Linker histone H1.b is polymorphic in grey partridge (Perdix perdix)

This study was aimed at characterizing allelic variations of erythrocyte histone H1.b by comparing the electrophoretic patterns of histone H1.b from individuals of grey partridge (Perdix perdix) population. As two alloforms, H1.b1 and H1.b2, were discerned in the screening gels, the histone H1.b was regarded to be a polymorphic protein encoded by a gene with two codominant alleles, b1 and b2, at a locus. The tested population was found to be at Hardy-Weinberg equilibrium (chi2 = 0.834, p = 0.361), with only a minor heterozygote deficiency (fixation index F = 0.136). Since the histone H1.b alloforms were identified in a two-dimensional gel containing sodium dodecyl sulfate, with no significant differences in their migration pattern in an one-dimensional acetic acid polyacrylamide gel, we assumed that the H1.b alloforms possessed a similar net charge and differed in their apparent molecular weights. A comparison of N-bromosuccinimide-cleaved and alpha-chymotrypsin-digested products of histone H1.b alloforms revealed slight differences in the velocity of C-terminal peptides and a similarity in migration of their N-terminal peptides in one-dimensional sodium dodecyl sulfate-polyacrylamide gel. Therefore, it seemed that the histone H1.b alloforms might differ in this amino acid sequence in a protein segment between N-bromosuccinimide cleavage site and the very C-terminus.     

Phenotypic variation of erythrocyte linker histone H1.c in a pheasant (Phasianus colchicus L.) population

Our goal was to characterize a phenotypic variation of the pheasant erythrocyte linker histone subtype H1.c. By using two-dimensional polyacrylamide gel electrophoresis three histone H1.c phenotypes were identified. The differently migrating allelic variants H1.c1 and H1.c2 formed either two homozygous phenotypes, c1 and c2, or a single heterozygous phenotype, c1c2. In the pheasant population screened, birds with phenotype c2 were the most common (frequency 0.761) while individuals with phenotype c1 were rare (frequency 0.043).     

Sequence variants of chicken linker histone H1.a

Two allelic isoforms (H1.a1 and H1.a2) of histone H1.a were identified within two conservative flocks (R11 and R55) of Rhode Island Red chickens. These proteins form three phenotypes: a1, a2 and a1a2. Birds with phenotype a1 were most common (frequency 0.825-0.980) while the a1a2 chickens appeared relatively rarely (0.017-0.175). The third phenotype a2, not detected in the tested populations, has only been revealed in progeny of the purpose-mated a1a2 birds. The polymorphism of histone H1.a was observed in all examined chicken tissues, so that the H1 preparations isolated from the lung, spleen, kidney and testis from the same individual exhibited identical phenotypes (a1, a2, or a1a2). This finding, together with inheritance data, supports the genetic nature of the H1.a polymorphism. As indicated by cleavages with alpha-chymotrypsin and protease V8, the H1.a1 and H1.a2 are two highly related proteins which differ within N-terminal part of their C-terminal tails. Only a single nonconservative amino acid substitution between both H1.a allelic isoforms was detected by Edman degradation: glutamic acid present at position 117 in histone H1.a1 was replaced by lysine in histone H1.a2. Furthermore, using microsequencing techniques we have found a sequence homology between the N- and C-terminal parts of an unknown minor protein H1.y, present in the phenotype a2, and similar regions of histone H1.b.     

The preferential binding of histone H1 to DNA scaffold-associated regions is determined by its C-terminal domain

Histone H1 preferentially binds and aggregates scaffold-associated regions (SARs) via the numerous homopolymeric oligo(dA).oligo(dT) tracts present within these sequences. Here we show that the mammalian somatic subtypes H1a,b,c,d,e and H1° and the male germline-specific subtype H1t, all preferentially bind to the Drosophila histone SAR. Experiments with the isolated domains show that whilst the C-terminal domain maintains strong and preferential binding, the N-terminal and globular domains show weak binding and poor specificity for the SAR. The preferential binding of SAR by the H1 molecule thus appears to be determined by its highly basic C-terminal domain. Salmine, a typical fish protamine, which could have its evolutionary origin in histone H1, also shows preferential binding to the SAR. The interaction of distamycin, a minor groove binder with high affinity for homopolymeric oligo(dA).oligo(dT) tracts, abolishes preferential binding of the C-terminal domain of histone H1 and protamine to the SAR, suggesting the involvement of the DNA minor groove in the interaction.     

Non-histone chromosomal protein HMG1 reduces the histone H5-induced changes in c.d. spectra of DNA: the acidic C-terminus of HMG1 is necessary for binding to H5

Chemical cross-linking was used to study the interaction between non-histone high-mobility-group (HMG)1 and histone H5 in free solution. The presence of acidic C-terminal domain in HMG1 was shown to be a prerequisite for HMG1 binding to histone H5. The objective of this communication is to ascertain whether HMG1 could affect the conformation of DNA associated with a linker histone H5. Complexes of histone H5 with chicken erythrocyte DNA or an alternating purine-pyrimidine polynucleotide poly[d(A-T)] were prepared at different molar ratios H5/DNA. Changes in DNA conformation in the complexes with histone H5 or H5/HMG1 were monitored by circular dichroism (c.d.). Depending on the molar ratio H5/poly[d(A-T)], under conditions limiting the complex aggregation, three distinct types of c.d. spectra were observed. The addition of HMG1 to H5-DNA complexes reduced in all cases the histone H5-induced conformational changes in poly[d(A-T)]. The sensitivity of H5-poly[d(A-T)] complexes to HMG1 was inversely proportional to the amount of H5 in the complex. The effect of HMG1 was not observed upon removal of the acidic C-terminal domain of HMG1.     

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.     

Primary structure of the two variants of a sperm-specific histone H1 from the annelid Platynereis dumerilii

The amino acid sequences of the two variants (H1a 121 residues and H1b 119 residues) of the sperm-specific histone H1 from the polychaete annelid Platynereis dumerilii have been completely established. Comparison of the sequences of these two variants shows one deletion of two residues in histone H1b and 22 substitents, of which most occur in the globular domain. The two variants differ highly in a sequence of nine residues adjacent to the conservative phenylalanine residue of histone H1 (64-72 in H1a, 62-70 in H1b) which makes H1a less hydrophobic than H1b. The small molecular size of Platynereis H1a and H1b is a unique feature among the histones H1 of which the size ranges between 189 residues (chicken erythrocyte H5) and 248 residues (sea urchin sperm H1). H1a and H1b have short N- and C-terminal basic domains but the size of the globular domain (approximately equal to 80 residues) is similar to that of other H1s. In the globular region the variant H1a exhibits a close relationship with somatic or sperm H1s whereas the variant H1b is more related to H5 histones.     

Solution NMR Structure and Histone Binding of the PHD Domain of Human MLL5

Mixed Lineage Leukemia 5 (MLL5) is a histone methyltransferase that plays a key role in hematopoiesis, spermatogenesis and cell cycle progression. In addition to its catalytic domain, MLL5 contains a PHD finger domain, a protein module that is often involved in binding to the N-terminus of histone H3. Here we report the NMR solution structure of the MLL5 PHD domain showing a variant of the canonical PHD fold that combines conserved H3 binding features from several classes of other PHD domains (including an aromatic cage) along with a novel C-terminal α-helix, not previously seen. We further demonstrate that the PHD domain binds with similar affinity to histone H3 tail peptides di- and tri-methylated at lysine 4 (H3K4me2 and H3K4me3), the former being the putative product of the MLL5 catalytic reaction. This work establishes the PHD domain of MLL5 as a bone fide ‘reader’ domain of H3K4 methyl marks suggesting that it may guide the spreading or further methylation of this site on chromatin.     

Multimerization and H3K9me3 Binding Are Required for CDYL1b Heterochromatin Association

Proteins containing defined recognition modules mediate readout and translation of histone modifications. These factors are thought to initiate downstream signaling events regulating chromatin structure and function. We identified CDYL1 as an interaction partner of histone H3 trimethylated on lysine 9 (H3K9me3). CDYL1 belongs to a family of chromodomain factors found in vertebrates. We show that three different splicing variants of CDYL1, a, b, and c, are differentially expressed in various tissues with CDYL1b being the most abundant variant. Although all three splicing variants share a common C-terminal enoyl-CoA hydratase-like domain, only CDYL1b contains a functional chromodomain implicated in H3K9me3 binding. A splicing event introducing an N-terminal extension right at the beginning of the chromodomain of CDYL1a inactivates its chromodomain. CDYL1c does not contain a chromodomain at all. Although CDYL1b displays binding affinity to methyl-lysine residues in different sequence context similar to chromodomains in other chromatin factors, we demonstrate that the CDYL1b chromodomain/H3K9me3 interaction is necessary but not sufficient for association of the factor with heterochromatin. Indeed, multimerization of the protein via the enoyl-CoA hydratase-like domain is essential for H3K9me3 chromatin binding in vitro and heterochromatin localization in vivo. In agreement, overexpression of CDYL1c that can multimerize, but does not interact with H3K9me3 can displace CDYL1b from heterochromatin. Our results imply that multimeric binding to H3K9me3 by CDYL1b homomeric complexes is essential for efficient chromatin targeting. We suggest that similar multivalent binding stably anchors other histone modification binding factors on their target chromatin regions.     

N- and C-terminal domains determine differential nucleosomal binding geometry and affinity of linker histone isotypes H1(0) and H1c

Eukaryotic linker or H1 histones modulate DNA compaction and gene expression in vivo. In mammals, these proteins exist as multiple isotypes with distinct properties, suggesting a functional significance to the heterogeneity. Linker histones typically have a tripartite structure composed of a conserved central globular domain flanked by a highly variable short N-terminal domain and a longer highly basic C-terminal domain. We hypothesized that the variable terminal domains of individual subtypes contribute to their functional heterogeneity by influencing chromatin binding interactions. We developed a novel dual color fluorescence recovery after photobleaching assay system in which two H1 proteins fused to spectrally separable fluorescent proteins can be co-expressed and their independent binding kinetics simultaneously monitored in a single cell. This approach was combined with domain swap and point mutagenesis to determine the roles of the terminal domains in the differential binding characteristics of the linker histone isotypes, mouse H1(0) and H1c. Exchanging the N-terminal domains between H1(0) and H1c changed their overall binding affinity to that of the other variant. In contrast, switching the C-terminal domains altered the chromatin interaction surface of the globular domain. These results indicate that linker histone subtypes bind to chromatin in an intrinsically specific manner and that the highly variable terminal domains contribute to differences between subtypes. The methods developed in this study will have broad applications in studying dynamic properties of additional histone subtypes and other mobile proteins.     

Natural allelic variation of duck erythrocyte histone H1b

In our previous work (J. Palyga, Genetic polymorphisms of histone H1. b in duck erythrocytes. Hereditas 114, 85-89, 1991) we reported a genetic polymorphism of duck erythrocyte histone H1.b. Here, we screened H1 preparations in a two-dimensional polyacrylamide gel to refine the distribution of allelic forms of H1.b in fifteen duck populations. We have revealed that the frequency of H1.b allelic variants was significantly different among many conservative and breeding duck groups. While b(1) and b(3) were common in all populations screened, the allele b(2), with a slightly lower apparent molecular weight, was confined mainly to brown-feathered ducks (Khaki Campbell and Orpington) and descendent lines.The C- and N-terminal peptides released upon cleavage with N-bromosuccinimide and Staphylococcus aureus protease V8 from duck allelic histones H1. b2 and H1.b3, respectively, migrated differently in the gel, probably as a result of potential amino acid variation in a C-terminal domain.     

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.     

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.     

Nucleosome compaction facilitates HP1γ binding to methylated H3K9

The α, β and γ isoforms of mammalian heterochromatin protein 1 (HP1) selectively bind to methylated lysine 9 of histone H3 via their chromodomains. Although the phenotypes of HP1-knockout mice are distinct for each isoform, the molecular mechanisms underlying HP1 isoform-specific function remain elusive. In the present study, we found that in contrast to HP1α, HP1γ could not bind tri-methylated H3 lysine 9 in a reconstituted tetra-nucleosomes when the nucleosomes were in an uncompacted state. The hinge region connecting HP1''s chromodomain and chromoshadow domain contributed to the distinct recognition of the nucleosomes by HP1α and HP1γ. HP1γ, but not HP1α, was strongly enhanced in selective binding to tri-methylated lysine 9 in histone H3 by the addition of Mg²⁺ or linker histone H1, which are known to induce compaction of nucleosomes. We propose that this novel property of HP1γ recognition of lysine 9 in the histone H3 tail in different nucleosome structures plays a role in reading the histone code.     

Molecular modeling of the chromatosome particle

In an effort to understand the role of the linker histone in chromatin folding, its structure and location in the nucleosome has been studied by molecular modeling methods. The structure of the globular domain of the rat histone H1d, a highly conserved part of the linker histone, built by homology modeling methods, revealed a three-helical bundle fold that could be described as a helix–turn–helix variant with its characteristic properties of binding to DNA at the major groove. Using the information of its preferential binding to four-way Holliday junction (HJ) DNA, a model of the domain complexed to HJ was built, which was subsequently used to position the globular domain onto the nucleosome. The model revealed that the primary binding site of the domain interacts with the extra 20 bp of DNA of the entering duplex at the major groove while the secondary binding site interacts with the minor groove of the central gyre of the DNA superhelix of the nucleosomal core. The positioning of the globular domain served as an anchor to locate the C-terminal domain onto the nucleosome to obtain the structure of the chromatosome particle. The resulting structure had a stem-like appearance, resembling that observed by electron microscopic studies. The C-terminal domain which adopts a high mobility group (HMG)-box-like fold, has the ability to bend DNA, causing DNA condensation or compaction. It was observed that the three S/TPKK motifs in the C-terminal domain interact with the exiting duplex, thus defining the path of linker DNA in the chromatin fiber. This study has provided an insight into the probable individual roles of globular and the C-terminal domains of histone H1 in chromatin organization.     

The histone domain of macroH2A1 contains several dispersed elements that are each sufficient to direct enrichment on the inactive X chromosome

Histone variants replace the core histones in a substantial fraction of nucleosomes, affecting chromatin structure and impacting chromatin-templated processes. In many instances incorporation of histone variants results in formation of specialized regions of chromatin. Proper localization of histone variants to distinct regions of the genome is critical for their function, yet how this specific localization is achieved remains unclear. macroH2A1 is enriched on the inactive X chromosome in female mammalian cells, where it functions to maintain gene silencing. macroH2A1 consists of a histone H2A-like histone domain and a large, globular C-terminal macro domain that is not present in other histone proteins. The histone domain of macroH2A1 is alone sufficient to direct enrichment on the inactive X chromosome when expressed in female cells, indicating that sequences important for correct localization lie in this domain. Here we investigate whether divergent sequences of the H2A variant macroH2A1 contribute to its correct localization. We mapped the regions of the macroH2A1 histone domain that are sufficient for localization to the inactive X chromosome using chimeras between H2A and the histone domain of macroH2A1. Multiple short sequences dispersed along the macroH2A1 histone domain individually supported enrichment on the inactive X chromosome when introduced into H2A. These sequences map to the surface of the macroH2A1/H2B dimer, but are buried in the crystal structure of the macroH2A1 containing nucleosome, suggesting that they may contribute to recognition by macroH2A1/H2B deposition factors.