Antibodies to histones in systemic lupus erythematosus: localization of prominent autoantigens on histones H1 and H2B.

By the technique of immunoblotting we have assessed the ability of sera from 24 patients with systemic lupus erythematosus to bind nuclear proteins. Of the 11 patients who had antibodies to histones, 10 had antibodies to histone H1 and 9 of these also had antibodies to histone H2B. Antibodies to the other histones (H2A, H3, and H4) were less apparent. Five of the 11 patients (and two others in the remainder of the sample of 24) also had antibodies to a small number of nonhistone proteins that are probably components of ribonucleoprotein particles, but there was no obvious correlation between the presence of antihistone antibodies and the known antiribonucleoprotein activity of these sera. Separate determinants on H1 and H2B were demonstrated by immunoblotting with affinity-purified anti-H1 and anti-H2B antibodies derived from serum that showed both specificities. The localization of the determinants within the histone polypeptide chains was shown by immunoblotting with large fragments produced by specific proteolytic or chemical cleavage of the histones. The strongest determinant on H1 was located within the COOH-terminal half, with a weaker determinant being present within the NH2-terminal half; the H2B determinant(s) was located entirely within the NH2-terminal half of the molecule. The selectivity with which the antihistone antibodies in systemic lupus erythematosus are produced against the more exposed histones in the nucleosome (and perhaps against the most exposed regions of these histones) is consistent with the involvement of intact chromatin structures as immunogens in this disease.     

Preparations of homeostatic thymus hormone consist predominantly of histones 2A and 2B and suggest additional histone functions.

The two major constituents in preparations of the homeostatic thymus hormone (HTH) were purified. Amino acid sequence analysis showed that the components (HTH alpha and HTH beta) are identical to histones H2A and H2B, suggesting the possibility that histones might have hitherto unrecognized occurrence and functions. If the HTH activities are not ascribed to the two histones in the preparation, they could only be derived from minor constituents present in minimal amounts. Therefore, the histone structures were scrutinized for properties of relevance in relation to hormone activities and for similarities with thymic hormones. Similarities between COOH-terminal regions of histones H2A, H2B, and H3 were noticed, as well as some similarities between NH2-terminal regions of histones and parts of recognized thymus hormones and related proteins. Potential signals, resembling cleavage sites in prohormones, are present in the histone structures, and further correlations with recently discovered ubiquitin functions may explain molecular mechanisms for actions of the HTH preparations. None of the observations is significant by itself, but the combined results suggest the hypothesis of different relationships and functions, including hormone-like activities, for some histones.     

The C2A domain of JFC1 binds to 3'-phosphorylated phosphoinositides and directs plasma membrane association in living cells

Phosphatidylinositol 3-kinase products play a central role in the regulation of several intracellular pathways via adaptor proteins that share the ability to bind to 3'-phosphoinositides with high affinity and specificity. JFC1 is a C2 domain-containing protein involved in cellular trafficking that has been shown to bind 3'-phosphoinositides in vitro. In this work, we demonstrate that the C2A domain of JFC1 is the module responsible for its binding to the plasma membrane via 3'-phosphoinositides in vivo. We show that the C2A domain of JFC1 is the only domain present in this protein that localizes to the plasma membrane in living cells. Moreover, the C2A domain of JFC1 binds 3'-phosphoinositides in vitro with similar specificity as that described for full-length JFC1, suggesting that the domain mediates the specific membrane localization of the full-length protein. Furthermore, the C2A domain of JFC1 colocalized with the pleckstrin homology domain of Akt in vivo, and both the JFC1 C2A domain and the full-length JFC1 dissociated from the membrane in the presence of PI 3-kinase specific inhibitors. We also show that the association of the C2A domain to the membrane is modulated by calcium. From these results we analyze possible mechanisms for the role of JFC1 in cellular trafficking.     

Recombinant kinesin motor domain binds to beta-tubulin and decorates microtubules with a B surface lattice.

We have expressed the recombinant squid kinesin head domain in Escherichia coli and studied its interaction with microtubules. The head is active as a microtubule-stimulated ATPase and binds to microtubules, but it does not support microtubule gliding by itself. The head binds to both microtubules and depolymerized tubulin. In each case the zero-length crosslinker 1-ethyl-3-[3-dimethylamino)propyl] carbodiimide induces a bond specifically to beta- but not alpha-tubulin. The head decorates brain microtubules with an 8-nm axial spacing. Thus the stoichiometry is one kinesin head per tubulin dimer. The lattice is that of flagellar B-tubules, implying that reassembled microtubules are not symmetric. Moreover, the A- and B-tubules of intact flagellar outer doublets are both decorated with a B lattice. This suggests that the B lattice is a general property of microtubules.     

Abnormal development of the cerebral cortex and cerebellum in the setting of lamin B2 deficiency

Nuclear lamins are components of the nuclear lamina, a structural scaffolding for the cell nucleus. Defects in lamins A and C cause an array of human diseases, including muscular dystrophy, lipodystrophy, and progeria, but no diseases have been linked to the loss of lamins B1 or B2. To explore the functional relevance of lamin B2, we generated lamin B2-deficient mice and found that they have severe brain abnormalities resembling lissencephaly, with abnormal layering of neurons in the cerebral cortex and cerebellum. This neuronal layering abnormality is due to defective neuronal migration, a process that is dependent on the organized movement of the nucleus within the cell. These studies establish an essential function for lamin B2 in neuronal migration and brain development.     

Evidence for a human histone gene cluster containing H2B and H2A pseudogenes.

Not all members of the human histone gene family are functional. We have isolated a human H2B pseudogene that contains alterations in the protein-coding sequences as well as in the 3' and 5' flanking sequences that preclude expression of a functional H2B histone protein. There are three modifications in the amino acid-coding region: a single-base deletion producing a frame shift, a single-base substitution resulting in a codon change from serine to tryptophan (an amino acid not present in histones), and the absence of a stop codon. Analysis of nucleotide sequences upstream from the AUG start signal indicates the absence of a "TATA" box and other putative consensus regulatory sequences. In the 3' flanking region, a highly conserved block of 22 nucleotides that exhibits hyphenated dyad symmetry is displaced downstream. Within the same genomic segment, the adjacent H2A histone gene is missing 12 nucleotides, resulting in a deletion of four amino acids in a highly conserved region of the protein.     

Colocalization of vertebrate lamin B and lamin B receptor (LBR) in nuclear envelopes and in LBR-induced membrane stacks of the yeast Saccharomyces cerevisiae.

We have expressed human lamin B and the chicken lamin B receptor (LBR), either separately or together, in yeast and have monitored the subcellular location of the expressed proteins by immunofluorescence microscopy, immunoelectron microscopy, and cell fractionation. At the light microscopic level, the heterologous lamin B localized to the yeast nuclear rim and at electron microscopic resolution was found subjacent to the yeast inner nuclear membrane. These data indicate that vertebrate lamin B was correctly targeted in yeast. Expression of the heterologous LBR, either alone or together with the heterologous lamin B, resulted in the formation of membrane stacks primarily adjacent to the nuclear envelope, but also projecting from the nuclear envelope into the cytoplasm or under the plasma membrane. Double immunoelectron microscopy showed colocalization of the heterologous lamin B and LBR in the yeast nuclear envelope and in the LBR-induced membrane stacks. Cell fractionation showed the presence of the heterologous lamin B and LBR in a subnuclear fraction enriched in nuclear envelopes. The heterologous lamin B was extracted at 8 M urea, but not at 4 M urea, thus behaving as a peripheral membrane protein and indistinguishable from assembled lamins. The heterologous LBR was not extracted by 8 M urea, indicating that it was integrated into the membrane. The observed colocalization and cofractionation are consistent with previously reported in vitro binding data and suggest that heterologous lamin B and LBR interact with each other when coexpressed in yeast.     

Contacts of the globular domain of histone H5 and core histones with DNA in a "chromatosome".

The globular domain of histone H5 is found to asymmetrically associate with a nucleosome core including the Xenopus borealis somatic 5S RNA gene. Histones H2A and H2B are required for association of histone H5. Strong crosslinking of the globular domain of histone H5 to the 5S DNA in the nucleosome occurs at a single site to one side of the dyad axis. This site is also in contact with the core histones, and the interactions of the core histones with 5S DNA change as a result of association of the globular domain of histone H5. We discuss evidence for an allosteric change in core histone-5S DNA interactions following the association of the linker histone in the nucleosome.     

A high-molecular-weight squid neurofilament protein contains a lamin-like rod domain and a tail domain with Lys-Ser-Pro repeats.

Previous studies have shown that two low molecular-weight neurofilament (NF) proteins (NF-60 and NF-70) from the squid Loligo pealei are translated from mRNAs that are splice variants of a single squid NF gene. In this study, we report the isolation and characterization of cDNA clones encoding a high-molecular-weight squid NF protein (NF-220), the mRNA of which derives from the same squid NF gene. All three proteins are identical in their amino-terminal and lamin-like rod domains but differ in their carboxyl-terminal tail regions. In contrast to the short tail domains of NF-60 and NF-70, the NF-220 protein has a longer tail domain containing an acidic cluster of amino acids immediately followed by repeated copies of the sequence motif Lys-Ser-Pro. The Lys-Ser-Pro domain is similar to that of mammalian medium NF (NF-M) and high NF (NF-H) proteins, where the serines are highly phosphorylated. Except for these Lys-Ser-Pro motifs, there is surprisingly little structural similarity between the squid NF-220 protein and mammalian NF-M and NF-H proteins. Furthermore, the location of introns in squid NF-220 protein shows that it is more closely related to nuclear lamins and type III intermediate-filament proteins than to vertebrate NF proteins.     

Rearrangement of the histone H2A C-terminal domain in the nucleosome.

Using zero-length covalent protein-DNA crosslinking, we have mapped the histone-DNA contacts in nucleosome core particles from which the C- and N-terminal domains of histone H2A were selectively trimmed by trypsin or clostripain. We found that the flexible trypsin-sensitive C-terminal domain of histone H2A contacts the dyad axis, whereas its globular domain contacts the end of DNA in the nucleosome core particle. The appearance of the histone H2A contact at the dyad axis occurs only in the absence of linker DNA and does not depend on the absence of linker histones. Our results show the ability of the histone H2A C-terminal domain to rearrange. This rearrangement might play a biological role in nucleosome disassembly and reassembly and the retention of the H2A-H2B dimer (or the whole octamer) during the passing of polymerases through the nucleosome.     

Linker histones H1 and H5 prevent the mobility of positioned nucleosomes.

We have previously identified a generally occurring short-range mobility of nucleosome cores on DNA in relatively low ionic strength conditions. Here we report that this mobility of histone octamers positioned on constructs of 5S rDNA is suppressed by the binding of histone H1 or H5 to the nucleosome. Histone H5 is the more potent inhibitor of nucleosome mobility, in accordance with its higher affinity for chromatin. We propose that this reversible restraint on chromatin dynamics may play a role in local regulation of processes that require access to the DNA.     

Histone H2A subtypes associate interchangeably in vivo with histone H2B subtypes.

The yeast Saccharomyces cerevisiae contains two primary sequence subtypes of histone H2B (H2B1 and H2B2) and of H2A (H2A1 and H2A2). Mutants in each of the H2B subtypes have been used to show previously that yeast cells lacking one or the other, but not both, of the H2B proteins are viable. Because H2A protein interacts in the nucleosome with H2B, we wished to determine whether specific H2A subtypes must interact with specific H2B subtypes. We describe experiments in which frameshift mutations were introduced into both of the H2A genes in vitro and the mutant genes integrated into the yeast genome, replacing the wild-type H2A genes by a subsequent recombination. Using these mutant (hta1- and hta2-) strains we find that neither H2A gene has a unique essential function during any phase of the yeast life cycle, although strains homozygous for hta1- grow more slowly. However, one functional H2A gene is required for viability because cells mutant in both H2A genes arrest at spore germination prior to bud separation. By combining these H2A mutations with the H2B mutations obtained previously, we show that all combinations of H2A and H2B subtypes produce viable cells. From these genetic experiments and electrophoretic analysis of the histone proteins of these mutants we conclude that the H2A subtypes can associate interchangeably with the H2B subtypes.     

Relationship between arsenic exposure and histone ubiquitination modifications of H2A and H2B in human peripheral blood leukocytes

正Objective To detect the modification levels of H2AK119 ubiquitination(H2AK119ub)and H2BK120ub,and to analyze the relationship between the levels of H2AK119ub,H2BK120ub and arsenic exposure.Methods A cross-sectional study was conducted in typical areas of drinking water type of endemic arsenicosis in