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Although the overall structure of the fifth histone (linker histone, H1) is understood, its location on the nucleosome is only partially defined. Whilst it is clear that H1 helps condense the chromatin fibre, precisely how this is achieved remains to be determined. H1 is not a general gene repressor in that although it must be displaced from transcription start sites for activity to occur, there is only partial loss along the body of genes. How the deposition and removal of H1 occurs in particular need of further study. Linker histones are highly abundant nuclear proteins about which we know too little., (Copyright © 2015. Published by Elsevier B.V.)

In the present review, we summarize advances in our knowledge on the role of the histone H1 family of proteins in breast cancer cells, focusing on their response to progestins. Histone H1 plays a dual role in gene regulation by hormones, both as a structural component of chromatin and as a dynamic modulator of transcription. It contributes to hormonal regulation of the MMTV promoter by stabilizing a homogeneous nucleosome positioning, which reduces basal transcription whereas at the same time promoting progesterone receptor binding and nucleosome remodeling. These combined effects enhance hormone dependent gene transcription, which eventually requires H1 phosphorylation and displacement. Various isoforms of histone H1 have specific functions in differentiated breast cancer cells and compact nucleosomal arrays to different extents in vitro. Genome-wide studies show that histone H1 has a key role in chromatin dynamics of hormone regulated genes. A complex sequence of enzymatic events, including phosphorylation by CDK2, PARylation by PARP1 and the ATP-dependent activity of NURF, are required for H1 displacement and gene de-repression, as a prerequisite for further nucleosome remodeling. Similarly, during hormone-dependent gene repression a dedicated enzymatic mechanism controls H1 deposition at promoters by a complex containing HP1γ, LSD1 and BRG1, the ATPase of the BAF complex. Thus, a broader vision of the histone code should include histone H1, as the linker histone variants actively participate in the regulation of the chromatin structure. How modifications of the core histones tails affect H1 modifications and vice versa is one of the many questions that remains to be addressed to provide a more comprehensive view of the histone cross-talk mechanisms., (Copyright © 2015 Elsevier B.V. All rights reserved.)

H1 linker histones are involved both in the maintenance of higher-order chromatin structure and in gene regulation. Histone H1 exists in multiple isoforms, is evolutionarily variable and undergoes a large variety of post-translational modifications. We review recent progress in the understanding of the folding and structure of histone H1 domains with an emphasis on the interactions with DNA. The importance of intrinsic disorder and hydrophobic interactions in the folding and function of the carboxy-terminal domain (CTD) is discussed. The induction of a molten globule-state in the CTD by macromolecular crowding is also considered. The effects of phosphorylation by cyclin-dependent kinases on the structure of the CTD, as well as on chromatin condensation and oligomerization, are described. We also address the extranuclear functions of histone H1, including the interaction with the β-amyloid peptide., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Linker histones are three domain proteins and consist of a structured (globular) domain, flanked by two likely non-structured NH2- and COOH-termini. The binding of the linker histones to the nucleosome was characterized by different methods in solution. Apparently, the globular domain interacts with the linker DNA and the nucleosome dyad, while the binding of the large and rich in lysines COOH-terminus results in "closing" the linker DNA of the nucleosome and the formation of the "stem" structure. What is the mode of binding of the linker histones within the chromatin fiber remains still elusive. Nonetheless, it is clear that linker histones are essential for both the assembly and maintenance of the condensed chromatin fiber. Interestingly, linker histones are post-translationally modified and how this affects both their binding to chromatin and functions is now beginning to emerge. In addition, linker histones are highly mobile in vivo, but not in vitro. No explanation of this finding is reported for the moment. The higher mobility of the linker histones should, however, have strong impact on their function. Linker histones plays an important role in gene expression regulation and other chromatin related process and their function is predominantly regulated by their posttranslational modifications. However, the detailed mechanism how the linker histones do function remains still not well understood despite numerous efforts. Here we will summarize and analyze the data on the linker histone binding to the nucleosome and the chromatin fiber and will discuss its functional consequences., (Copyright © 2015 Elsevier B.V. All rights reserved.)

H1 linker histones are key chromatin architectural proteins facilitating the formation of higher order chromatin structures. The H1 family constitutes the most heterogeneous group of histone proteins, with eleven non-allelic H1 variants in mammals. H1 variants differ in their biochemical properties and exhibit significant sequence divergence from one another, yet most of them are highly conserved during evolution from mouse to human. H1 variants are differentially regulated during development and their cellular compositions undergo dramatic changes in embryogenesis, gametogenesis, tissue maturation and cellular differentiation. As a group, H1 histones are essential for mouse development and proper stem cell differentiation. Here we summarize our current knowledge on the expression and functions of H1 variants in mammalian development and stem cell differentiation. Their diversity, sequence conservation, complex expression and distinct functions suggest that H1s mediate chromatin reprogramming and contribute to the large variations and complexity of chromatin structure and gene expression in the mammalian genome., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Chromatin-related proteins have emerged as important players in the initiation and maintenance of several types of cancer. In addition to the established role of histone-modifying enzymes and chromatin remodelers in promoting and sustaining malignant phenotypes, recent findings suggest that the basic components of chromatin, the histone proteins, also suffer severe alterations in cancer and may contribute to the disease. Histopathological examination of clinical samples, characterization of the mutational landscape of various types of cancer and functional studies in cancer cell lines have highlighted the linker histone H1 both as a potential biomarker and a driver in cancer. This review summarizes H1 abnormalities in cancer identified by various approaches and critically discusses functional implications of such alterations, as well as potential mechanisms through which they may contribute to the disease., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Many metazoan cell types differentially express multiple non-allelic amino acid sequence variants of histone H1. Although early work revealed that H1 variants, collectively, are phosphorylated during interphase and mitosis, differences between individual H1 variants in the sites they possess for mitotic and interphase phosphorylation have been elucidated only relatively recently. Here, we review current knowledge on the regulation and function of interphase H1 phosphorylation, with a particular emphasis on how differences in interphase phosphorylation among the H1 variants of mammalian cells may enable them to have differential effects on transcription and other chromatin processes., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Histone H1 is a structural component of chromatin that may have a role in the regulation of chromatin dynamics. Unlike core histones, the linker histone H1 family is evolutionarily diverse and many organisms have multiple H1 variants or subtypes, distinguishable between germ-line and somatic cells. In mammals, the H1 family includes seven somatic H1 variants with a prevalence that varies between cell types and over the course of differentiation, H1.1 to H1.5 being expressed in a replication-dependent manner, whereas H1.0 and H1X are replication-independent. Until recently, it has not been known whether the different variants had specific roles in the regulation of nuclear processes or were differentially distributed across the genome. To address this, an increasing effort has been made to investigate divergent features among H1 variants, regarding their structure, expression patterns, chromatin dynamics, post-translational modifications and genome-wide distribution. Although H1 subtypes seem to have redundant functions, several reports point to the idea that they are also differently involved in specific cellular processes. Initial studies investigating the genomic distribution of H1 variants have started to suggest that despite a wide overlap, different variants may be enriched or preferentially located at different chromatin types, but this may depend on the cell type, the relative abundance of the variants, the differentiation state of the cell, or whether cells are derived from a neoplastic process. Understanding the heterogeneity of the histone H1 family is crucial to elucidate their role in chromatin organization, gene expression regulation and other cellular processes.

The dynamic interaction of nucleosome binding proteins with their chromatin targets is an important element in regulating the structure and function of chromatin. Histone H1 variants and High Mobility Group (HMG) proteins are ubiquitously expressed in all vertebrate cells, bind dynamically to chromatin, and are known to affect chromatin condensation and the ability of regulatory factors to access their genomic binding sites. Here, we review the studies that focus on the interactions between H1 and HMGs and highlight the functional consequences of the interplay between these architectural chromatin binding proteins. H1 and HMG proteins are mobile molecules that bind to nucleosomes as members of a dynamic protein network. All HMGs compete with H1 for chromatin binding sites, in a dose dependent fashion, but each HMG family has specific effects on the interaction of H1 with chromatin. The interplay between H1 and HMGs affects chromatin organization and plays a role in epigenetic regulation., (Published by Elsevier B.V.)

Background: Linker histone H1 is a structural component of chromatin. It exists as a family of related proteins known as variants and/or subtypes. H1.1, H1.2, H1.3, H1.4 and H1.5 are present in most somatic cells, whereas other subtypes are mainly expressed in more specialized cells., Scope of Review: H1 subtypes have been shown to have unique functions in chromatin structure and dynamics. This can occur at least in part via specific post-translational modifications of distinct H1 subtypes. However, while core histone modifications have been extensively studied, our knowledge of H1 modifications and their molecular functions has remained for a long time limited to phosphorylation. In this review we discuss the current state of knowledge of linker histone H1 modifications and where possible highlight functional differences in the modifications of distinct H1 subtypes., Major Conclusions and General Significance: H1 histones are intensely post-translationally modified. These modifications are located in the N- and C-terminal tails as well as within the globular domain. Recently, advanced mass spectrometrical analysis revealed a large number of novel histone H1 subtype specific modification sites and types. H1 modifications include phosphorylation, acetylation, methylation, ubiquitination, and ADP ribosylation. They are involved in the regulation of all aspects of linker histone functions, however their mechanism of action is often only poorly understood. Therefore systematic functional characterization of H1 modifications will be necessary in order to better understand their role in gene regulation as well as in higher-order chromatin structure and dynamics., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Eukaryotic genomes are structured in the form of chromatin with the help of a set of five small basic proteins, the histones. Four of them are highly conserved through evolution, form the basic unit of the chromatin, the nucleosome, and have been intensively studied and are well characterized. The fifth histone, histone H1, adds to this basic structure through its interaction at the entry/exit site of DNA in the nucleosome and makes an essential contribution to the higher order folding of the chromatin fiber. Histone H1 is the less conserved histone and the less known of them. Though for long time considered as a general repressor of gene expression, recent studies in Drosophila have rejected this view and have contributed to uncover important functions on genome stability and development. Here we present some of the most recent data obtained in the Drosophila model system and discuss how the lessons learnt in these studies compare and could be applied to all other eukaryotes.

Linker histones H1 are ubiquitous chromatin proteins that play important roles in chromatin compaction, transcription regulation, nucleosome spacing and chromosome spacing. H1 function in DNA and chromatin structure stabilization is well studied and established. The current paradigm of linker histone mode of function considers all other cellular roles of linker histones to be a consequence from H1 chromatin compaction and repression. Here we review the multiple processes regulated by linker histones and the emerging importance of protein interactions in H1 functioning. We propose a new paradigm which explains the multi functionality of linker histones through linker histones protein interactions as a way to directly regulate recruitment of proteins to chromatin., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Glucocorticoids are known to induce or repress the expression of a wide variety of genes with roles in various biological processes such as the circadian clock and the stress response. We studied the changes in the levels of two histone H3 post-translational modifications associated with active chromatin, H3 trimethylated at lysine 4 (H3K4me3) and H3 acetylated at lysines 9/14 (H3K9/14ac), that take place in the promoters of two glucocorticoid early response genes, Per1 and Sgk1, during their induction by the synthetic glucocorticoid, dexamethasone. Sgk1 mediates the effects of acute and chronic stress on the prefrontal cortex and other parts of the brain, while Per1 is a core circadian clock gene whose expression is strongly induced by the increased levels of blood-borne glucocorticoids that accompany acute and chronic stress. Here we show that dexamethasone rapidly increases the levels of H3K4me3 and H3K9/14ac in the promoters of both genes. Furthermore, the effect of dexamethasone on these genes, regarding both mRNA levels and the abundance of H3K4me3 and H3K9/14ac in their promoters, can be inhibited by the presence of nicotinamide, a metabolic molecule which has been shown to possess anxiolytic properties., (Copyright © 2014 Elsevier B.V. All rights reserved.)

FACT is a roughly 180 kDa heterodimeric protein complex important for managing the properties of chromatin in eukaryotic cells. Chromatin is a repressive barrier that plays an important role in protecting genomic DNA and regulating access to it. This barrier must be temporarily removed during transcription, replication, and repair, but it also must be rapidly restored to the original state afterwards. Further, the properties of chromatin are dynamic and must be adjusted as conditions dictate. FACT was identified as a factor that destabilizes nucleosomes in vitro, but it has now also been implicated as a central factor in the deposition of histones to form nucleosomes, as an exchange factor that swaps the histones within existing nucleosomes for variant forms, and as a tether that prevents histones from being displaced by the passage of RNA polymerases during transcription. FACT therefore plays central roles in building, maintaining, adjusting. and overcoming the chromatin barrier. This review summarizes recent results that have begun to reveal how FACT can promote what appear to be contradictory goals, using a simple set of binding activities to both enhance and diminish the stability of nucleosomes. This article is part of a Special Issue entitled: Histone chaperones and Chromatin assembly.

Chromatin is a dynamic complex of DNA and proteins that regulates the flow of information from genome to end product. The efficient recognition and faithful repair of DNA damage, particularly double-strand damage, is essential for genomic stability and cellular homeostasis. Imperfect repair of DNA double-strand breaks (DSBs) can lead to oncogenesis. The efficient repair of DSBs relies in part on the rapid formation of foci of phosphorylated histone H2AX (γ-H2AX) at each break site, and the subsequent recruitment of repair factors. These foci can be visualized with appropriate antibodies, enabling low levels of DSB damage to be measured in samples obtained from patients. Such measurements are proving useful to optimize treatments involving ionizing radiation, to assay in vivo the efficiency of various drugs to induce DNA damage, and to help diagnose patients with a variety of syndromes involving elevated levels of γ-H2AX. We will survey the state of the art of utilizing γ-H2AX in clinical settings. We will also discuss possibilities with other histone post-translational modifications. The ability to measure in vivo the responses of individual patients to particular drugs and/or radiation may help optimize treatments and improve patient care. This article is part of a Special Issue entitled: Chromatin in time and space., (Published by Elsevier B.V.)

Numerous studies of nucleosome positioning have shown that nucleosomes almost invariably adopt one of several alternative overlapping positions on a short DNA fragment in vitro. We define such a set of overlapping positions as a "position cluster", and the 5S RNA gene positioning sequence is presented as an example. The notable exception is the synthetic 601-sequence, which can position a nucleosome perfectly in vitro, though not in vivo. Many years ago, we demonstrated that nucleosome position clusters are present on the CUP1 and HIS3 genes in native yeast chromatin. Recently, using genome-wide paired-end sequencing of nucleosomes, we have shown that position clusters are the general rule in yeast chromatin, not the exception. We argue that, within a cell population, one of several alternative nucleosomal arrays is formed on each gene. We show how position clusters and alternative arrays can give rise to typical nucleosome occupancy profiles, and that position clusters are disrupted by transcriptional activation. The centromeric nucleosome is a rare example of perfect positioning in vivo. It is, however, a special case, since it contains the centromeric histone H3 variant instead of normal H3. Perfect positioning might be due to centromeric sequence-specific DNA binding proteins. Finally, we point out that the existence of position clusters implies that the putative nucleosome code is degenerate. We suggest that degeneracy might be a crucial point in the debate concerning the code. This article is part of a Special Issue entitled: Chromatin in time and space., (Published by Elsevier B.V.)

To study the structural and functional changes accompanying the integration of histone H5 into the nucleosome structure, linear DNA species have been employed with a terminal promoter for bacteriophage T7 RNA polymerase followed by tandem repeats of a 207-bp nucleosome positioning sequence. The oligonucleosomes assembled from 12-repeat DNA and saturating amounts of core histone octamer plus histone H5 are compacted, in the presence of 1 mM free magnesium ions, to the level of the 30-nm fiber. Under these ionic conditions the efficiency in RNA synthesis and the size distribution of RNA chains obtained with this template are the same as those corresponding to the template without H5, indicating that the 30-nm fiber stabilized by H5 does not impair RNA elongation. Therefore, under our experimental conditions, incorporation of one molecule of histone H5 per nucleosome does not affect elongation of RNA even when a folded structure is produced. However, elongation is inhibited by binding of an excess of H5.

Histone H4 is one of the most conserved proteins known. The very low rate of nonsynonymous substitution in H4 suggests that it fulfills an essential function in virtually all eukaryotes. While the majority of histone H4 sequences differ only slightly from the general consensus H4 sequence, yeast and Tetrahymena sequences diverge substantially from both the consensus and from each other. This study demonstrates that despite this divergence, when Saccharomyces cerevisiae cells are forced to use the Tetrahymena thermophila histone H4 protein, they are viable although they have a reduced growth rate, are temperature-sensitive relative to wild-type, have a lengthened G2 phase, and show a dramatic repression of mating. An amino acid replacement at position 33 in the protein improves the growth rate of these cells growing at temperatures above 28 degrees C. This replacement changes a proline to a serine and is a further divergence from both the Tetrahymena thermophila and Saccharomyces cerevisiae histone H4 sequences. Thus, the replacement and expression of a non wild-type histone H4 in yeast offers measurable effects on cell growth, identifying amino acids required for optimal yeast functioning.

H1 exerts a specific in vitro inhibitory effect on enzymic DNA methylation. The experiments reported in this paper were undertaken in order to assess whether the lower methylation level found in internucleosomal DNA compared to core DNA is the in vivo consequence of the well-known localization of this histone in the linker region, as opposed to a possible deficiency of CpG dinucleotides in linker DNA. The methyl-accepting ability of H1-depleted oligonucleosomes from human placenta and of the corresponding core particles were assayed by addition of purified DNA methyltransferase, using S-adenosylmethionine as the methyl group donor. We have found that approx. 80% of newly-incorporated methyl groups are localized in linker DNA, which is indeed a good potential substrate for enzymic DNA methylation. Addition of quasi-physiological amounts of H1 to H1-depleted oligonucleosomes markedly reduced their methyl-accepting ability, while exerting a re-condensing effect on these particles, as revealed by the distortions of their circular dichroism spectra.

Ustilago maydis topoisomerase I relaxes superhelical DNA in the absence of any co-factors. The reaction reaches a defined end-point proportional to the amount of enzyme added and an analysis of the reaction by Hill plot transformation indicates that at least two molecules of topoisomerase must interact with the DNA to catalyze relaxation. The addition of purified Ustilago histone H1 reduces the stoichiometric amount of topoisomerase I required by 50%. H1 histone may function to enhance DNA relaxation through a cooperative mechanism. The purified HMG-like protein from Ustilago also enhances DNA relaxation mediated by the topoisomerase. Whereas H1 stimulates topo I-mediated DNA relaxation through a processive mode, the HMG-like protein enhances through a distributive mechanism. Taken together, these results demonstrate that the interaction of chromosomal proteins with topoisomerase can influence DNA topology, and mechanisms are proposed to explain this enhancement.

Crude preparations of histones had insulin-like actions in isolated adipocytes. This activity was attributed to the arginine-rich histones, H3 and H4. The metabolic effects of purified H3 and H4 on isolated adipocytes were similar to those of insulin in a number of respects. Like insulin, H3 and H4 stimulated the incorporation of both glucose and pyruvate in isolated cells and stimulated intercellular oxidation of glucose; in contrast, the lipolytic agents ACTH and isoproterenol actually inhibited the incorporation of pyruvate into adipocytes. In contrast to the effects of the lipolytic hormones, the effects of H3 and H4, like insulin, were not blocked by the presence of adenosine deaminase in the medium. The same concentrations of phenylarsine oxide were required to inhibit the stimulation of glucose incorporation whether by insulin or by histones. Furthermore, the addition of H4 or insulin to isolated adipocytes resulted in the increased phosphorylation of 17 kDa phosphoproteins as detected by two-dimensional electrophoresis. The insulin-like effect of the active histones was specific to their structure. Lysine-rich histones (H1, H2A and H2B), various polycations, and proteolytic fragments of purified H3 or H4 were all inactive. It is unknown whether this phenomenon might imply a physiological function for such endogenous molecules; however, a comparison of the detailed effects of insulin and histones might be informative in terms of common intracellular transduction systems.

The potential of nucleosome assembly along the sequence of a plasmid carrying the long terminal repeat (LTR) and its flanking region of Moloney murine leukemia virus was analyzed by in vitro reconstitution experiments with histones from chicken erythrocytes. The results of electrophoretic mobility-shift and micrococcal nuclease-digestion assays indicated that the plasmid DNA contained four preferred sites for nucleosome formation. However, all of these sites were mapped on the vector moiety but not on the LTR moiety. Computer analysis of the sequences in the four preferred sites, each spanning about 150 bp, indicated that short runs of (dA,dT) containing two kinds of triplets, AAA/TTT and AAT/ATT, occurred frequently. Furthermore, many of these triplets tended to occur in the same side of the DNA helix, suggesting that DNA curvature was involved in the preferred sites for nucleosome assembly. Consistent was the observation that DNA fragments carrying these preferred sites showed anomalous electrophoretic mobilities at a low temperature.

The effect of binding a high mobility group protein (HMG 17) on the stability and conformation of acetylated and control HeLa high molecular weight core chromatin (stripped of H1 and non-histone chromosomal proteins) was studied by circular dichroism and thermal-denaturation measurements. Previously it had been shown that conformational differences exist between native whole chromatin derived from butyrate-treated (acetylated) and control HeLa cells and that these conformational differences disappear by removing H1 and non-histone chromosomal proteins ( Reczek , P.R., Weissman , D., Huvos , P.E. and Fasman, G.D. (1982) Biochemistry 21, 993-1002). The circular dichroism spectra and the thermal denaturation profiles of control and acetylated core chromatin were found to be similar. The circular dichroism properties of HMG 17 reconstituted highly acetylated and control core chromatin indicated the same alteration of chromatin structure at low ionic strength (1 mM sodium phosphate/0.25 mM EDTA, pH 7.0). The magnitudes of the decrease in ellipticity were proportional to the amount of HMG 17 bound and were found to be the same for both the acetylated and control core chromatin. Thermal denaturation profiles confirmed this change in structure induced by HMG 17 on control and highly acetylated core chromatin. The thermal denaturation profiles, which were resolved into three component transitions, exhibited a shifting of hyperchromicity from the lower melting transitions to the higher melting transitions, with a concomitant rise in Tm, on HMG 17 binding to both control and acetylated chromatin. The natures of the interactions of HMG 17 at higher ionic strength (50 mM NaCl/0.25 mM EDTA/1 mM sodium phosphate, pH 7.0) with acetylated and control core chromatin were slightly different, as measured by circular dichroism; however, a decrease in ellipticity was observed for both samples upon binding of HMG 17. These observations suggest that acetylation coupled with HMG 17 binding to core chromatin does not loosen chromatin structure. HMG 17 binding to control and acetylated core chromatin produces an overall stabilization and compaction of chromatin structure.

Equilibrium and hydrodynamic studies on the complex of actinomycin D with H1-H5 depleted, 175 basepair nucleosomes are reported. By spectral titration the intrinsic affinities of actinomycin D for nucleosomes and for DNA are found strictly comparable. Sedimentation analysis shows that actinomycin can apparently unfold the nucleosome, like ethidium bromide and daunomycin, but it does so at a much lower bound drug to DNA molar ratio (about 1 drug molecule to 45 basepairs). Since about four bound actinomycin molecules are able to induce the reversible conformational transition of a nucleosome, it is suggested that the sites of interaction may correspond to the kinked DNA sites evidenced by Klug and collaborators (Richmond, T.J., Finch, J.T., Rushton, B., Rhodes, D. and Klug, A. (1984) Nature 311, 532-537) in the structure of the nucleosome. A relevance of these findings to the interaction of actinomycin with "active chromatin" is also suggested.

Two protein phosphatases were isolated from rat liver nuclei. The enzymes, solubilized from crude chromatin by 1 M NaCl, were resolved by column chromatography on Sephadex G-150, DEAE-Sepharose and heparin-Sepharose. The phosphorylase phosphatase activity of one of the enzymes (inhibitor-sensitive phosphatase) was inhibited by heat-stable phosphatase inhibitor proteins and also by histone H1. This phosphatase had a molecular weight of approx. 35,000 both before and after 4 M urea treatment. Its activity was specific for the beta-subunit of phosphorylase kinase. Pretreatment with 0.1 mM ATP inhibited the enzyme only about 10%, and it did not require divalent cations for activity. On the basis of these properties, this nuclear enzyme was identified as the catalytic subunit of phosphatase 1. The other phosphatase (polycation-stimulated phosphatase) was insensitive to inhibition by inhibitor 1, and it was stimulated 10-fold by low concentrations of histone H1 (A0.5 = 0.6 microM). This enzyme had a molecular weight of approx. 70,000 which was reduced to approx. 35,000 after treatment with 4 M urea. It dephosphorylated both the alpha- and beta-subunits of phosphorylase kinase. The enzyme was inhibited more than 90% by preincubation with 0.1 mM ATP and did not require divalent cations for activity. On the basis of these properties, this nuclear enzyme was identified as phosphatase 2A.

Digestion of sea urchin sperm nuclei with micrococcal nuclease yields nucleosomal monomer fragments of 151 and 164 base pairs. Prior trypsin treatment of the sperm chromatin does not alter the size of these monomer DNA fragments despite the fact that the H1 histone is reduced to a limit globular peptide of about 83 residues. Heterologous reconstitution experiments show that this peptide is capable of protecting an extra 22 base pairs beyond the core particle in a chromatosome. Nuclease digestion of reconstitutes from DNA and sperm core histones yields a core monomer of about 141 base pairs. It is concluded that this sperm chromatin contains a chromatosome of 164 bp essentially similar to that observed in the more usual chromatins. Edman degradation of the H1 limit peptide shows its sequence to be closely analogous to the corresponding peptide of calf H1 and chicken H5. Circular dichroism studies of histone H1 from the sperm of three sea urchin species demonstrate the presence of trypsin-sensitive helical regions outside the globular domain that are absent in calf H1 and chicken H5.