The primary structure of a minor isoform (H1.2) of histone H1 from the nematode Caenorhabditis elegans.

The complete amino acid sequence of a minor isoform (H1.2) of histone H1 from the nematode Caenorhabditis elegans was determined. The amino acid chain consists of 190 residues and has a blocked N-terminus. Histone subtype H1.2 is 17 residues shorter than the major isoform H1.1, mainly as the result of deletions of short peptide fragments. Considerable divergence from isoform H1.1 has occurred in the N-terminal domain and the very C-terminus of the molecule, but the central globular domain and most of the C-terminal domain, including two potential phosphorylation sites, have been well conserved. Secondary-structure predictions for both H1 isoforms reveal a high potential for helix formation in the N-terminal region 1-33 of isoform H1.1 whereas the corresponding region in isoform H1.2 has low probability of being found in alpha-helix. No major differences in secondary structure are predicted for other parts of both H1 subtypes. The aberrant conformation of isoform H1.2 may be indicative of a significantly different function.     

Histone H3 N-terminal mutations allow hyperactivation of the yeast GAL1 gene in vivo.

Recent work has shown that the yeast histone H4 N-terminus, while not essential for viability, is required for repression of the silent mating loci and activation of GAL1 and PHO5 promoters. Because histone H3 shares many structural features with histone H4 and is intimately associated with H4 in the assembled nucleosome, we asked whether H3 has similar functions. While the basic N-terminal domain of H3 is found to be non-essential (deletion of residues 4-40 of this 135 amino acid protein allows viability), its removal has only a minor effect on mating. Surprisingly, both deletions (of residues 4-15) and acetylation site substitutions (at residues 9, 14 and 18) within the N-terminus of H3 allow hyperactivation of the GAL1 promoter as well as a number of other GAL4-regulated genes including GAL2, GAL7 and GAL10. To a limited extent glucose repression is also alleviated by H3 N-terminal deletions. Expression of another inducible promoter, PHO5, is shown to be relatively unaffected. We conclude that the H3 and H4 N-termini have different functions in both the repression of the silent mating loci and in the regulation of GAL1.     

Differential distribution of lysine and arginine residues in the closely related histones H1 and H5. Analysis of a human H1 gene

A human H1 histone gene and its flanking sequences were isolated from a human gene library using a fragment of the duck H5 histone gene as a hybridization probe. The primary structure of this human H1 histone (as deduced from the nucleotide sequence of the gene) reveals a close homology of H1 and H5 histones and fits the three-domain organization of all members of the H1 histone family. Within this protein organization, the C-terminal domain of H1 differs from the arginine-rich H5 in its distribution of the basic amino acids: the C-terminal domain of the human H1 shows only one arginine and most of the H5 specific arginine positions show lysine instead.     

The primary structure of histone H3 from the nematode Caenorhabditis elegans

The complete amino acid sequence of histone H3 (135 residues) from the nematode Caenorhabditis elegans has been established. Microheterogeneity occurs at positions 96 and 100 of the chain. The sequences of the nematode H3 isoforms are very similar to the major chain of calf thymus H3 with which they show 4 substitutions in total. The major variant has cysteine in position 96. This is the first report of cysteine in this position in H3 from non-mammalian tissue. An exceptional methylation site has been detected at position 79. Various other sites of secondary modification are of a conservative nature.     

Precise elimination of the N-terminal domain of histone H1.

The proteinase from mouse submaxillary gland was used to cleave total calf thymus histone H1 between residues 32 and 33. The C-terminal peptide, comprising residues 33 to the C-terminus, was purified and identified by amino acids analysis and Edman degradation. Spectroscopic characterization by n.m.r. for tertiary structure and by c.d. for secondary structure shows the globular domain of the parent histone H1 to be preserved intact in the peptide. It has therefore lost only the N-terminal domain and is a fragment of histone H1 comprising the globular plus C-terminal domains only. Precise elimination of only the N-terminal domain makes the fragment suitable for testing domain function in histone H1.     

Mutations in both the structured domain and N-terminus of histone H2B bypass the requirement for Swi-Snf in yeast.

The chromatin elements targeted by the ATPdependent, Swi-Snf nucleosome-remodeling complex are unknown. To address this question, we generated mutations in yeast histone H2B that suppress phenotypes associated with the absence of Swi-Snf. Sin- (Swi-Snf-independent) mutations occur in residues involved in H2A-H2B dimer formation, dimer- tetramer association, and in the H2B N-terminus. The strongest and most pleiotropic Sin- mutation removed 20 amino acid residues from the H2B N-terminus. This mutation allowed active chromatin to be formed at the SUC2 locus in a snf5Delta mutant and resulted in hyperactivated levels of SUC2 mRNA under inducing conditions. Thus, the H2B N-terminus may be an important target of Swi-Snf in vivo. The GCN5 gene product, the catalytic subunit of several nuclear histone acetytransferase complexes that modify histone N-termini, was also found to act in conjunction with Swi-Snf. The phenotypes of double gcn5Deltasnf5Delta mutants suggest that histone acetylation may play both positive and negative roles in the activity of the Swi-Snf-remodeling factor.     

Identification of a non-basic domain in the histone H4 N-terminus required for repression of the yeast silent mating loci.

We have shown previously that a stretch of four charged residues (16-19) at the histone H4 N-terminus is involved in repression of the yeast silent mating loci. One of these residues, Lys16, is a site for acetylation, which may prevent repression of the silent mating loci. In this paper we ask whether other sequences in histone H4, possibly in conjunction with H3 residues, are required for repression. We find that even in combination, the other seven acetylatable lysines in H3 and H4 do not function in repression. In contrast, we have found that an adjacent relatively uncharged domain (residues 21-29) is required for repression and that single amino acid insertions and deletions in this region are extremely detrimental. We propose that the basic and non-basic domains together form a DNA (or protein) induced amphipathic alpha-helix required in the formation of a repressive chromatin structure.     

Studies on the role and mode of operation of the very-lysine-rich histone H1 in eukaryote chromatin. The isolation of the globular and non-globular regions of the histone H1 molecule.

Digestion of calf thymus H1 histone with thrombin cleaves the molecule at the sequence -(Pro)-Lys-Lys-Ala-, corresponding to a point approximately 122 residues from the N-terminus (about 56% along the molecule). The N-terminal fragment is shown by proton nuclear magnetic resonance (NMR) to possess the globular structure of the intact histome H1 molecule, whereas the C-terminal fragment appears to possess little or no structure. The N-terminal fragment separates into two peaks on an ion-exchange column, one of which is shown to originate from a single subfraction of calf thymus histone H1 and the other to originate from the other subfractions, by detailed comparison of the NMR spectra. It thus seems that the structure of the H1 histone in solution under physiological conditions consists of a globular head with a highly basic random coil tail. It is suggested that the globular head has a specific binding site on the subunit structure of the chromosome.     

Localization of lysine residue in histone H3 forming the thymine-lysine cross-link after UV-irradiation of deoxyribonucleoprotein

A thymine-modified derivative of histone H3 formed as a result of thermal treatment of UV-irradiated (lambda = 254 nm) solution of deoxyribonucleoprotein from calf thymus at low ionic strength was isolated. The peptides obtained by tryptic hydrolysis of modified histone H3 were separated by high pressure liquid chromatography. The amino acid sequence of the peptide containing a lysine residue with covalently linked thymine was determined by the Edman method. It was found that Lys localized at the N-terminus of the histone H3 molecule interacts with DNA within the composition of the deoxyribonucleoprotein.     

The primary structure of histone H4 from the nematode Caenorhabditis elegans

1. The complete amino acid sequence of histone H4 from the nematode Caenorhabditis elegans has been established. 2. The polypeptide chain consists of 102 amino acids and has a completely alpha-N-blocked serine at residue 1. 3. The sequence differs from vertebrate H4 in position 73 by substitution of cysteine for threonine. 4. Lysine in position 20 is monomethylated.     

dBigH1, a second histone H1 in Drosophila,and the consequences for histone fold nomenclature

Recently, Pérez-Montero and colleagues (Developmental cell, 26: 578–590, 2013) described the occurrence of a new histone H1 variant (dBigH1) in Drosophila. The presence of unusual acidic amino acid patches at the N-terminal end of dBigH1 is in contrast to the arginine patches that exist at the N- and C-terminal domains of other histone H1-related proteins found in the sperm of some organisms. This departure from the strictly lysine-rich composition of the somatic histone H1 raises a question about the true definition of its protein members. Their minimal essential requirements appear to be the presence of a lysine- and alanine–rich, intrinsically disordered C-terminal domain, with a highly helicogenic potential upon binding to the linker DNA regions of chromatin. In metazoans, specific targeting of these regions is further achieved by a linker histone fold domain (LHFD), distinctively different from the characteristic core histone fold domain (CHFD) of the nucleosome core histones.     

Identification of a 34 amino acid stretch within the C-terminus of histone H1 as the DNA-condensing domain by site-directed mutagenesis

The C-terminus of histone H1 is necessary for the folding of polynucleosomal arrays into higher-order structure(s) and contains octapeptide repeats each having DNA binding S/TPKK motifs. These repeat motifs were earlier shown to mimic the DNA/chromatin-condensing properties of the C-terminus of histone H1 (Khadake, J. R., and Rao, M. R. S. (1995) Biochemistry 36, 1041-1051). In the present study, we have generated a series of C-terminal mutants of rat histone H1d and studied their DNA-condensation properties. The single proline to alanine mutation in the S/TPKK motifs either singly or in combination resulted in only a 20% decrease in the DNA-condensation property of histone H1. Deletion of all the three S/TPKK motifs resulted in a 45% decrease in DNA condensation. When the three octapeptide repeats encompassing the S/TPKK motifs were deleted, there was again a 45% decrease in DNA condensation. On the other hand, when the entire 34 amino acid stretch (residue 145-178) was deleted, there was nearly a 90% decrease in DNA condensation brought about by histone H1d. Interestingly, deletion of the 10 amino acid spacer between the octapeptide repeats (residues 161-170) also reduced the DNA condensation by 70%. Deletion of the region (residues 115-141) immediately before the 34 amino acid stretch and after the globular domain and the region (residues 184-218) immediately after the 34 amino acid stretch had only a marginal effect on DNA condensation. The importance of the 34 amino acid stretch, including the 10 amino acid spacer, was also demonstrated with the recombinant histone H1d C-terminus. We have also determined the induced alpha-helicity of histone H1 and its various mutants in the presence of 60% trifluoroethanol, and the experimentally determined induced helical contents agree with the theoretical predictions of secondary structural elements in the C-terminus of histone H1d. Thus, we have identified a 34 amino acid stretch in the C-terminus of histone H1d as the DNA-condensing domain.     

Crystal Structure of the Human SUV39H1 Chromodomain and Its Recognition of Histone H3K9me2/3

SUV39H1, the first identified histone lysine methyltransferase in human, is involved in chromatin modification and gene regulation. SUV39H1 contains a chromodomain in its N-terminus, which potentially plays a role in methyl-lysine recognition and SUV39H1 targeting. In this study, the structure of the chromodomain of human SUV39H1 was determined by X-ray crystallography. The SUV39H1 chromodomain displays a generally conserved structure fold compared with other solved chromodomains. However, different from other chromodomains, the SUV39H1 chromodomain possesses a much longer helix at its C-terminus. Furthermore, the SUV39H1 chromodomain was shown to recognize histone H3K9me2/3 specifically.     

Dissection of the unusual structural and functional properties of the variant H2A.Bbd nucleosome

The histone variant H2A.Bbd appeared to be associated with active chromatin, but how it functions is unknown. We have dissected the properties of nucleosome containing H2A.Bbd. Atomic force microscopy (AFM) and electron cryo-microscopy (cryo-EM) showed that the H2A.Bbd histone octamer organizes only approximately 130 bp of DNA, suggesting that 10 bp of each end of nucleosomal DNA are released from the octamer. In agreement with this, the entry/exit angle of the nucleosomal DNA ends formed an angle close to 180 degrees and the physico-chemical analysis pointed to a lower stability of the variant particle. Reconstitution of nucleosomes with swapped-tail mutants demonstrated that the N-terminus of H2A.Bbd has no impact on the nucleosome properties. AFM, cryo-EM and chromatin remodeling experiments showed that the overall structure and stability of the particle, but not its property to interfere with the SWI/SNF induced remodeling, were determined to a considerable extent by the H2A.Bbd docking domain. These data show that the whole H2A.Bbd histone fold domain is responsible for the unusual properties of the H2A.Bbd nucleosome.     

Anomalous nuclease digestion of Holothuria sperm chromatin containing histone H1 variants

We have investigated the micrococcal nuclease digestion of chromatin from the spermatozoa of the sea cucumber Holothuria tubulosa. This chromatin contains minor protein variants related to histone H1 with a high proportion of basic amino acids. One of these variants, protein phi 0, represents about 4% of the total histones. It is 78 amino acids long and its amino acid composition and sequence are related to the very basic C-terminal region of histone H1. The presence of these proteins induces an unusual digestion pattern. Oligonucleosomal particles which are soluble at 150 mM NaCl are depleted of protein phi 0 and they are also defective in histone H1. A low percentage of the insoluble material can be solubilized at lower NaCl concentrations (50 mM). These oligonucleosomal particles show a very peculiar protein content, since at early digestion times, they contain histone H1 and protein phi 0 exclusively. We conclude that these particles arise from a cooperative displacement of core histones by protein phi 0 and histone H1. These results show that minor changes in histone H1 complement can result in the formation of artifactual particles upon microccocal nuclease digestion. These observations may be of interest in other systems which contain H1 variants.     

H1 family histones in the nucleus. Control of binding and localization by the C-terminal domain

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.     

dBigH1, a second histone H1 in Drosophila,and the consequences for histone fold nomenclature

Recently, Pérez-Montero and colleagues (Developmental cell, 26: 578–590, 2013) described the occurrence of a new histone H1 variant (dBigH1) in Drosophila. The presence of unusual acidic amino acid patches at the N-terminal end of dBigH1 is in contrast to the arginine patches that exist at the N- and C-terminal domains of other histone H1-related proteins found in the sperm of some organisms. This departure from the strictly lysine-rich composition of the somatic histone H1 raises a question about the true definition of its protein members. Their minimal essential requirements appear to be the presence of a lysine- and alanine–rich, intrinsically disordered C-terminal domain, with a highly helicogenic potential upon binding to the linker DNA regions of chromatin. In metazoans, specific targeting of these regions is further achieved by a linker histone fold domain (LHFD), distinctively different from the characteristic core histone fold domain (CHFD) of the nucleosome core histones.     

Modification of the lysine residues of histones H1 and H5: Effects on structure and on the binding to chromatin

The extensive modification of histone H1 from calf thymus with the amino-group reagent dimethylmaleic anhydride (over 35 lysine residues modified per molecule) produces no effect on its secondary structure detectable by circular dichroism (far UV). Fluorescence and circular dichroism (near-UV) of the modified histone show variations in the local environment of its sole tyrosine residue. These changes are reversed on regeneration of the modified amino groups. While histone H1 is easily dissociated with this reagent from calf thymus or chicken erythrocyte chromatin, a much stronger treatment is needed to liberate histone H5 from erythrocyte chromatin. This difference appears to be related to the higher arginine content of histone H5.