Interaction of MAR-sequences with nuclear matrix proteins.

The recent discovery of DNA sequences responsible for the specific attachment of chromosomal DNA to the nuclear skeleton (MARs/SARs) was an important step towards our understanding of the functional and structural organization of eukaryotic chromatin [Mirkovitch et al.: Cell 44:273-282, 1984; Cockerill and Garrard: Cell 44:273-282, 1986]. A most important question, however, remains the nature of the matrix proteins involved in the specific binding of the MARs. It has been shown that topoisomerase II and histone H1 were capable of a specific interaction with SARs by the formation of precipitable complexes [Adachi et al.: EMBO J8:3997-4006, 1989; Izaurralde et al.: J Mol Biol 210:573-585, 1989]. Here, applying a different approach, we were able to "visualize" some of the skeletal proteins recognizing and specifically binding MAR-sequences. It is shown that the major matrix proteins are practically the same in both salt- and LIS-extracted matrices. However, the relative MAR-binding activity of the individual protein components may be different, depending on the method of matrix preparation. The immunological approach applied here allowed us to identify some of the individual MAR-binding matrix proteins. Histone H1 and nuclear actin are shown to be not only important components of the matrix, but to be involved in a highly efficient interaction with MAR-sequences as well. Evidence is presented that proteins recognized by the anti-HMG antibodies also participate in MAR-interactions.     

The relationship between adenosine diphosphate-ribosylation and mammary gland differentiation

Poly(adenosine diphosphate [ADP]-ribosyl)ation, although associated with differentiation in many systems, exhibited a reciprocal relationship with mammary gland differentiation, and both the synthetic and degradatory pathways complemented each other in this regard. Poly(ADP-ribosyl)synthetase activity declined during pregnancy and lactation, while poly(ADP-ribose) degradatory activity rose late in pregnancy and peaked during lactation. In explant cultures, similar changes occurred and appeared to be under separate hormonal control; prolactin suppressed the synthetase activity, whereas insulin stimulated the poly(ADP-ribosyl)glycohydrolase activity. This latter effect may be mediated by a decline in cAMP levels for the following reasons: the glycohydrolase is known to be inhibited by cAMp, insulin decreased cAMP concentrations in mammary explants by 70%, and cholera toxin blocked the effects of insulin on poly(ADP-ribose) degradation. This reciprocal relationship between poly(ADP-ribosyl)ation and mammary gland differentiation is further supported by pharmacological studies: in the presence of insulin, cortisol, and prolactin, an inhibitor of the synthetase stimulated alpha-lactalbumin three-fold over hormone stimulation alone. However, this inhibitor was unable to induce differentiation in the absence of prolactin. Therefore, although there is a close association between a decline in enzyme activity and mammary differentiation, the data are insufficient to support a causal relationship.     

HMG1 Domains: The Victims of the Circumstances

The method of circular dichroism (CD) was used to compare DNA behavior during its interaction with linker histone H1 and with nonhistone chromosomal protein HMG1 at different ionic strength and at different protein content in the system. The role of the negatively charged C-terminal segment of HMG1 was analyzed using recombinant protein HMG1-(A+B), which lacks the C-terminal amino acid sequence. The -type CD spectra were common for DNA interaction with histone H1, but no spectra of this type were observed in HMG1–DNA systems even at high ionic strength. The CD spectrum of the truncated recombinant protein at high salt concentration somewhat resembled the ⁺-type spectrum. Two very intense positive bands were located near 215 nm and near 272 nm, and the whole CD spectrum was positive. The role of the C-terminal part of HMG1 in the formation of ordered DNA–protein complexes is discussed.     

Somatic H1 histone accumulation and germ layer determination in amphibian embryos

The induction of mesoderm and/or endoderm from prospective ectoderm and dorsalization of the marginal zone mesoderm may be linked to inhibition of cell cycling and DNA synthesis in early amphibian embryos. In turn, this may lead to reduction of somatic H1 histone accumulation. A greater number of cell cycles and rounds of DNA synthesis characterizes the induction of neural tissue. This is correlated with an increase of somatic H1 histone accumulation. The number of rounds of DNA replication may regulate the level of H1 histone accumulation and this may have a role in germ layer determination.     

Linker histone subtypes and their allelic variants

Members of histone H1 family bind to nucleosomal and linker DNA to assist in stabilization of higher‐order chromatin structures. Moreover, histone H1 is involved in regulation of a variety of cellular processes by interactions with cytosolic and nuclear proteins. Histone H1, composed of a series of subtypes encoded by distinct genes, is usually differentially expressed in specialized cells and frequently non‐randomly distributed in different chromatin regions. Moreover, a role of specific histone H1 subtype might be also modulated by post‐translational modifications and/or presence of polymorphic isoforms. While the significance of covalently modified histone H1 subtypes has been partially recognized, much less is known about the importance of histone H1 polymorphic variants identified in various plant and animal species, and human cells as well. Recent progress in elucidating amino acid composition‐dependent functioning and interactions of the histone H1 with a variety of molecular partners indicates a potential role of histone H1 polymorphic variation in adopting specific protein conformations essential for chromatin function. The histone H1 allelic variants might affect chromatin in order to modulate gene expression underlying some physiological traits and, therefore could modify the course of diverse histone H1‐dependent biological processes. This review focuses on the histone H1 allelic variability, and biochemical and genetic aspects of linker histone allelic isoforms to emphasize their likely biological relevance.     

Replication timing and cell differentiation

Cell differentiation may depend in part upon a type of unbalanced growth in which several cell cycles occur with a reduced level of total protein synthesis. During this period the synthesis of the chromatin protein HMG-I/Y is reduced since its synthesis is correlated with that of total protein. The synthesis of histone H1 shows less reduction since its synthesis is entrained with that of DNA. This greater reduction of HMG-I/Y than of histone H1 is thought to delay or prevent replicon initiations within AT-enriched isochores. This shifts their time of replication from early to late S phase. This may restrict certain pathways of cell differentiation in multipotent progenitor cells and allow one particular type of differentiation.     

The effects of histones H1o,H5 and HMG proteins on cell division of cultured murine erythroleukemia cells

Summary In the present study the effect of histones H1^o and H5, and the nonhistone chromatin proteins HMG 1, 2, 14 and 17 (the high mobility group proteins), as well as the acidic peptide fragments of HMG 1 and 2 and polyglutamate, on cell division and differentation of cultured murine erythroleukemia (Friend) cells has been investigated. It was found that histones H1^o and H5, the acidic peptide fragments of HMG 1 and 2, HMG 14 and 17 and sodium polyglutamate stimulated cell division at a concentration of 10 g/ml. None of the H1^o, H5 or HMG protein preparations induced hemoglobin synthesis, as judged by benzidine staining.     

Interactions of MCP1 with components of the replication machinery in mammalian cells

Eukaryotic DNA replication starts with the assembly of a pre-replication complex (pre-RC) at replication origins. We have previously demonstrated that Metaphase Chromosome Protein 1 (MCP1) is involved in the early events of DNA replication. Here we show that MCP1 associates with proteins that are required for the establishment of the pre-replication complex. Reciprocal immunoprecipitation analysis showed that MCP1 interacted with Cdc6, ORC2, ORC4, MCM2, MCM3 and MCM7, with Cdc45 and PCNA. Immunofluorescence studies demonstrated the co-localization of MCP1 with some of those proteins. Moreover, biochemical studies utilizing chromatin-immunoprecipitation (ChIP) revealed that MCP1 preferentially binds replication initiation sites in human cells. Interestingly, although members of the pre-RC are known to interact with some hallmarks of heterochromatin, our co-immunoprecipitation and immunofluorescence analyses showed that MCP1 did not interact and did not co-localize with heterochromatic proteins including HP1β and MetH3K9. These observations suggest that MCP1 is associated with replication factors required for the initiation of DNA replication and binds to the initiation sites in loci that replicate early in S-phase. In addition, immunological assays revealed the association of MCP1 forms with histone H1 variants and mass spectrometry analysis confirmed that MCP1 peptides share common sequences with H1.2 and H1.5 subtypes.     

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

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

Rabbit anti-HMG-17 antibodies recognize similar epitopes on the HMG-17 molecule as lupus autoantibodies. Relation with histone H1 defined epitopes.

HMG-17 is a nucleosomal protein which is an immune target of autoantibodies in systemic lupus erythematosus (SLE) and other autoimmune diseases. Autoantibody production in SLE is believed to result from autoantigen specific immune stimulation and subsequently, it is expected that antigenic determinants recognized by SLE autoantibodies and induced antibodies by immunization are quite similar. To examine this issue, rabbits were immunized with purified HMG-17. The produced antiserum showed cross reactivity on blots and in inhibition ELISA with histone H1, even after its affinity purification with immobilized HMG-17. Finally, purification of the antiserum over H1 absorbed on nitrocellulose membrane produced specific anti-HMG-17 antibodies in the supernatant and anti-HMG-17/H1 antibodies that were bound to H1. SLE sera positive for HMG-17 had also cross reactivity with H1, and following the same procedure as before we received HMG-17 specific SLE autoantibodies and anti-HMG-17/H1 autoantibodies. Using the multipin epitope mapping technology, 19 overlapping 15-mer HMG-17 peptides and six 15-peptides, corresponding to known epitopes of histone H1, were synthesized. Four major epitopes were identified on the HMG-17 molecule, reactive with induced anti-HMG-17 antibodies, and these were the same as major autoepitopes In SLE. The sequence 25-51 of HMG-17, part of its DNA-binding domain, was recognized by the anti-HMG-17/H1 antibodies that were bound to H1. These antibodies recognized also defined epitopes of H1. Our results show that SLE autoantibodies can be directed against the same or similar epitopes as do IgGs evoked during the active immunization of animals, and provide additional evidence that autosensitization with an autoantigen might be operative. The possibility that the same or similar epitopes are found on different molecules (in this study HMG-17 and H1) supports the fact that there are rules by which nature selects the most dominant immunodeterminant to a given protein, which often represents functional or structural sites in the autoantigen.     

Distinct effects of histone H1 and non-histone chromosomal proteins on the structure of nucleosomes and the chromatin fibre in solution

In this study we attempt to differentiate between the effects of the non-histone chromosomal proteins and histone H1 on the structure of the nucleosomes and the chromatin fibre in solution. The properties of chromatin preparations with different histone H1 and non-histone protein compositions were compared using circular dichroism and flow linear dichroism and the following conclusions were drawn. When histone H1 is absent the non-histone proteins partially prevent the unfolding of the nucleosomes at low ionic strength. The complete blocking of this unfolding, however, is accomplished only in the presence of histone H1. The non-histone proteins do not affect the orientation of the nucleosomes along the fibre axis. Only histone H1 can maintain the positive anisotropy of the chromatin fibre.