Identification of histone H2B as a regulated plasminogen receptor

Tethering of plasminogen to cell surfaces controls plasmin formation and, thereby, influences pericellular proteolysis and cell migration. Modulation of cellular plasminogen binding sites provides a mechanism for regulation of these events. In this study, two distinct models, phorbol ester-stimulated adhesion of U937 monocytoid cells and culturing of peripheral blood neutrophils, treatments which modulate plasminogen binding sites, have been examined to determine the molecular basis for the upregulation of plasminogen receptors. Membranes were isolated from cell populations, with and without upregulated plasminogen binding capacities, and analyzed by [(125)I]plasminogen ligand blotting of gel transfers. Approximately 15 different [(125)I]plasminogen-binding proteins were discerned in the membrane fractions, and only relatively minor differences in the intensities of individual bands were noted in the different cell populations. The notable exception was the presence of a 17 kDa band, which was selectively and markedly enhanced in the membranes from cells with enhanced plasminogen binding capacities. The 17 kDa protein was isolated from both cell types, and amino acid sequencing of peptide fragments identified the same protein, histone H2B. Increased expression of histone H2B was observed on stimulated U937 cells and cultured neutrophils by confocal microscopy with an antibody raised to the carboxy-terminal octopeptide sequence of histone H2B. This antibody or its Fab fragments substantially decreased the level of binding of plasminogen to these cultured neutrophils and stimulated U937 cells that exhibited elevated levels of binding but not to nonstimulated cells. Thus, histone H2B represents a regulated plasminogen receptor, which contributes significantly to the plasminogen binding capacity of cells.     

Antigenic structure of histone H2B

Antigenic determinants of histone H2B were localized using a series of 23 overlapping fragments of H2B obtained either by chemical and enzymatic cleavage of the histone or by solid-phase peptide synthesis. The ability of peptides to bind H2B antibodies was measured in an enzyme-linked immunosorbent assay, using antisera directed against calf thymus and chicken erythrocyte H2B as well as four anti H2B monoclonal antibodies obtained from autoimmune mice. Seven antigenic determinants were localized in the H2B molecule in the vicinity of residues 1-11, 6-18, 15-25, 26-35, 50-65, 94-113 and 114-125. Two of these determinants (residues 6-18 and 26-35) were revealed only through the binding properties of antibodies isolated from autoimmune mice. The usual correlation between hydrophilicity and antigenicity was found to hold for four of the epitopes, and the N- and C-termini of H2B were both antigenically active.     

On the mechanism of interaction between histone II B1 and DNA and histone II B2 and DNA

A mechanism is suggested at the molecular level whereby histone IIB₂ can act as a cross-link between two (or possibly three) adjacent and parallel strands of DNA double helix some 40 Å apart. Application of Prothero's rule and the Lewis probability functions indicate the probable locations of three a-helices and a number of β-turns. This, coupled with the requirement that the tertiary conformation of the histone be complementary to the DNA molecules and for as many basic groups as possible to bind to phosphate oxygens, allows us to suggest, on the basis of model building using accurate space-filling (CPK) models, a complex conformation that achieves this.A similar process applied to histone IIB₁, whose complete amino acid sequence is also known, shows the location of five probable a-helices, a number of β-turns, and a segment of β-pleated sheet. The basic amino acids are gathered in four groupings. Model building experiments suggest that histone IIB₁ forms a complex strut joining four parallel strands of DNA double helix that form a diamond with diameters 100 and 40 Å. In both these models the purpose and function of a fair proportion of the individual amino acids can be specified.This paper is the third and last of a series in this Journal in which models are presented for the tertiary conformation and function of all five histones of known (in whole or in part) amino acid sequence. This suggests that all five are concerned in packing the long DNA double helix, which may be in a “square helix” form, into the confined space of the chromosome. The hypotheses may be tested by a direct investigation of nucleoprotein in situ to see if these 40, 70, and 100 Å interhelical distances can be detected by biophysical methods.     

Absence of histone H2B in nucleosomes containing histone TH2B and interaction of immunoglobulin with nucleosomes

Nucleosomes containing histone TH2B were isolated from chromatin subunits of rat testis nuclei (MNT) by incubating with anti-TH2B immunoglobulin (IgTH2B) which was covalently attached to agarose gels. Electrophoretic separation of histones of these isolated nucleosomes revealed that histone H2B was completely absent, suggesting that histone TH2B, the variant of H2B, existed in nucleosomes only as TH2B X TH2B and that TH2B X H2B was not likely to exist in chromatin. Sucrose gradient ultracentrifugation of mixtures of MNT and IgTH2B revealed that when excess amounts of immunologically active IgTH2B were present, complexes of higher sedimentation coefficients than MNT X IgTH2B were formed, but with limited amounts of active IgTH2B, only MNT X IgTH2B was formed. When purified IgTH2B was coated on polystyrene tubes and incubated with MNT, those MNT immobilized by the tube-coated IgTH2B adsorbed IgTH2B from diluted antiserum during subsequent incubation. Those results suggested the absence of steric hindrance in the binding of IgTH2B to MNT X IgTH2B. When MNT was coated on polystyrene tubes and incubated with DNase and then with dilute anti-TH2B antiserum, it was found that DNase digestion increased the binding of immunoglobulin to the tubes approximately 76%. Interaction of chromatin subunits of rat liver nuclei (MNL) with anti-TH2B antiserum was negligible, but DNase digestion of MNL coated on tubes was followed by considerable interaction with anti-TH2B antiserum. Those results indicated DNase unmasked at least part of the determinants encased by DNA. Anti-H2B immunoglobulin (IgH2B) interacted with histone H2B and TH2B to the same extent, and interacted significantly to a lesser extent with either MNT or MNL. DNase digestion of MNT and MNL increased binding of IgH2B approximately 170 and 117%, respectively.     

Functional analysis of histone methyltransferase g9a in B and T lymphocytes

Lymphocyte development is controlled by dynamic repression and activation of gene expression. These developmental programs include the ordered, tissue-specific assembly of Ag receptor genes by V(D)J recombination. Changes in gene expression and the targeting of V(D)J recombination are largely controlled by patterns of epigenetic modifications imprinted on histones and DNA, which alter chromatin accessibility to nuclear factors. An important component of this epigenetic code is methylation of histone H3 at lysine 9 (H3K9me), which is catalyzed by histone methyltransferases and generally leads to gene repression. However, the function and genetic targets of H3K9 methyltransferases during lymphocyte development remain unknown. To elucidate the in vivo function of H3K9me, we generated mice lacking G9a, a major H3K9 histone methyltransferase, in lymphocytes. Surprisingly, lymphocyte development is unperturbed in G9a-deficient mice despite a significant loss of H3K9me2 in precursor B cells. G9a deficiency is manifest as modest defects in the proliferative capacity of mature B cells and their differentiation into plasma cells following stimulation with LPS and IL-4. Precursor lymphocytes from the mutant mice retain tissue- and stage-specific control over V(D)J recombination. However, G9a deficiency results in reduced usage of Iglambda L chains and a corresponding inhibition of Iglambda gene assembly in bone marrow precursors. These findings indicate that the H3K9me2 epigenetic mark affects a highly restricted set of processes during lymphocyte development and activation.     

The amino acid sequence of wheat histone H2B(2). A core histone with a novel repetitive N-terminal extension

Two of the four electrophoretic histone H2B variants present in wheat embryos have been isolated. The complete primary structure of the H2B(2) variant has been deduced from sets of overlapping peptides generated by CNBr cleavage, Staphylococcus aureus V8 protease, endoproteinase Arg-C, the post-proline cleaving enzyme, chymotrypsin and cleavage in dilute acid. A minimum of 17 peptides were required to establish the sequence. This variant has a blocked N terminus and comprises a total of 149 amino acids. The C-terminal two-thirds of the protein are highly homologous to vertebrate H2B. In contrast, the N-terminal third is entirely different and contains an N-terminal extension of 23 residues in which the sequence Ala-Glu-Lys or variants are repeated several times. This region is also highly homologous to the H2B from Tetrahymena pyriformis. It shows in addition similarities to wheat H2A(1) and bovine H1.     

The structure of ubiquitinated histone H2B.

Ubiquitinated histone H2B (uH2B) has been purified from both calf and pig thymus by exclusion chromatography in 7 M urea. Digestion of uH2B with Staphylococcus aureus V8 protease yielded the peptide 114-125 containing the ubiquitin moiety. Further digestion of this peptide with trypsin removed the ubiquitin and three H2B residues from the N-terminus. Edman degradations of both peptides established that ubiquitin is attached to the epsilon-amino group of lysine 120 in both calf and pig uH2B by an iso-peptide bond to the C-terminal glycine 76 of ubiquitin.     

Function of nucleophosmin/B23, a nucleolar acidic protein, as a histone chaperone

We previously identified and purified a nucleolar phosphoprotein, nucleophosmin/B23, as a stimulatory factor for replication from the adenovirus chromatin. We show here that nucleophosmin/B23 functions as a histone chaperone protein such as nucleoplasmin, TAF-I, and NAP-I. Nucleophosmin/B23 was shown to bind to histones, preferentially to histone H3, to mediate formation of nucleosome, and to decondense sperm chromatin. These activities of B23 were dependent on its acidic regions as other histone chaperones, suggesting that B23/nucleophosmin is a member of histone chaperone proteins.     

Sequence of histone 2B of Drosophila melanogaster.

The complete sequence of histone 2B of Drosophila has been determined by using an improved Beckman sequenator. Comparing these data with those previously published by other investigators on the histone 2B of calf [Iwai, K., Hayashi, H., & Ishikawa, K. (1972) J. Biochem. (Tokyo) 72, 357--367], trout [Koostra, A., & Bailey, G. S. (1978) Biochemistry 17, 2504--2510], and Patella (a limpet) [van Helden, P. D., Strickland, W. N., Brandt, W. F., & von Holt, C. (1979) Eur. J. Biochem. 93, 71--78], it is possible to assess the evolutionary stability of this protein. There is little conservation of sequence in the N-terminal portion of the molecule (residues 1--26 numbering according to calf H2B), while the remainder of the protein, which we designate the C-terminal portion, is highly conserved. In the region of 27--125 residues, there are 9 substitutions in the composite data among the 98 positions, 8 of them conservative. These data indicate that very different selective pressures operate on the two different portions of the H2B molecule, implying the existence of two well-defined regions. Studies on the structure of the nucleosome by others have suggested that the C-terminal portion of H2B is involved in histone-histone interactions while the N-terminal portion is a relatively free "tail" binding to DNA. The sequence data indicate that the function of the C-terminal region of H2B requires considerable sequence specificity while that of the N-terminal region does not.