Histone Affinity Research Articles

Pygo-F773W Mutation Reveals Novel Functions beyond Wnt Signaling in Drosophila.

Pygopus (Pygo) has been identified as a specific nuclear co-activator of the canonical Wingless (Wg)/Wnt signaling pathway in Drosophila melanogaster. Pygo proteins consist of two conserved domains: an N-terminal homologous domain (NHD) and a C-terminal plant homologous domain (PHD). The PHD's ability to bind to di- and trimethylated lysine 4 of histone H3 (H3K4me2/3) appears to be independent of Wnt signaling. There is ongoing debate regarding the significance of Pygo's histone-binding capacity. Drosophila Pygo orthologs have a tryptophan (W) > phenylalanine (F) substitution in their histone pocket-divider compared to vertebrates, leading to reduced histone affinity. In this research, we utilized CRISPR/Cas9 technology to introduce the Pygo-F773W point mutation in Drosophila, successfully establishing a viable homozygous Pygo mutant line for the first time. Adult mutant flies displayed noticeable abnormalities in reproduction, locomotion, heart function, and lifespan. RNA-seq and cluster analysis indicated that the mutation primarily affected pathways related to immunity, metabolism, and posttranslational modification in adult flies rather than the Wnt signaling pathway. Additionally, a reduction in H3K9 acetylation levels during the embryonic stage was observed in the mutant strains. These findings support the notion that Pygo plays a wider role in chromatin remodeling, with its involvement in Wnt signaling representing only a specific aspect of its chromatin-related functions.

Moderation of Structural DNA Properties by Coupled Dinucleotide Contents in Eukaryotes

Dinucleotides are known as determinants for various structural and physiochemical properties of DNA and for binding affinities of proteins to DNA. These properties (e.g., stiffness) and bound proteins (e.g., transcription factors) are known to influence important biological functions, such as transcription regulation and 3D chromatin organization. Accordingly, the question arises of how the considerable variations in dinucleotide contents of eukaryotic chromosomes could still provide consistent DNA properties resulting in similar functions and 3D conformations. In this work, we investigate the hypothesis that coupled dinucleotide contents influence DNA properties in opposite directions to moderate each other's influences. Analyzing all 2478 chromosomes of 155 eukaryotic species, considering bias from coding sequences and enhancers, we found sets of correlated and anti-correlated dinucleotide contents. Using computational models, we estimated changes of DNA properties resulting from this coupling. We found that especially pure A/T dinucleotides (AA, TT, AT, TA), known to influence histone positioning and AC/GT contents, are relevant moderators and that, e.g., the Roll property, which is known to influence histone affinity of DNA, is preferably moderated. We conclude that dinucleotide contents might indirectly influence transcription and chromatin 3D conformation, via regulation of histone occupancy and/or other mechanisms.

Enhanced nucleosome assembly at CpG sites containing an extended 5-methylcytosine analogue.

Methylation of cytosine to 5-methylcytosine (mC) at CpG sites is a prevalent reversible epigenetic mark in vertebrates established by DNA methyltransferases (MTases); the attached methyl groups can alter local structure of DNA and chromatin as well as binding of dedicated proteins. Nucleosome assembly on methylated DNA has been studied extensively, however little is known how the chromatin structure is affected by larger chemical variations in the major groove of DNA. Here, we studied the nucleosome formation in vitro on DNA containing an extended 5mC analog, 5-(6-azidohex-2-ynyl)cytosine (ahyC) installed at biological relevant CpG sites. We found that multiple ahyC residues on 80-Widom and Hsp70 promoter DNA fragments proved compatible with nucleosome assembly. Moreover, unlike mC, ahyC increases the affinity of histones to the DNA, partially altering nucleosome positioning, stability, and the action of chromatin remodelers. Based on molecular dynamics calculations, we suggest that these new features are due to increased DNA flexibility at ahyC-modified sites. Our findings provide new insights into the biophysical behavior of modified DNA and open new ways for directed design of synthetic nucleosomes.

Modified Histone Peptides Linked to Magnetic Beads Reduce Binding Specificity.

Histone post-translational modifications are small chemical changes to the histone protein structure that have cascading effects on diverse cellular functions. Detecting histone modifications and characterizing their binding partners are critical steps in understanding chromatin biochemistry and have been accessed using common reagents such as antibodies, recombinant assays, and FRET-based systems. High-throughput platforms could accelerate work in this field, and also could be used to engineer de novo histone affinity reagents; yet, published studies on their use with histones have been noticeably sparse. Here, we describe specific experimental conditions that affect binding specificities of post-translationally modified histones in classic protein engineering platforms and likely explain the relative difficulty with histone targets in these platforms. We also show that manipulating avidity of binding interactions may improve specificity of binding.

Thermodynamic modeling of genome-wide nucleosome depleted regions in yeast

Nucleosome positioning in the genome is essential for the regulation of many nuclear processes. We currently have limited capability to predict nucleosome positioning in vivo, especially the locations and sizes of nucleosome depleted regions (NDRs). Here, we present a thermodynamic model that incorporates the intrinsic affinity of histones, competitive binding of sequence-specific factors, and nucleosome remodeling to predict nucleosome positioning in budding yeast. The model shows that the intrinsic affinity of histones, at near-saturating histone concentration, is not sufficient in generating NDRs in the genome. However, the binding of a few factors, especially RSC towards GC-rich and poly(A/T) sequences, allows us to predict ~ 66% of genome-wide NDRs. The model also shows that nucleosome remodeling activity is required to predict the correct NDR sizes. The validity of the model was further supported by the agreement between the predicted and the measured nucleosome positioning upon factor deletion or on exogenous sequences introduced into yeast. Overall, our model quantitatively evaluated the impact of different genetic components on NDR formation and illustrated the vital roles of sequence-specific factors and nucleosome remodeling in this process.

High throughput screening of novel histone deacetylase inhibitors for epigenetic therapy of primary brain tumors

The complex interplay between histone deacetylases (HDACs) and histone acetyltransferases (HATs) plays a crucial role in the regulation of gene expression. By removing and adding acetyl groups to histone proteins, the affinity of histones and DNA is tightly regulated. As key players of epigenetic and posttranslational modification, HDACs therefore represent a promising therapeutic target in cancer harboring distinct epigenomes from normal cells. Consequently, HDAC inhibitors (HDACi) are increasingly evaluated in clinical trials, with four HDACi already approved by the FDA for treating lymphomas and multiple myelomas.

Abstract 5258: Inhibition of DNA methyltransferases and histone methyltransferases by plant flavones

Abstract Methylation of DNA and histone protein is an epigenetic modification mediated by DNA methyltransferases (DNMTs) and histone methyltransferases (HMTs) critically involved in methylation and regulation of affinity binding between the histones and DNA backbone. HMTs catalyze the transfer of one, two, or three methyl groups to lysine and arginine residues of histone proteins. The HMTs-mediated increase in histone affinity to DNA causes chromatin condensation, preventing transcription; whereas DNMTs methylate cytosine residues within gene promoters resulting in transcriptional repression. DNMT and HMT activities are reported to be associated with signal transduction, cell growth and death, as well as with the pathogenesis of various human diseases including cancer. Dietary plant flavones can affect epigenetic modifications accumulated over time and have shown health-beneficial effects. However, the epigenetic response on DNMT and HMT elicited by plant flavones has not been elucidated. Through in silico protein-ligand docking studies and molecular studies with plant flavones viz. chrysin, apigenin and luteolin and their effect on DNA and histone methylation was assessed. The ligands were individually docked into the pocket of DNMT and EZH2 using Glide in XP (extra precision) mode (Schrodinger, LLC). Binding of ligands with proteins were evaluated using GlideScore, which is an empirically derived scoring function. Virtual screening approach using the model of the catalytic site of DNMT and EZH2 demonstrated that plant flavones tethered at both ends inside the catalytic pocket of DNMT and EZH2 by means of hydrogen bonding. Flavones exhibited a high docking rank (Glide score) in the order of chrysin (-5.8), apigenin (-6.4), luteolin (-7.4) compared with the pharmacological inhibitor, 5-Aza-2′-deoxycytidine (-4.2). Notably, all three flavones inhibited EZH2, having a high docking rank compared to the known pharmacological inhibitor, 3-Deazaneplanocin A (DZNep). The docking rank for chrysin was -10.07, apigenin -9.73 and luteolin -11.23, compared to -7.62 for DZNep. Epigenetic studies performed with plant flavones demonstrated reversal of hypermethylation of cytosine bases in the DNA and prevented methylation of cytosine in the GC-rich promoter sequence incubated with MSsI enzyme. Furthermore, decrease protein expression of EZH2 and trimethylation of H3K27 was noted in prostate cancer cells treated with these plant flavones. Taken together, our results suggest that plant flavone can alter DNMT and HMT activities and methylation of DNA and histone proteins that regulate epigenetic modifications providing significant health-effects and prevent various pathological processes involved in the development of cancer. Citation Format: Rajnee Kanwal, Manish Datt, Sanjay Gupta. Inhibition of DNA methyltransferases and histone methyltransferases by plant flavones. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5258.

Constitutive hyperactivity of histone deacetylases enhances radioresistance in Lepidopteran Sf9 insect cells

BackgroundLepidopteran insect cells withstand multifold higher radiation doses and suffer far less DNA damage despite carrying numerous structural/functional homologies with mammalian cells. Since DNA–histone interactions significantly influence radiation-induced DNA damage, we investigated the role of histones in insect cell radioresistance. MethodsModified comet assay was used to assess the γ-radiation-induced DNA damage following serial histone depletion by varied salt concentrations. Acid–Urea–Triton (AUT) gel analysis combined with in silico predictions was used to compare mammalian and insect histones and acetylation status while HDAC activity was assessed/modified for studying the latter's role in radioresistance. Cell death was measured by morphological analysis and flow cytometry. ResultsHigh-salt extraction pattern from Sf9 nuclei suggested stronger DNA–histone affinity as the two core histones H2A/H2B could be extracted at much higher (2M) concentration as compared to 1.2M NaCl in mammalian (AA8) cells. Electrophoretic mobility of unirradiated Sf9 cells remained unaltered at all salt concentrations (0.14M–2M NaCl), and radiation-induced DNA damage increased only by 2M-NaCl pre-treatment. In silico analysis confirmed excellent conservation of Lepidopteran H2A/H2B sequence with human histones including comparable N-terminal lysine residues, yet these had ~60% lower acetylation. Importantly, insect cells showed ~70% higher histone deacetylase activity whose inhibition by Trichostatin-A reversed hypo-acetylation state and caused significant radiosensitization, thereby confirming the protective contribution of reduced acetylation. ConclusionOur study reveals that the hypo-acetylated state of well-conserved core histones, maintained by considerable HDAC activity, contributes significantly in Lepidopteran radioresistance. General SignificanceThis investigation shows constitutively high activity of HDACs as a potential radioprotective mechanism existing in insect cells.

Heparan Sulfate Modulates Neutrophil and Endothelial Function in Antibacterial Innate Immunity.

Recently, we showed that endothelial heparan sulfate facilitates entry of a bacterial pathogen into the central nervous system. Here, we show that normal bactericidal activity of neutrophils is influenced by the sulfation pattern of heparan sulfate. Inactivation of heparan sulfate uronyl 2-O-sulfotransferase (Hs2st) in neutrophils substantially reduced their bactericidal activity, and Hs2st deficiency rendered mice more susceptible to systemic infection with the pathogenic bacterium group B Streptococcus. Specifically, altered sulfation of heparan sulfate in mutant neutrophils affected formation of neutrophil extracellular traps while not influencing phagocytosis, production of reactive oxygen species, or secretion of granular proteases. Heparan sulfate proteoglycan(s) is present in neutrophil extracellular traps, modulates histone affinity, and modulates their microbial activity. Hs2st-deficient brain endothelial cells show enhanced binding to group B Streptococcus and are more susceptible to apoptosis, likely contributing to the observed increase in dissemination of group B Streptococcus into the brain of Hs2st-deficient mice following intravenous challenge. Taken together, our data provide strong evidence that heparan sulfate from both neutrophils and the endothelium plays important roles in modulating innate immunity.

DNA shape dominates sequence affinity in nucleosome formation.

Nucleosomes provide the basic unit of compaction in eukaryotic genomes, and the mechanisms that dictate their position at specific locations along a DNA sequence are of central importance to genetics. In this Letter, we employ molecular models of DNA and proteins to elucidate various aspects of nucleosome positioning. In particular, we show how DNA's histone affinity is encoded in its sequence-dependent shape, including subtle deviations from the ideal straight B-DNA form and local variations of minor groove width. By relying on high-precision simulations of the free energy of nucleosome complexes, we also demonstrate that, depending on DNA's intrinsic curvature, histone binding can be dominated by bending interactions or electrostatic interactions. More generally, the results presented here explain how sequence, manifested as the shape of the DNA molecule, dominates molecular recognition in the problem of nucleosome positioning.

Fitness landscape for nucleosome positioning

Histone-DNA complexes, so-called nucleosomes, are the building blocks of DNA packaging in eukaryotic cells. The histone-binding affinity of a local DNA segment depends on its elastic properties and determines its accessibility within the nucleus, which plays an important role in the regulation of gene expression. Here, we derive a fitness landscape for intergenic DNA segments in yeast as a function of two molecular phenotypes: their elasticity-dependent histone affinity and their coverage with transcription factor binding sites. This landscape reveals substantial selection against nucleosome formation over a wide range of both phenotypes. We use it as the core component of a quantitative evolutionary model for intergenic DNA segments. This model consistently predicts the observed diversity of histone affinities within wild Saccharomyces paradoxus populations, as well as the affinity divergence between neighboring Saccharomyces species. Our analysis establishes histone binding and transcription factor binding as two separable modes of sequence evolution, each of which is a direct target of natural selection.

Computational analysis suggests a highly bendable, fragile structure for nucleosomal DNA

Eukaryotic chromosomal DNA coils around histones to form nucleosomes. Although histone affinity for DNA depends on DNA sequence patterns, how nucleosome positioning is determined by them remains unknown. Here, we show relationships between nucleosome positioning and two structural characteristics of DNA conferred by DNA sequence. Analysis of bendability and hydroxyl radical cleavage intensity of nucleosomal DNA sequences indicated that nucleosomal DNA is bendable and fragile and that nucleosome positional stability was correlated with characteristics of DNA. This result explains how histone positioning is partially determined by nucleosomal DNA structure, illuminating the optimization of chromosomal DNA packaging that controls cellular dynamics.

Effects of carriers on transgene expression from plasmids containing a DNA sequence with high histone affinity

The intranuclear disposition of plasmid DNA is highly important for transgene expression. The effects of a left-handedly curved sequence with high histone affinity on transgene expression were examined in COS-7 cells with two kinds of carriers (Lipofectamine Plus and TransIT-LT1). Three plasmids containing the curved sequence at different positions were transfected. The transgene expression was affected by the position of the left-handedly curved sequence, and the sequence at appropriate locations enhanced the expression from plasmid DNAs. However, the position effects on the expression differed from those obtained by electroporation of the same plasmid DNAs in a naked form. In addition, the degree of expression enhancement seemed to depend on the carriers. These results suggest that the left-handedly curved sequence with high histone affinity could increase the transgene expression from a plasmid delivered with carriers.

Role of Jade-1 in the Histone Acetyltransferase (HAT) HBO1 Complex

Regulation of global chromatin acetylation is important for chromatin remodeling. A small family of Jade proteins includes Jade-1L, Jade-2, and Jade-3, each bearing two mid-molecule tandem plant homology domain (PHD) zinc fingers. We previously demonstrated that the short isoform of Jade-1L protein, Jade-1, is associated with endogenous histone acetyltransferase (HAT) activity. It has been found that Jade-1L/2/3 proteins co-purify with a novel HAT complex, consisting of HBO1, ING4/5, and Eaf6. We investigated a role for Jade-1/1L in the HBO1 complex. When overexpressed individually, neither Jade-1/1L nor HBO1 affected histone acetylation. However, co-expression of Jade-1/1L and HBO1 increased acetylation of the bulk of endogenous histone H4 in epithelial cells in a synergistic manner, suggesting that Jade1/1L positively regulates HBO1 HAT activity. Conversely, small interfering RNA-mediated depletion of endogenous Jade resulted in reduced levels of H4 acetylation. Moreover, HBO1-mediated H4 acetylation activity was enhanced severalfold by the presence of Jade-1/1L in vitro. The removal of PHD fingers affected neither binding nor mutual Jade-1-HBO1 stabilization but completely abrogated the synergistic Jade-1/1L- and HBO1-mediated histone H4 acetylation in live cells and in vitro with reconstituted oligonucleosome substrates. Therefore, PHDs are necessary for Jade-1/1L-induced acetylation of nucleosomal histones by HBO1. In contrast to Jade-1/1L, the PHD zinc finger protein ING4/5 failed to synergize with HBO1 to promote histone acetylation. The physical interaction of ING4/5 with HBO1 occurred in the presence of Jade-1L or Jade-3 but not with the Jade-1 short isoform. In summary, this study demonstrates that Jade-1/1L are crucial co-factors for HBO1-mediated histone H4 acetylation.

Epigenetics in Male Germ Cells

Epigenetics in Male Germ Cells

The use of Quantitative Agarose Gel Electrophoresis for rapid analysis of the integrity of protein–DNA complexes

Recent biochemical studies evaluated the affinity of histones to DNA in the context of nucleosome core particle (NCP). These have indicated a concentration-dependence for nucleosome stability. However, when studying chromatin the preferred templates are nucleosome arrays (NA) and not the NCP. Biochemical methods are poorly suited for structural analysis of chromatin. To overcome that technical hindrance, and investigate the effect of concentration on stability of the histone–DNA interactions, we have applied the multigel Quantitative Agarose Gel Electrophoresis (QAGE) method to in vitro-assembled nucleosomal arrays. The results demonstrated the method to be extremely valuable for the evaluation of the effect of low concentration on NA. However, QAGE is a fairly time-demanding and complex method. To maximize the efficiency of use of this technology, we devised a protocol that allowed for multiple sets of templates to be analyzed simultaneously. Briefly, samples can be loaded at regular intervals and analyzed individually for their molecular composition. The technique presented in this study describes the calibration steps and proof of concept necessary to validate the use of multiple loading of multigel to evaluate the composition of nucleosomal arrays as a function of concentration.

The location of the left-handedly curved DNA sequence affects exogenous DNA expression in vivo

The intranuclear disposition of a plasmid is extremely important for transgene expression. The effects of a left-handedly curved sequence with high histone affinity on plasmid expression were examined in vivo. A naked luciferase-plasmid was delivered into mouse liver by a hydrodynamics-based injection, and the luciferase activities were quantitated at various time points. The location of the left-handedly curved sequence determined the transgene expression, without affecting the amount of intranuclear exogenous DNA. The plasmid containing the curved sequence at the location that results in the exposure of the TATA box out of the nucleosome core showed the highest expression. These results suggest that sequences with high histone affinity could control transgene expression from plasmids in vivo.

Analyses of linker histone – chromatin interactions in situThis paper is one of a selection of papers published in this Special Issue, entitled 27th International West Coast Chromatin and Chromosome Conference, and has undergone the Journal's usual peer review process.

Recent studies, using cytometric techniques based on fluorescence microscopy, have provided new information on how linker histones interact with chromatin in vivo or in situ. In particular, the use of green fluorescent proteins (GFPs) has enabled detailed studies of how individual H1 subtypes, and specific motifs in them, interact with chromatin in vivo. Furthermore, the development of cytochemical methods to study the interaction between linker histones and chromatin using DNA-binding fluorochromes as indirect probes for linker histone affinity in situ, in combination with highly sensitive and specific analytical methods, has provided additional information on the interactions between linker histones and chromatin in several cell systems. Such results verified that linker histones have a substantially higher affinity for chromatin in mature chicken erythrocytes than in frog erythrocytes, and they also indicated that the affinity decreased during differentiation of the frog erythrocytes. Furthermore, in cultured human fibroblasts, the linker histones showed a relatively high affinity for chromatin in interphase, whereas it showed a significantly lower affinity in highly condensed metaphase chromosomes. This method also enables the analysis of linker histone affinity for chromatin in H1-depleted fibroblasts reconstituted with purified linker histones. No consistent correlation between linker histone affinity and chromatin condensation has so far been detected.

Intermediate filament proteins with nuclear functions: NuMA, lamin-like proteins and MFP1.

The -helical coiled coil is one of the principal subunit oligomerisation motifs in proteins, consisting of two or more -helices that twist around one another to form a supercoil. Coiled-coil proteins can be classified into two general categories. The first one comprises short coiledcoil domains called leucine zippers that form homoand hetero-dimers, as those found in transcription factors. The second class contains long coiled-coil domains with high polymerisation potential, which form a central rod configuration. The latter constitutes the intermediate filament (IF) protein family. The IF proteins are involved in structural organisation of cells, acting as scaffolds, fixing other proteins to solid state cell components, and also as cytoskeletal motors. They are also involved in molecular recognition and signal transduction (Burkhard et al., 2001). The IF proteins constitute a family of more than 40 different molecular species, classified into six main types (Coulombe et al., 2001); the group V corresponds to the lamins, nuclear proteins that play a role in nuclear organisation and replication (Schirmer et al., 2001). Some IFs associate with centromeres, nuclear matrix (NM), chromatin and the nuclear pore complex, and have DNA binding activity, histone affinity and similarity to NM proteins (Traub and Shoeman, 1994). In contrast to animals, only a reduced number of IFs have been identified in plants. Our previous ultrastructural studies had demonstrated a nucleoskeleton in plants containing both an organised structure underlying the nuclear envelope or lamina (Fig. 1A) (Moreno Diaz De La Espina, 1995) and an internal nucleoskeleton made up from branched knobbed 15–25 nm filaments, with a periodic organisation similar to those of the animal nucleoskeleton, as well as associated aggregates of different sizes (Fig. 1B,C) (Yu and Moreno Diaz De La Espina, 1999). Although from the 1990s we have made some contributions to the identification of IF and coiled-coil proteins in the plant matrix, such as lamins, NuMA and MFP1 (Fig. 1D–F), the molecular composition of the core filaments remains unknown. Nuclear mitotic apparatus protein (NuMA) is a 200– 240 kD coiled-coil protein first identified in animals. NuMA is a prominent component of nuclear core filaments. During mitosis, it relocates to the mitotic spindle and associates with motor proteins, contributing to microtubule stabilisation and spindle organisation. Although its functions during mitosis are well established, studies on its nuclear function are scarce (Taimen et al., 2000). Using an immunological approach, three onion homologues of NuMA, of 210, 220 and 230 kD (Fig. 1D), were identified in the filaments of its nucleoskeleton (Fig. 1G) and in the mitotic spindle (Yu and Moreno Diaz De La Espina, 1999). The potential of NuMA oligomers to form higher order structures in animals (Harborth et al., 1999) and the localisation of AcNuMA in core filaments, suggest its structural role in plant nuclear organisation. Lamin-like proteins with conserved Mr and pI values, antigenically related to animal A and B lamins, were detected in the lamina and the internal NM of several plants (Fig. 1E,H) (see Moreno Diaz De La Espina, 1995 for a review). In spite of all their similarities and of the important nuclear functions played by lamins (Schirmer et al., 2001), lamin genes have not been identified in plants as yet, suggesting that they could have distinctly related nucleoskeletal proteins that functionally replace lamins. Recently, Gindullis et al. (2002) reported a novel family of long coiled-coil proteins from plants, which represents a good candidate for the abovementioned fact. * Corresponding author. Tel.: +34-91-561-1800; fax: +34-91-562-7518. E-mail address: smoreno@cib.csic.es (S. Moreno Diaz De La Espina). Cell Biology International 27 (2003) 233–235 Cell Biology International

Linker Histone Subtype Composition and Affinity for Chromatin in Situ in Nucleated Mature Erythrocytes

The replacement linker histones H1(0) and H5 are present in frog and chicken erythrocytes, respectively, and their accumulation coincides with cessation of proliferation and compaction of chromatin. These cells have been analyzed for the affinity of linker histones for chromatin with cytochemical and biochemical methods. Our results show a stronger association between linker histones and chromatin in chicken erythrocyte nuclei than in frog erythrocyte nuclei. Analyses of linker histones from chicken erythrocytes using capillary electrophoresis showed H5 to be the subtype strongest associated with chromatin. The corresponding analyses of frog erythrocyte linker histones using reverse-phase high performance liquid chromatography showed that H1(0) dissociated from chromatin at somewhat higher ionic strength than the three additional subtypes present in frog blood but at lower ionic strength than chicken H5. Which of the two H1(0) variants in frog is expressed in erythrocytes has thus far been unknown. Amino acid sequencing showed that H1(0)-2 is the only H1(0) subtype present in frog erythrocytes and that it is 100% acetylated at its N termini. In conclusion, our results show differences between frog and chicken linker histone affinity for chromatin probably caused by the specific subtype composition present in each cell type. Our data also indicate a lack of correlation between linker histone affinity and chromatin condensation.