromatin R. I. M. J.L. M.A. M. Suau P. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). we the between histones and those of and this domain a the binding of the the recovery of the histone that the This that the N-terminal domain not on the binding to of the recovery of the histone H1 variants suggested a between the of the and binding the globular domains are this is with the C-terminal domain. However, this was not with the variant histone we the content of the residues in the CTD, histone H1.0 has the residues in amino than the binding histones residues in amino and residues in amino The presence of of the phosphorylation sites in histone and that these have on the binding The to histone H1 binding of these amino and the are the of the C terminus of histone to the of histone in a in the binding of the protein. These that the are not the of the affinity of histone H1 binding. Histone is the most histone in most cell and of the subtypes, with histone This may histone a very for in the DNA. Histone has recently been as a that is required for the of the of A. S. J. T. L. 2003; 114: Full Text Full Text PDF PubMed Scopus Google Scholar). Histone H1.0 has been the most histone H1 variant from mammalian species. and in terminally differentiated and has to the that it is in the terminal differentiation It was recently that histone H1.0 is required in the terminal differentiation of cells P. B. E. A. M. T. S. J. Biol. 2002; Google Scholar). H1.0 mice are in this cell We find that histone H1.0 to chromatin with an is its preference for regions of the genome and its in heterochromatin. in regions of the that it is in the of and regions of the with cells that it may function to repress expression of that are required, such as those associated with cell cycle In the of study show that the C-terminal which the linker histone H1 variants from each also binding to the This domain was shown to for chromatin in studies using reconstituted (30Allan J. Mitchell T. Harborne N. Bohm L. Crane-Robinson C. J. Mol. Biol. 1986; 187: 591-601Crossref PubMed Scopus (263) Google Scholar), and we have this using live cells (33Hendzel M.J. Lever M.A. Crawford E. Th'ng J.P. J. Biol. Chem. 2004; 279: 20028-20034Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). The of these regions is by the between the and sequences in the of the lysine residues and the phosphorylation These differences in the C terminus most likely the functions of the variants in mammalian cells and their distinct roles in the development of

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