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.     

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.     

Interaction of chromatin with a histone H1 containing swapped N- and C-terminal domains

Although the details of the structural involvement of histone H1 in the organization of the nucleosome are quite well understood, the sequential events involved in the recognition of its binding site are not as well known. We have used a recombinant human histone H1 (H1.1) in which the N- and C-terminal domains (NTD/CTD) have been swapped and we have reconstituted it on to a 208-bp nucleosome. We have shown that the swapped version of the protein is still able to bind to nucleosomes through its structurally folded wing helix domain (WHD); however, analytical ultracentrifuge analysis demonstrates its ability to properly fold the chromatin fibre is impaired. Furthermore, FRAP analysis shows that the highly dynamic binding association of histone H1 with the chromatin fibre is altered, with a severely decreased half time of residence. All of this suggests that proper binding of histone H1 to chromatin is determined by the simultaneous and synergistic binding of its WHD–CTD to the nucleosome.     

Carassius auratus-originated recombinant histone H1 C-terminal peptide as gene delivery material

The effective delivery of exogenous genes into eukaryotic cells is important for fundamental and biotechnological research. Protein-based gene delivery including histone proteins has recently emerged as a powerful technique for non-viral DNA transfer. Histones are DNA-binding proteins that function in DNA packaging and protection. In particular, histone H1 is largely responsible for the stabilization of higher-order chromatin structures. Several studies have examined the use of full-length histone H1-mediated gene transfer, and a few studies have investigated the use of C-terminal histone H1 fragments as gene-transfer materials. Previously, we cloned a novel histone H1 cDNA from the goldfish Carassius auratus and found that a recombinant histone H1 C-terminal short peptide (H1C) of 61 amino acids has comparable DNA binding and protection functions as full-length histone H1. In the present work, we successfully expressed and purified soluble recombinant H1C in an Escherichia coli expression system using a hexahistidine tag fusion strategy and providing tRNAs for rare codons. We confirmed its DNA-binding ability and found that this H1C peptide had similar or higher transfection efficiency in mammalian cells (human 293T and mouse NIH/3T3) than the widely used agent lipofectamine. Therefore, we suggest that this novel goldfish-derived recombinant histone H1 C-terminal short peptide could be used as a peptide-based gene-transfer mediator.     

Prediction of an HMG-box fold in the C-terminal domain of histone H1: insights into its role in DNA condensation

In eukaryotes, histone H1 promotes the organization of polynucleosome filaments into chromatin fibers, thus contributing to the formation of an important structural framework responsible for various DNA transaction processes. The H1 protein consists of a short N-terminal "nose," a central globular domain, and a highly basic C-terminal domain. Structure prediction of the C-terminal domain using fold recognition methods reveals the presence of an HMG-box-like fold. We recently showed by extensive site-directed and deletion mutagenesis studies that a 34 amino acid segment encompassing the three S/TPKK motifs, within the C-terminal domain, is responsible for DNA condensing properties of H1. The position of these motifs in the predicted structure corresponds exactly to the DNA-binding segments of HMG-box-containing proteins such as Lef-1 and SRY. Previous analyses have suggested that histone H1 is likely to bend DNA bound to the C-terminal domain, directing the path of linker DNA in chromatin. Prediction of the structure of this domain provides a framework for understanding the higher order of chromatin organization.     

Monoclonal antibody with specificity to a conserved epitope in the C-terminal domain of histone H1 variants.

A monoclonal type M-immunoglobulin (IgM) was generated in mice against a nuclease-urea extract of HeLa metaphase chromosomes. This antibody stains metaphase chromosomes from a variety of mammalian cultured cell types by indirect immunofluorescence. Antibody 12C7 reacts by western transfer technique with histone H1 in all the cell lines tested. The antibody cross-reacts with H1, and H1(0) in human cells. Proteolytic digestions of H1 suggest that the epitope is localized in the carboxy-terminal domain of the histone H1 molecule. Digestion with trypsin demonstrates that the antibody 12C7 does not react with the globular domain of histone H1. The C-terminal domain of H1 subtypes therefore seems to have a conserved determinant which does exist in H1, H1(0), and probably in H5. This antibody has applications in studying the role of that domain of H1 in processes like chromosome condensation and variations in chromatin structure which influence gene expression.     

Structure of allelic variants of subtype 5 of histone H1 in pea Pisum sativum L

The pea genome contains seven histone H1 genes encoding different subtypes. Previously, the DNA sequence of only one gene, His1, coding for the subtype H1-1, had been identified. We isolated a histone H1 allele from a pea genomic DNA library. Data from the electrophoretic mobility of the pea H1 subtypes and their N-bromosuccinimide cleavage products indicated that the newly isolated gene corresponded to the H1-5 subtype encoded by His5. We confirmed this result by sequencing the gene from three pea lines with H1-5 allelic variants of altered electrophoretic mobility. The allele of the slow H1-5 variant differed from the standard allele by a nucleotide substitution that caused the replacement of the positively charged lysine with asparagine in the DNA-interacting domain of the histone molecule. A temperature-related occurrence had previously been demonstrated for this H1-5 variant in a study on a worldwide collection of pea germplasm. The variant tended to occur at higher frequencies in geographic regions with a cold climate. The fast allelic variant of H1-5 displayed a deletion resulting in the loss of a duplicated pentapeptide in the C-terminal domain.     

A highly conserved sequence in H1 histone genes as an oligonucleotide hybridization probe: Isolation and sequence of a duck H1 gene

A 3.5-kb HindIII fragment of a histone gene cluster was isolated from a recombinant phage out of a duck genomic library. This DNA contains a duck H1 gene and its flanking sequences. The hybridization probe, which was used to screen for the H1 gene, had been designed on the basis of a comparative analysis of available H1 gene and protein data. Most H1 histones contain repeated motifs in their C-terminal domain, and these form part of an octapeptide (ser pro lys lys ala lys lys pro) that is highly conserved in many H1 histone proteins. A comparison of the duck H1 described here with two different published chicken H1 histone sequences reveals conservative amino acid exchanges at 22 (of 217 and 218, respectively) positions. The homology is maintained at the flanking sequences, and includes the putative H1 histone gene-specific signal structures and the established 3' stem and loop structures and the CAAGA box. The duck H1 gene and its flanking sequence have been found in identical arrangements in two recombinant bacteriophages, but minor sequence variations and genomic Southern blotting after HindIII digestion suggest that we have either isolated alleles of this genome segment or that the gene described may occur twice per haploid duck genome.     

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.     

Characteristics of limited trypsinolysis of histone H5 in a solution and as a component of chromatin with different degrees of compactness

Trypsinolysis of histone H5 in solution and as a component of chromatin with different level of compactization was studied. It was demonstrated that the existence of supernucleosomal organization leads to a significant decrease of the degradation rate of histones H1 and H5 in comparison with histones H2A, H2B, H3 and H4. Analysis of trypsinolysis electrophoretic spectra of histone H5 revealed the existence of protease-resistant fragments in chromatin, but not in solution. These fragments contain not only the globular domain of histone H5 but also small-sized unstructured N- and/or C-terminal regions. The peptides were identified with the help of an immune serum specific for the globular region of histone H5. The possible role of resistant fragments in the nucleosomal organization of chromatin is discussed.     

Histone H2A and H4 N-terminal Tails Are Positioned by the MEP50 WD Repeat Protein for Efficient Methylation by the PRMT5 Arginine Methyltransferase

The protein arginine methyltransferase PRMT5 is complexed with the WD repeat protein MEP50 (also known as Wdr77 or androgen coactivator p44) in vertebrates in a tetramer of heterodimers. MEP50 is hypothesized to be required for protein substrate recruitment to the catalytic domain of PRMT5. Here we demonstrate that the cross-dimer MEP50 is paired with its cognate PRMT5 molecule to promote histone methylation. We employed qualitative methylation assays and a novel ultrasensitive continuous assay to measure enzyme kinetics. We demonstrate that neither full-length human PRMT5 nor the Xenopus laevis PRMT5 catalytic domain has appreciable protein methyltransferase activity. We show that histones H4 and H3 bind PRMT5-MEP50 more efficiently compared with histone H2A(1–20) and H4(1–20) peptides. Histone binding is mediated through histone fold interactions as determined by competition experiments and by high density histone peptide array interaction studies. Nucleosomes are not a substrate for PRMT5-MEP50, consistent with the primary mode of interaction via the histone fold of H3-H4, obscured by DNA in the nucleosome. Mutation of a conserved arginine (Arg-42) on the MEP50 insertion loop impaired the PRMT5-MEP50 enzymatic efficiency by increasing its histone substrate K_m, comparable with that of Caenorhabditis elegans PRMT5. We show that PRMT5-MEP50 prefers unmethylated substrates, consistent with a distributive model for dimethylation and suggesting discrete biological roles for mono- and dimethylarginine-modified proteins. We propose a model in which MEP50 and PRMT5 simultaneously engage the protein substrate, orienting its targeted arginine to the catalytic site.     

Structure and expression of the human gene encoding testicular H1 histone (H1t)

The gene coding for the human H1t histone, a testis-specific H1 subtype, was isolated from a genomic library using a human somatic H1 gene as a hybridization probe. The corresponding mRNA is not polyadenylated and encodes a 206-amino-acid protein. Sequence analysis and S1 nuclease mapping of the human H1t gene reveals that the 5' flanking region contains several consensus promoter elements, as described for somatic, i.e., S-phase-dependent H1 subtype genes. The 3' region includes the stem-and-loop structure necessary for mRNA processing of most histone mRNAs. Northern blot analysis with RNAs from different human tissues and cell lines revealed that only testicular RNA hybridized with this gene probe.     

Condensation of DNA by the C-terminal domain of histone H1. A circular dichroism study

The condensation of DNA by the C-terminal domain of histone H1 has been studied by circular dichroism in physiological salt concentration (0.14 M NaF). As the intact H1 molecule, its C-terminal domain induces the so-called psi state of DNA that is characterized by a nonconservative circular dichroism spectrum which is currently attributed to ordered aggregation of the DNA molecules. On a molar basis, intact H1 and its C-terminal domain give spectra of similar intensity. Neither the globular domain of H1 nor an N-terminal fragment, that includes both the globular and N-terminal domains, has any effect on the conservative circular dichroism of DNA. From these results it is concluded that the condensation of DNA mediated by histone H1 is mainly due to its C-terminal domain. The effect of the salt concentration and the size of DNA molecules on the circular dichroism of the complexes are also examined.     

Histones H1(0) and H5 share common epitopes with RNA polymerase II

We report here the cross-reaction of RNA polymerase II antiserum with histones H1(0) and H5 and the complementary cross-reactions of antisera to the globular domain of histone H1(0) (GH1(0)) and histone H5 (GH5) with RNA polymerase II. Immunoblotting of RNA polymerase II antiserum with fragments of histone H1(0) localized the cross-reaction at the junction of the globular and C-terminal domains of histone H1(0). The structural homology implied by these cross-reactions is interesting in light of reports that suggest H1(0) may play a role in differentiation and development.     

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.     

Disruptor of telomeric silencing-1 is a chromatin-specific histone H3 methyltransferase

Yeast disruptor of telomeric silencing-1 (DOT1) is involved in gene silencing and in the pachytene checkpoint during meiotic cell cycle. Here we show that the Dot1 protein possesses intrinsic histone methyltransferase (HMT) activity. When compared with Rmt1, another putative yeast HMT, Dot1 shows very distinct substrate specificity. While Rmt1 methylates histone H4, Dot1 targets histone H3. In contrast to Rmt1, which can only modify free histones, Dot1 activity is specific to nucleosomal substrates. This was also confirmed using native chromatin purified from yeast cells. We also demonstrate that, like its mammalian homolog PRMT1, Rmt1 specifically dimethylates an arginine residue at position 3 of histone H4 N-terminal tail. In surprising contrast, methylation by Dot1 occurs in the globular domain of nucleosomal histone H3. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis suggests that H3 lysine 79 is trimethylated by Dot1. The intrinsic nucleosomal histone H3 methyltransferase activity of Dot1 is certainly a key aspect of its function in gene silencing at telomeres, most likely by directly modulating chromatin structure and Sir protein localization. In agreement with a role in regulating localization of histone deacetylase complexes like SIR, an increase of bulk histone acetylation is detected in dot1- cells.     

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.     

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.