Developmental and tissue expression patterns of histone macroH2A1 subtypes

MacroH2A is a novel nucleosomal core histone that contains a large nonhistone region and a region that closely resembles a full length histone H2A. We have cloned a cDNA that contains the entire coding region of macroH2A1.2, one of the two identified subtypes of macroH2A1. MacroH2A1.2 was found to differ from the other known subtype, macroH2A1.1, in a single segment of the nonhistone region. MacroH2A1 specific antibodies revealed relatively high levels of both subtypes in adult liver and kidney. MacroH2A1.1 was much lower in fetal liver and kidney in comparison to their adult counterparts, and was not detected in adult thymus and testis, tissues with active cell division and differentiation. Both subtypes were present at very low levels or absent from mouse embryonic stem cells maintained in an undifferentiated state by growth in the presence of leukemia inhibitory factor. MacroH2A1.2 increased when the embryonic stem cells were induced to differentiate in vitro, while macroH2A1.1 remained undetectable. These results support the idea that macroH2A1.1 and macroH2A1.2 are functionally distinct, and suggest that changes in their expression may play a role in developmentally regulated changes in chromatin structure and function. J. Cell. Biochem. 65:107–113. © 1997 Wiley-Liss, Inc.     

Calculated flexibility of histone proteins correlate with mammalian histone H1 subtype.

We have calculated the polypeptide flexibility index for mammalian histone H1 sequences obtained from the National Center for Biotechnology Information Histone Sequence Database. This database contains over 1000 histone protein entries, from various species, compiled from SWISS_PROT, PIR, the Protein Data Bank (PDB), and CDS translations from GenBank. Histone H1 proteins were analyzed because of their critical role in chromatin structure and gene expression. Flexibility calculations revealed that histone subtype H1.0, which accumulates during terminal differentiation, has the highest flexibility index of all mammalian H1 subtypes. Other mammalian H1 subtypes had lower flexibility indices, including the human H1.2 subtype whose mRNA contains both a hairpin loop sequence and a poly(A) addition sequence. Histone mRNAs containing both of these structures have been shown to be expressed prior to and after terminal differentiation, yet these proteins do not necessarily accumulate in the chromatin of terminally differentiated cells. H1.2 and the H1.t have the lowest flexibility index (most ridged) of all human H1 subtypes. All human H1 proteins of the replication dependent subtypes have intermediate values for their flexibility indices.     

Quantitative changes in the expression of histone H1 and H2B subtypes and their relationship to the differentiation of mouse embryonal carcinoma cells

The pattern of histones from several mouse embryonal carcinoma cell (ECC) lines, differentiated cell lines, and adult organs was analyzed using acid-urea gels containing Triton X-100 and long SDS-gel electrophoresis. All cell lines had comparable histone types except for a unique H2B-like component that was found only in the ECC line PCC4. The mouse histone H1 has four different subtypes (H1a, H1b, H1c, and H1d), as resolved in SDS-gel electrophoresis. The expression of the four subtypes was shown to be cell line specific. Subtypes H1a and H1d are present in approximately the same relative amounts in all cell lines investigated. Subtype H1b is found in higher relative amounts than subtype H1c in ECC lines and testis. The ratio of H1b and H1c is reversed in differentiated cell lines and in kidney, white blood cells, liver and spleen. All four subtypes of H1 are phosphorylated although to a different extent in different cell lines. In ECC lines, subtypes H1b and especially H1d incorporate most of a ³²P label, whereas H1c is predominately phosphorylated in differentiated parietal endoderm cell lines. These data indicate that H1 subtypes differ depending on the stage of cell differentiation. Difference in ratio between H1 subtypes and in phosphorylation might influence the chromatin configuration and thus gene expression in these cells.     

Increased histone H1(0) expression in differentiating mouse erythroleukemia cells is related to decreased cell proliferation.

The amount of histone H1(0) increases relative to other H1 subtypes in terminally differentiated cells, and its expression has been associated with the onset of differentiation. We have studied the kinetics of H1(0) accumulation in mouse erythroleukemia (MEL) cells and found that the levels of H1(0) reflect the rate of cell proliferation rather than the state of differentiation. This suggests that changes in the relative amount of H1(0) during MEL cell differentiation are primarily a consequence of cell cycle arrest.     

The relative proportion of H1° and A24 is reversed in oligodendrocytes during rat brain development

1. The histone complement of oligodendrocyte chromatin at different stages of brain development was studied after acid extraction of nuclei. 2. HCl-soluble proteins were analyzed by different electrophoretic techniques. 3. Our results show an increase in the concentration of histone H1(0) with differentiation. 4. The increase in H1(0) is accompanied by a concomitant decrease in the total amount of the ubiquitinated form of histone H2A (A24).     

Binding Dynamics of Disordered Linker Histone H1 with a Nucleosomal Particle

Linker histone H1 is an essential regulatory protein for many critical biological processes, such as eukaryotic chromatin packaging and gene expression. Mis-regulation of H1s is commonly observed in tumor cells, where the balance between different H1 subtypes has been shown to alter the cancer phenotype. Consisting of a rigid globular domain and two highly charged terminal domains, H1 can bind to multiple sites on a nucleosomal particle to alter chromatin hierarchical condensation levels. In particular, the disordered H1 amino- and carboxyl-terminal domains (NTD/CTD) are believed to enhance this binding affinity, but their detailed dynamics and functions remain unclear. In this work, we used a coarse-grained computational model, AWSEM-DNA, to simulate the H1.0b-nucleosome complex, namely chromatosome. Our results demonstrate that H1 disordered domains restrict the dynamics and conformation of both globular H1 and linker DNA arms, resulting in a more compact and rigid chromatosome particle. Furthermore, we identified regions of H1 disordered domains that are tightly tethered to DNA near the entry-exit site. Overall, our study elucidates at near-atomic resolution the way the disordered linker histone H1 modulates nucleosome’s structural preferences and conformational dynamics.     

A novel role for the Aurora B kinase in epigenetic marking of silent chromatin in differentiated postmitotic cells

Combinatorial modifications of the core histones have the potential to fine-tune the epigenetic regulation of chromatin states. The Aurora B kinase is responsible for generating the double histone H3 modification tri-methylated K9/phosphorylated S10 (H3K9me3/S10ph), which has been implicated in chromosome condensation during mitosis. In this study, we have identified a novel role for Aurora B in epigenetic marking of silent chromatin during cell differentiation. We find that phosphorylation of H3 S10 by Aurora B generates high levels of the double H3K9me3/S10ph modification in differentiated postmitotic cells and also results in delocalisation of HP1beta away from heterochromatin in terminally differentiated plasma cells. Microarray analysis of the H3K9me3/S10ph modification shows a striking increase in the modification across repressed genes during differentiation of mesenchymal stem cells. Our results provide evidence that the Aurora B kinase has a role in marking silent chromatin independently of the cell cycle and suggest that targeting of Aurora B-mediated phosphorylation of H3 S10 to repressed genes could be a mechanism for epigenetic silencing of gene expression.     

Subtype-specific cyclic AMP-dependent histone H1 phosphorylation at the differentiation of mouse neuroblastoma cells.

At the initial phase of cell differentiation in mouse neuroblastoma (N18) induced by dibutyrylcyclic AMP (dbcAMP), an additional site of histone H1 was extensively phosphorylated. Forskolin and various phosphodiesterase inhibitors also induced both cell differentiation and H1 phosphorylation at the identical site. The phosphorylation preferentially occurred in a single H1 subtype (H1c) among the five (H1a-e) fractionated by high performance liquid chromatography. The three H1 subtypes of N18 (H1c, H1d, and H1e) were phosphorylated in vitro, and their amino acid sequences of the phosphopeptides were identical to the known sequence of rabbit H1 peptides containing a serine 37 residue. However, the amount of H1a and H1b phosphorylations was negligible. The serine residue was replaced by threonine residue in H1a, and H1b did not have a homologous peptide. The tryptic phosphopeptides of H1 in N18 were identical to that in rat liver H1 induced by glucagon (Langan, T.A. (1969) Proc. Natl. Acad. Sci. USA 64, 1276-1283). The results indicate that 1) the response of H1 subtypes to cAMP-dependent protein kinase in vivo and in vitro is H1 subtype-specific, and 2) the H1c phosphorylation may play an important role in the restrictive area of chromatin in both cell differentiation and hormonal stimulation mediated by cAMP.     

Somatic histone H1 microinjected into fertilized mouse eggs is transported into the pronuclei but does not disrupt subsequent preimplantation development

We injected somatic subtypes of histone H1 into newly fertilized mouse eggs, which do not naturally contain this chromosomal protein, and examined the fate of the injected protein and its effect on preimplantation development of recipient eggs. Rhodamine-labelled H1 injected into the cytoplasm of 53 eggs was transported into the pronuclei in 51 cases, and this nuclear accumulation could be detected within 15 min of injection. Unlabelled histone H1, which was detected using immunofluorescence, was also transported following microinjection to the pronuclei, where it colocalized with the chromatin and remained associated with the nuclei following cleavage to the two-cell stage. Nuclear accumulation of injected H1 was inhibited when injected eggs were incubated in the presence of drugs that prevent mitochondrial electron transport or glycolysis, which indicates that nuclear transport occurs through an energy-dependent process, as previously observed in tissue culture cells. To determine whether the presence of somatic H1 in early embryonic nuclei would influence subsequent development, fertilized eggs were injected with an approximately physiological quantity (1–5 pg) of somatic H1 or, as controls, with another small basic protein, cytochrome c. Fifty-three eggs were injected with cytochrome c, of which 51 divided to the two-cell stage, and 32 (60%) reached the blastocyst stage, after 5 days in culture. One hundred and eleven eggs were injected with somatic H1, of which 95 divided to the two-cell stage, and 53 (48%) reached the blastocyst stage, after 5 days in culture. The two groups did not differ statistically (X², P > 0.1) with respect to the fraction of injected embryos that developed to the blastocyst stage. These results show that, although mouse embryos lack the somatic subtypes of histone H1 until the four-cell stage of development, they are able to progress through preimplantation development when these subtypes are present beginning at the one-cell stage. This may imply that the distinctive chromatin composition that characterizes early embryos of a variety of species is not essential for early development in mammals. © 1996 Wiley-Liss, Inc.     

Histone H1 Subtypes Differentially Modulate Chromatin Condensation without Preventing ATP-Dependent Remodeling by SWI/SNF or NURF

Although ubiquitously present in chromatin, the function of the linker histone subtypes is partly unknown and contradictory studies on their properties have been published. To explore whether the various H1 subtypes have a differential role in the organization and dynamics of chromatin we have incorporated all of the somatic human H1 subtypes into minichromosomes and compared their influence on nucleosome spacing, chromatin compaction and ATP-dependent remodeling. H1 subtypes exhibit different affinities for chromatin and different abilities to promote chromatin condensation, as studied with the Atomic Force Microscope. According to this criterion, H1 subtypes can be classified as weak condensers (H1.1 and H1.2), intermediate condensers (H1.3) and strong condensers (H1.0, H1.4, H1.5 and H1x). The variable C-terminal domain is required for nucleosome spacing by H1.4 and is likely responsible for the chromatin condensation properties of the various subtypes, as shown using chimeras between H1.4 and H1.2. In contrast to previous reports with isolated nucleosomes or linear nucleosomal arrays, linker histones at a ratio of one per nucleosome do not preclude remodeling of minichromosomes by yeast SWI/SNF or Drosophila NURF. We hypothesize that the linker histone subtypes are differential organizers of chromatin, rather than general repressors.     

Early Evolution of Histone Genes: Prevalence of an ‘Orphon’ H1 Lineage in Protostomes and Birth-and-Death Process in the H2A Family

The study of histone evolution has experienced a rebirth, for two main reasons: the identification of new essential histone variants responsible for regulating chromatin dynamics and the subsequent contradictions posed by this variability as it pertains to their long-term evolution process. Although different evolutionary models (e.g., birth-and-death evolution, concerted evolution) may account for the observed divergence of histone genes, conclusive evidence is lacking (e.g., histone H1) or totally nonexistent (e.g., histone H2A). While most of the published work has focused on deuterostomes, very little is known about the diversification and functional differentiation mechanisms followed by histone protein subtypes in protostomes, for which histone variants have only been recently described. In this study, we identify linker and core histone genes in three clam species. Our results demonstrate the prevalence of an 'orphon' H1 lineage in molluscs, a group in which the protostome H1 and sperm nuclear basic proteins are on the verge of diversification. They share an early monophyletic origin with vertebrate-specific variants prior to the differentiation between protostomes and deuterostomes. Given the intringuing evolutionary features of the histone H1 family, we have evaluated the relative importance of gene conversion, point mutation, and selection in maintaining the diversity found among H2A subtypes in eukaryotes. We show evidence for the first time that the long-term evolution of this family is not subject to concerted evolution but, rather, to a gradual evolution following a birth-and-death model under a strong purifying selection at the protein level.     

Isolation and characterisation of five histone H1 subtypes from adult rat liver

A procedure is described for quantitative purification of H10 and five H1-1 subtypes--named H1-1a to e--from adult rat liver by reverse-phase high-pressure liquid chromatography. Milligram amounts of each fraction have been obtained. The H1-1a subtype shows a very high lysine content (34%) and H1-1d subtype has an amino-acid composition close to that of H10, but its electrophoretic mobility is different. Salt dependent folding of these subtypes has been studied by circular dichroism. In the presence of 2 or 10 mM sodium phosphate buffers at pH 7.5, H1-1a shows the lowest alpha-helix content. In phosphate-buffer containing 1 M NaCl the number of residues in alpha-helix for all the subtypes rises to 9-10%. Partial cleavage of these subtypes by endoproteinase Glu-C produce three main peptides arising from C-terminal domains. The interaction of the H1-1 subtypes with 196 basepairs linear DNA, purified from rat liver chromatin by high-pressure ion-exchange liquid chromatography, has for consequences a modification of the patterns of digestion: partial proteolysis of the H1-1a and H1-1b subtypes shows differences in the presence or in absence of DNA; on the contrary, H1-1c and H1-1d seem to have the same organization. So these subtypes may play a role in the differential packing of specific region of chromatin.     

Linker histone subtypes differ in their effect on nucleosomal spacing in vivo

Linker histone H1 is located on the surface of the nucleosome where it interacts with the linker DNA region and stabilizes the 30-nm chromatin fiber. Vertebrates have several different, relatively conserved subtypes of H1; however, the functional reason for this is unclear. We have previously shown that H1 can be reconstituted in Xenopus oocytes, cells that lack somatic H1, by cytosolic mRNA injection and incorporated into in vivo assembled chromatin. Using this assay, we have expressed individual H1 subtypes in the oocytes to study their effect on chromatin structure using nucleosomal repeat length (NRL) as readout. We have compared chicken differentiation-specific histone H5, Xenopus differentiation-specific xH1(0) and the somatic variant xH1A as well as the ubiquitously expressed human somatic subtypes hH1.2, hH1.3, hH1.4 and hH1.5. This shows that all subtypes, except for human H1.5, result in a saturable increase in NRL. hH1.4 results in an increase of approximately 13-20 bp as does xH1(0) and xH1A. chH5 gives rise to the same or slightly longer increase compared to hH1.4. Interestingly, both hH1.2 and hH1.3 show a less extensive increase of only 4.5-7 bp in the NRL, thus yielding the shortest increase of the studied subtypes. We show for the first time in an in vivo system lacking H1 background that ubiquitously expressed and redundant H1 subtypes that coexist in most types of cells of higher eukaryotes differ in their effects on the nucleosomal spacing in vivo. This suggests that H1 subtypes have different roles in the organization and functioning of the chromatin fiber.     

Histone H1 subtype synthesis in neurons and neuroblasts.

Rat cerebral cortex neurons contain the five histone H1 subtypes H1a-e and the subtype H1 zero present in other mammalian somatic tissues. The four subtypes H1a-d decay exponentially during postnatal development and are partially or totally replaced by H1e that becomes the major H1 subtype in adults. H1 zero accumulates in a period restricted to neuronal terminal differentiation. Here we study the synthesis of the H1 subtypes in cortical neurons and their neuroblasts by in vivo labeling with [14C]lysine. The subtype synthesis pattern of neuroblasts has been determined by labeling gravid rats during the period of proliferation of cortical neurons and synthesis in neurons has been studied by postnatal labeling. The subtype H1a is synthesized in neuroblasts but not in neurons and is therefore rapidly removed from neuronal chromatin. The synthesis of H1b and H1d is much lower in neurons than in neuroblasts so that these subtypes are replaced to a large extent during postnatal development. H1c is synthesized at levels much higher than the other subtypes both in neurons and neuroblasts, but its very high turnover, about one order of magnitude faster than that of H1e in neurons, favors its partial replacement during postnatal development. Comparison of the synthesis rates of H1 zero in newborn and 30-day-old rats shows that the accumulation of H1 zero in differentiating neurons is due to an increased level of synthesis.     

Changes in H1 complement in differentiating rat-brain cortical neurons

Neuronal nuclei have a low H1 content. A stoichiometry of 0.47 molecule/nucleosome, on average, is calculated for rat brain cortical neurons by comparing its H1 content with that of liver nuclei. The H1 fraction of rat cerebral cortex neurons has been resolved into five subtypes, H1a--e, that have the same mobility as the unphosphorylated H1 forms of other rat tissues. The subtypes H1a--d decay exponentially during postnatal development and are substituted to different extents by H1e. The higher replacement rate is shown by H1a with an apparent half-lifetime of about 5 days. The corresponding values for H1b, H1c and H1d are 11, 21 and 15 days. Several conclusions can be drawn from the observation of postnatal changes in H1 subtype proportions. The low H1 content of neuronal nuclei does not imply the presence of notable peculiarities in subtype composition or in subtype substitution pattern. There is turnover of H1 in differentiating neurons once cell proliferation and DNA replication have ceased. The relative rates of synthesis and/or degradation of the subtypes differ in germinal cells and in neurons. Comparison with previous results on H1 degrees accumulation also shows that in cortical neurons the regulation of the subtypes H1a--e differs from that of H1 degrees.     

Normal and neoplastic human cells have different histone H1 compositions

The H1 histone is the least conserved of the five major groups of histone proteins. There are as many as five subtypes of H1 histone (1-3). These H1 subtypes occur in different amounts in different animal species and also show tissue specificity (1-3). Normal and neoplastic tissues from the same animal, e.g. rat or calf, contain the same H1 subtypes but in different relative amounts (4, 5). Because H1 subtypes exhibit tissue specificity, it is therefore difficult to identify the changes in their composition that are associated with neoplasia reported in studies utilizing tissues of different origins (4, 5). Tissue culture cells and their in vitro transformed neoplastic counterparts, on the other hand, offer an excellent system in which to study these changes because the cells are derived from the same origin. We have examined normal and neoplastic human cells and found a relationship between the H1 composition and the ability of the cells to form tumors in nude mice. The ratio of H1A to H1B in normal human cells is considerably lower than that in neoplastic cells and this ratio increases with the increased ability of the cells to form tumors in nude mice.