Parathymosin affects the binding of linker histone H1 to nucleosomes and remodels chromatin structure

Linker histone H1 is the major factor that stabilizes higher order chromatin structure and modulates the action of chromatin-remodeling enzymes. We have previously shown that parathymosin, an acidic, nuclear protein binds to histone H1 in vitro and in vivo. Confocal laser scanning microscopy reveals a nuclear punctuate staining of the endogenous protein in interphase cells, which is excluded from dense heterochromatic regions. Using an in vitro chromatin reconstitution system under physiological conditions, we show here that parathymosin (ParaT) inhibits the binding of H1 to chromatin in a dose-dependent manner. Consistent with these findings, H1-containing chromatin assembled in the presence of ParaT has reduced nucleosome spacing. These observations suggest that interaction of the two proteins might result in a conformational change of H1. Fluorescence spectroscopy and circular dichroism-based measurements on mixtures of H1 and ParaT confirm this hypothesis. Human sperm nuclei challenged with ParaT become highly decondensed, whereas overexpression of green fluorescent protein- or FLAG-tagged protein in HeLa cells induces global chromatin decondensation and increases the accessibility of chromatin to micrococcal nuclease digestion. Our data suggest a role of parathymosin in the remodeling of higher order chromatin structure through modulation of H1 interaction with nucleosomes and point to its involvement in chromatin-dependent functions.     

Influence of linker histone H1 on chromatin remodeling.

Chromatin-remodeling complexes have been a central area of focus for research dealing with accessing cellular DNA sequestered in chromatin. Although the linker histone H1 plays a major role in promoting and maintaining higher-order chromatin structure, it has been noticeably absent from assays utilizing chromatin-remodeling enzymes. This review focuses on two ATP-dependent chromatin-remodeling complexes, Drosophila ISWI and mammalian SWI/SNF, that have been assayed using chromatin templates containing histone H1.     

Modulation of chromatin function through linker histone H1 variants

In this review, the structural aspects of linker H1 histones are presented as a background for characterization of the factors influencing their function in animal and human chromatin. The action of H1 histone variants is largely determined by dynamic alterations of their intrinsically disordered tail domains, posttranslational modifications and allelic diversification. The interdependent effects of these factors can establish dynamic histone H1 states that may affect the organization and function of chromatin regions.     

Interaction between non-histone chromatin protein HMGB1 and linker histone H1

The fundamental possibility of interactions between non-histone chromatin protein HMGB1 and linker histone H1 in solutions with different ionic strengths was studied by intrinsic UV fluorescence, far and near UV CD, and spectrophotometry. The data we obtained allow us to assume that the increase in the histone H1 content in HMGB1 solutions with low ionic strengths is accompanied by the destruction of HMGB1 associates. The interactions between HMGB1 and H1 proteins increase the number of ordered regions in the protein molecules and causes slight changes in the tertiary structure of the protein.     

Interaction between non-histone chromatin protein HMGB1 and linker histone H1

The fundamental possibility of interaction between non-histone chromatin protein HMGB1 and linker histone H1 was studied in the solutions with different ionic strength by intrinsic UV-fluorescence, far and near-UV CD and spectrophotometry. The obtained data allow us to assume that the increase of histone H1 content in the HMGB1 solutions in a low ionic strength is accompanied by the destruction of HMGB1 associates. The interaction between proteins of HMGB1 and H1 causes the increase in the number of ordered regions in the protein molecules and the minor changes in their tertiary structure.     

Removal of histone H1 exposes linker DNA in chromatin to DNAse I

After removal of histone H1 about 40% of DNA in chromatin acquires the sensitivity of naked DNA to DNAse I. Digestion of H1-depleted chromatin with DNAse I leads to a qualitative change in the digestion pattern, generating DNA fragments of approx. 200 b.p. and multiples, similar to those obtained with micrococcal nuclease. Both effects are reversed upon reconstitution of purified H1 to H1-depleted chromatin.     

Accessibility of histone H1° and its structural domains to antibody binding in extended and folded chromatin

The aim of this work was to study the accessibility of histone H1° and its structural domains to antibody binding in high molecular mass chromatin fragments of different conformations. Three types of specific antibody populations were used: (1) anti-H1° which reacted with antigenic determinants situated along the whole polypeptide chain, (2) anti-GH5 or anti-GH1° which recognized epitopes located in the globular region of H1° and (3) anti-C-tail antibodies reacting specifically with fragment 99–193 of the protein molecule. The immunoreactivity of the chromatin-bound antigen was investigated by solid-phase ELISA performed on glutaraldehyde-cross-linked chromatin and by an inhibition assay carried out with native chromatin in solution. The results of both methods were unidirectional and showed that: (1) the accessibility of H1° did not change with the compaction of the fiber; (2) the G-domain was not accessible to antibodies either in the relaxed or in the condensed state of the fragments, (3) the binding of the C-terminus-specific antibodies was different for isolated monosomes and for the chromatin fiber and (4) the degree of exposure of the epitopes of H1° in chromatin was much less than that of histone H1.Abbreviations ELISA Enzyme-Linked Immunosorbent Assay - G-domain Globular domain - IgG Immunoglobulin G - SDS Sodium Dodecylsulphate     

A major nucleolar protein, nucleolin, induces chromatin decondensation by binding to histone H1

Using circular dichroism to probe the extent of DNA condensation in chromatin, we have demonstrated that a major nucleolar protein, nucleolin can decondense chromatin. By means of various binding assays we show that nucleolin has a strong affinity for histone H1 and that the phosphorylated N-terminal domain, rich in lengthy stretches of acidic amino acids, is responsible for this ionic interaction. Additional experiments clearly demonstrate that nucleolin is unable to act as a nucleosome core assembly or disassembly factor and hence has little affinity for the core histone octamer. We propose that this nucleolar protein induces chromatin decondensation by binding to histone H1, and that nucleolin can therefore be regarded as a protein of the high-mobility-group type.     

Oocyte-specific linker histone H1foo is an epigenomic modulator that decondenses chromatin and impairs pluripotency

Mammalian oocytes contain the histone H1foo, a distinct member with low sequence similarity to other members in the H1 histone family. Oocyte-specific H1foo exists until the second embryonic cell stage. H1foo is essential for oocyte maturation in mice; however, the molecular function of this H1 subtype is unclear. To explore the function of H1foo, we generated embryonic stem (ES) cells ectopically expressing H1foo fused to an EGFP (H1foo-ES). Interestingly, ectopic expression of H1foo prevented normal differentiation into embryoid bodies (EBs). The EB preparations from H1foo-ES cells maintained the expression of pluripotent marker genes, including Nanog, Myc and Klf9, and prevented the shift of the DNA methylation profile. Because the short hairpin RNA-mediated knockdown of H1foo-EGFP recovered the differentiation ability, H1foo was involved in preventing differentiation. Furthermore, ChIP analysis revealed that H1foo-EGFP bound selectively to a set of hypomethylated genomic loci in H1foo-ES, clearly indicating that these loci were targets of H1foo. Finally, nuclease sensitivity assay suggested that H1foo made these target loci decondensed. We concluded that H1foo has an impact on the genome-wide, locus-specific epigenetic status.     

Oocyte-specific linker histone H1foo is an epigenomic modulator that decondenses chromatin and impairs pluripotency

Mammalian oocytes contain the histone H1foo, a distinct member with low sequence similarity to other members in the H1 histone family. Oocyte-specific H1foo exists until the second embryonic cell stage. H1foo is essential for oocyte maturation in mice; however, the molecular function of this H1 subtype is unclear. To explore the function of H1foo, we generated embryonic stem (ES) cells ectopically expressing H1foo fused to an EGFP (H1foo-ES). Interestingly, ectopic expression of H1foo prevented normal differentiation into embryoid bodies (EBs). The EB preparations from H1foo-ES cells maintained the expression of pluripotent marker genes, including Nanog, Myc and Klf9, and prevented the shift of the DNA methylation profile. Because the short hairpin RNA-mediated knockdown of H1foo-EGFP recovered the differentiation ability, H1foo was involved in preventing differentiation. Furthermore, ChIP analysis revealed that H1foo-EGFP bound selectively to a set of hypomethylated genomic loci in H1foo-ES, clearly indicating that these loci were targets of H1foo. Finally, nuclease sensitivity assay suggested that H1foo made these target loci decondensed. We concluded that H1foo has an impact on the genome-wide, locus-specific epigenetic status.     

On the binding of histone H1 in chromatin

Nucleosomal subunits isolated from rabbit thymus nuclei in 0.04 M K₂SO₄-0.02 M Tris, pH 7.4 were devoid of histone H1, while whole chromatin prepared in the same buffer contained the full complement of histone H1. The question is asked why histone H1 dissociates from the subunits but not from the high molecular weight material. We propose that, at physiological salt concentrations, histone H1 is not bound to linker DNA as depicted in the current models; rather, alternate attachment sites, present only in the polymer, are involved.     

Change in linker DNA conformation upon histone H1.5 binding to nucleosome: Fluorescent microscopy of single complexes

A method for fluorescently labeled DNA synthesis, which makes it possible to assemble mononucleosomes with 40 bp linkers, was developed. Cy3 and Cy5 labels were introduced into the linkers at distances of 10 bp before the first nucleotide and 15 bp after the last nucleotide of the nucleosome positioning DNA sequence, respectively. Without histone H1.5, f luorescence microscopy of single complexes revealed two equally probable states of nucleosomes. The states were different in the linker conformation: the open one with the energy transfer efficiency (E) between the labels of 0.06 and the closed one with E = 0.37, when the linkers are brought together. Binding of histone H1.5 with nucleosomes occurs in a range of nanomolar concentrations, and the complex formation rate is significantly higher as compared with its dissociation rate. The significant convergence of the DNA linkers (E = 0.73) is observed in these complexes together with the higher conformation uniformity in the region where the labels are localized. The developed nucleosomal constructs represent highly sensitive f luorescent sensors that can be used for the analysis of structural linker rearrangements. Also, in combination with microscopy of single complexes, they are suitable for studying the structure of complexes of nucleosomes with different chromatin architectural proteins.     

Prothymosin alpha modulates the interaction of histone H1 with chromatin.

Prothymosin alpha (ProTalpha) is an abundant acidic nuclear protein that may be involved in cell proliferation. In our search for its cellular partners, we have recently found that ProTalpha binds to linker histone H1. We now provide further evidence for the physiological relevance of this interaction by immunoisolation of a histone H1-ProTalpha complex from NIH 3T3 cell extracts. A detailed analysis of the interaction between the two proteins suggests contacts between the acidic region of ProTalpha and histone H1. In the context of a physiological chromatin reconstitution reaction, the presence of ProTalpha does not affect incorporation of an amount of histone H1 sufficient to increase the nucleosome repeat length by 20 bp, but prevents association of all further H1. Consistent with this finding, a fraction of histone H1 is released when H1-containing chromatin is challenged with ProTalpha. These results imply at least two different interaction modes of H1 with chromatin, which can be distinguished by their sensitivity to ProTalpha. The properties of ProTalpha suggest a role in fine tuning the stoichiometry and/or mode of interaction of H1 with chromatin.     

Mutual arrangement of histone H1 molecules in extended chromatin. Chymotryptic digestion of cross-linked H1 histone dimers

Mutual arrangement of histone H1 molecules in chromatin extended in low salt-EDTA buffer and additionally in the presence of urea was studied by means of reversible cross-linking combined with chymotryptic digestion. In the chromatins tested, the chymotryptic halves of H1 were cross-linked in all possible combinations; i.e., C-C, C-N and N-N. The results imply that the mutual arrangement of H1 histones is determined by the structure of extended nucleosomal chain, rather than chromatin superstructure.     

Phosphorylation of the linker histone H1 by CDK regulates its binding to HP1alpha

Two key components of mammalian heterochromatin that play a structural role in higher order chromatin organization are the heterochromatin protein 1alpha (HP1alpha) and the linker histone H1. Here, we show that these proteins interact in vivo and in vitro through their hinge and C-terminal domains, respectively. The phosphorylation of H1 by CDK2, which is required for efficient cell cycle progression, disrupts this interaction. We propose that phosphorylation of H1 provides a signal for the disassembly of higher order chromatin structures during interphase, independent of histone H3-lysine 9 (H3-K9) methylation, by reducing the affinity of HP1alpha for heterochromatin.     

Determinants of histone H1 mobility and chromatin binding in living cells

The dynamic interaction of chromatin-binding proteins with their nucleosome binding sites is an important element in regulating the structure and function of chromatin in living cells. Here we review the major factors regulating the intranuclear mobility and chromatin binding of the linker histone H1, the most abundant family of nucleosome-binding proteins. The information available reveals that multiple and diverse factors modulate the interaction of H1 with chromatin at both a local and global level. This multifaceted mode of modulating the interaction of H1 with nucleosomes is part of the mechanism that regulates the dynamics of the chromatin fiber in living cells.