Anti-histone H1 Antibodies Research Articles

Observation of intracellular protein localization area in a single neuron using gold nanoparticles with a scanning electron microscope

The localization areas of intracellular proteins in rat cortical neurons were visualized using a scanning electron microscope (SEM) coupled with a focused ion beam (FIB) system. To obtain a clear contrast in the SEM images, gold nanoparticles (GNPs) were bound to specific intracellular proteins by antigen-antibody reactions. By obtaining a cross section of the desired location of the neurons by FIB milling under the SEM imaging condition, it was possible to observe the proteins inside the cells as clear bright spots. When a neuron was stained with anti-tau and anti-histone H1 antibodies, the bright spots were localized in the cross section of the axon and the nucleus, respectively. It was confirmed that targeted proteins in a single neuron on a substrate could be successfully identified. The development of FIB/SEM observation with immunological GNP staining will offer important information for the stable growth of neurons on various substrate structures, since the elongation and turning of axons on the substrates are activated by the redistribution of intracellular proteins.

ANTI-HISTONE H1 IgGs POSSESS PROLIFERATIVE ACTIVITY TOWARDS HUMAN T-LEUKAEMIA CEM CELLS

Anti-histone H1 IgGs were purified from blood serum of systemic lupus erythematosus patients by precipitation of serum proteins with 50% ammonium sulfate followed by a sequential affinity chromatography on Protein G-Sepharose and histone H1-Sepharose columns. To avoid contamination with other proteins, anti-histone H1 IgGs were subjected to strongly acidic pH 2.0 during gel filtration through HPLC column. The effects of the anti-histone H1 IgGs on cell viability and cell cycle were tested by MTS-assay and flow cytometry, correspondingly. The cross-reactivity of the anti-histone H1 antibodies towards heterogenetic and cellular antigens was evaluated by Western-blot analysis. It was found that incubation of CEM cells with the HPLC-purified anti-histone H1 IgGs resulted in significant stimulation of cell growth by 46% after 48 h of incubation. These IgGs possess an antigenic poly-specificity to positively charged heterogenetic antigens and different cellular antigens. FITC-labeled and biotinylated anti-histone H1 IgGs are internalized by CEM cells and preferentially accumulated in the cytoplasm. The anti-histone H1 IgGs are shown to internalize human T-leukemia CEM and stimulate their proliferation. These IgGs are polyspecific toward cellular antigens.

Significance of Post-Liver Transplantation Induced Anti-Histone H1 Antibody for Prevention of Chronic Rejection and Liver Fibrosis.

Objectives: Post-transplant chronic rejection and fibrosis is still the major two obstacles to obtain long-term survival in orthotopic liver transplantation (OLT). In order to prevent these complications, in clinical settings, administration of low-dose immunosuppressive drugs for long-term surviving OLT patients is preferred rather than complete withdrawal of immunosuppressant (drug-free). We have clinically and experimentally demonstrated that immunosuppressive anti-nuclear histone H1antibody(Ab) can be observed not only in a drug-free OLT patient but also in patients treated with a low-dose immunosuppressant.Our previous study has also demonstrated that anti-hitone H1 Ab lead to immature dendritic cells and to subsequently induce regulatory T cells. Methods: In the present study, we explored the role of histone H1 during hepatic fibrogenesis and investigated the functional effects of histone H1 gene silencing inhepatic stellate cells (HpSCs) activation. Results: We found that histone H1 was upregulated during liver fibrosis and that its expression was closely correlated with the deposition of collagen fibers. Inhibition of histone H1 by small interfering RNA (siRNA) in HpSCs suppressed synthesis of α-smooth muscle action (SMA) and collagen type I. Conclusions: These results suggest that histone H1 may implicate in the development of liver fibrosis and it may be novel therapeutic target for repressing liver fibrosis. Additionally, post-transplant elevation of immunosuppressiveanti-histone H1 Ab in OLT patients treated with low-dose immunosuppressant may contribute to not only inhibit chronic rejection but alsorepress liver fibrosis.

Anti‐histone H1 IgGs from blood serum of systemic lupus erythematosus patients are capable of hydrolyzing histone H1 and myelin basic protein

Novel hydrolytic activity of the anti-histone H1 antibodies (Ab) toward histone H1 and myelin basic protein (MBP) was shown. Blood serum of ten patients with clinically diagnosed systemic lupus erythematosus (SLE), and nine healthy donors (control) were screened for the anti-histone H1 antibody- and anti-MBP antibody-mediated specific proteolytic activity. IgGs were isolated by chromatography on Protein G-Sepharose, and four of ten SLE patients appeared to possess IgGs that were capable of cleaving both histone H1 and MBP. Such activity was confirmed to be an intrinsic property of the IgG molecule, since it was preserved at gel filtration at alkaline and acidic pH. At the same time, proteolytic activity was absent in the sera-derived Ab of all healthy donors under control. Anti-histone IgGs were purified by the affinity chromatography on histone H1-Sepharose. Their cross-reactivity toward cationic proteins (histones, lysozyme, and MBP) and their capability of hydrolyzing histone H1 and MBP were detected. However, these IgGs were not cleaving core histones, lysozyme, or albumin. Capability of cleaving histone H1 and MBP was preserved after additional purification of anti-histone H1 IgGs by the HPLC gel filtration. The protease activity of anti-histone H1 IgG Ab was inhibited by serine protease inhibitors.

Immunological aspects and therapeutic significance of an autoantibody against histone H1 in a rat model of concanavalin A‐induced hepatitis

We previously demonstrated the immunosuppressive activity of anti-histone H1 autoantibody induced in experimental and clinical liver allograft tolerance. This study aimed to explore the immunological aspects of anti-histone H1 autoantibody in liver injury induced by concanavalin A (Con A). To establish a Con A-hepatitis model, 20 mg/kg Con A was intravenously injected into rats, after which liver function and histopathological analyses were performed. In this model, anti-histone H1 autoantibody was transiently induced in the sera during the natural recovery stage, 3-7 days after Con A injection. To evaluate the therapeutic significance of anti-histone H1 autoantibody, a polyclonal antibody against histone H1 was intraperitoneally injected immediately after Con A injection. We found that injection of anti-histone H1 antibody could reduce Con A-induced liver damage. Further mechanical analyses revealed that anti-histone H1 antibody altered the intracellular activation of mitogen-activated protein kinase, nuclear factor-kappaB and calcineurin via T-cell receptor signalling, suggesting that anti-histone H1 antibody may protect the liver from Con A-induced injury by inhibiting activation of effector T cells. These findings suggest that anti-histone H1 autoantibody may be a natural immune regulatory factor that protects inflamed livers suffering from autoimmune hepatitis and may lead to T-cell unresponsiveness through the selective regulation of mitogen-activated protein kinase/nuclear factor-kappaB and calcineurin signalling.

Detection and characterization of IgG-and sIgA-abzymes capable of hydrolyzing histone H1

Immunoglobulins IgG and sIgA actively hydrolyzing histone H1 have been detected on analyzing proteolytic activity of antibodies isolated by chromatography on Protein A-agarose from blood serum of patients with multiple sclerosis and from colostrum of healthy mothers. These antibodies hydrolyze other histones less actively and virtually failed to cleave lysozyme of chicken egg. By gel filtration at acidic pH and subsequent analysis of protease activity of chromatographic fractions, it was shown that IgG and sIgA molecules were responsible for hydrolysis of histone H1. Anti-histone H1 antibodies of IgG and sIgA classes were purified by affinity chromatography on histone H1-Sepharose from catalytically active antibody preparations. The protease activity of anti-histone H1 IgG antibodies was inhibited by serine proteinase inhibitors, whereas anti-histone H1 sIgA antibodies were insensitive to inhibitors of serine, asparagine, and cysteine proteases.

The role of a nuclear protein, histone H1, on signalling pathways for the maturation of dendritic cells

We have demonstrated previously that liver allograft tolerance is associated with the immunosuppressive activity of anti-histone H1 autoreactive antibodies induced in the serum of liver transplantation. Furthermore, we and others have shown that nuclear proteins such as histone H1 and high mobility group box 1 play an important role in maturation of dendritic cells (DCs), although the precise mechanisms are still unknown. In the present study, we focus upon the significance of histone H1 on DCs in terms of the intracellular signalling pathway of DCs. Our immunostaining and immunoblot studies demonstrated that histone H1 was detected in cytoplasm and culture supernatants upon the activation of DCs. Histone H1 blockage by anti-histone H1 antibody down-regulated the intracellular activation of mitogen-activated protein kinases (MAPKs) (p38) and IkappaBalpha of DCs, and inhibited DC activity in the proliferation of CD4+ T cells. On the other hand, the addition of histone H1 without endotoxin stimulation up-regulated major histocompatibility complex class II, the CD80 and CD86 surface markers of DCs and the activation of MAPKs (p38 and extracellular-regulated kinase 1/2) and IkappaBalpha. These results suggest that the translocation of histone H1 from nuclei to cytoplasm and the release of their own histone H1 are necessary for the maturation of DCs and the activation for T lymphocytes.

Engulfed cell remnants, and not cells undergoing apoptosis, constitute the LE-cell phenomenon

The LE cell has been one of the first immunological signs of active systemic lupus erythematosus, included into the ACR criteria. LE cells consist of a phagocyte engulfing material of disputed origin, which was interpreted as either cellular remnants from necrotic cells or as early apoptotic cells. It is well established that LE cell formation is dependent on autoantibodies against the linker histone H1. In view of this fact, we investigated whether anti-histone H1 antibodies and LE cell positive sera bound to cells where apoptosis had been induced by gliotoxin or actinomycin D or which were necrotic after heating. Necrotic cell remnants, but not (early) apoptotic cells were bound by anti-histone H1 antibodies and LE cell positive sera, establishing that the process of LE cell formation, which is dependent on anti-H1 binding, leads to engulfment of necrotic (or late apoptotic) material, but not of early apoptotic cells.

Pretreatment of Acetylsalicylic Acid Promotes Tumor Necrosis Factor-related Apoptosis-inducing Ligand-induced Apoptosis by Down-regulating BCL-2 Gene Expression

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to be selective in the induction of apoptosis in cancer cells with minimal toxicity to normal tissues. However, not all cancers are sensitive to TRAIL-mediated apoptosis. Thus, TRAIL-resistant cancer cells must be sensitized first to become responsive to TRAIL. In this study, we observed that pretreatment by acetylsalicylic acid (ASA) augmented TRAIL-induced apoptotic death in human prostate adenocarcinoma LNCaP and human colorectal carcinoma CX-1 cells. Western blot analysis showed that pretreatment of ASA followed by TRAIL treatment activated caspases (8, 9, and 3) and cleaved poly(ADP-ribose) polymerase, the hallmark feature of apoptosis. Most interestingly, at least 12 h of pretreatment with ASA was prerequisite for promoting TRAIL-induced apoptosis and was related to down-regulation of BCL-2. Biochemical analysis revealed that ASA inhibited NF-kappaB activity, which is known to regulate BCL-2 gene expression, by dephosphorylating IkappaB-alpha and inhibiting IKKbeta activity but not by affecting the HER-2/neu phosphatidylinositol 3-kinase-Akt signal pathway. Overexpression of BCL-2 suppressed the promotive effect of ASA on TRAIL-induced apoptosis and changes in mitochondrial membrane potential. Taken together, our studies suggested that ASA-promoted TRAIL cytotoxicity is mediated through down-regulating BCL-2 and by decreasing mitochondrial membrane potential.

Cross-recognition between histones and La/SSB may account for anti-DNA reactivity in SLE patients

Antibodies to La/SSB are detected in sera of patients with primary Sjogren's syndrome (pSS) and systemic lupus erythematosus (SLE). The vast majority of anti-La/SSB positive sera contain antibodies directed towards a linear B-cell epitope of La/SSB spanning the sequence 349-364aa (pep349-364). The aim of this study was to evaluate the fluctuation of antibody levels to major B-cell epitopes of La/SSB over time and investigate for their possible crossreactions. Sequential sera from 15 SLE and 15 pSS patients, followed from 3 to 10 years were obtained. All patients with SLE were positive for anti-Ro/SSA, anti-La/SSB and anti-dsDNA antibodies and patients with pSS were positive for anti-Ro/SSA and anti-La/SSB antibodies. Sera from 30 patients with SLE without anti-La/SSB antibodies and 30 healthy individuals served as disease and negative control respectivelly. All sera tested for the presence of anti-pep349-364 antibodies, using a specific ELISA. Specific anti-pep349-364 IgG was purified from sera of SLE patients and evaluated for cross reactivity against dsDNA and histones. In all SLE sera the levels of anti-pep349-364 antibodies varied in time and fluctuated in parallel with anti dsDNA antibodies. Anti-pep349-364 IgG purified from 7 SLE patients. Five out of 7 were found to react with calf thymus DNA in ELISA. All purified (7/7) anti-pep349-364 IgG preparations reacted with histone H1 and failed to produce a positive immunofluorescence pattern in Crithidia luciliae anti-dsDNA assay which lacks histones. Competative inhibition experiments demonstrated that histone H1 could inhibit completely the binding of anti-pep349-364 IgG to pep349-364 while pep349-364 inhibited by 70% the binding of anti-pep349-364 IgG to histone H1. These findings indicate that a subgroup of SLE patients possess cross-reacting anti-histone H1 antibodies and anti-pep349-364 antibodies, which can be faulty considered as anti-dsDNA reactivity in regular ELISA techniques.

The effects of anti-histone H1 antibody on immune cells responsible for rejection reaction

We previously demonstrated the immunosuppressive activity of anti-histone H1 autoreactive antibodies (Ab) transiently induced in serum of a rat tolerogenic orthotopic liver transplantation (OLT) model. In the present study, we investigated the effects of anti-histone H1 Ab on dendritic cells (DCs), T-cells, lymphokine-activated killer (LAK) cells, and human natural killer (NK) cells. The effects of anti-histone H1 Ab on Concanavalin A (ConA) blast, on rat DC cytokine profiles and phenotypes, and on T-cells, LAK cells, and human NK cells were examined by flow cytometry and RT-PCR. The cytotoxicity of LAK and NK cells pretreated with anti-histone H1 Ab was assayed. The addition of anti-histone H1 Ab to ConA blast inhibited the proliferation of 5-(6)-carboxy-fluorescein succinimidyl ester (CFSE)-labeled lymphocytes without toxicity but increased the population of CD4 +CD25 + T-cells. DCs treated with anti-histone H1 Ab expressed lower levels of CD80/CD86, IL-1β, and IL-6. The addition of anti-histone H1 Ab to LAK culture decreased the percentages of NKR-P1 populations and down-regulated levels of inducible nitric oxide synthase (iNOS), IL-2, and INF-γ in RT-PCR. The cytotoxicity of LAK and NK cells was lower when pretreated with anti-histone H1 Ab than when pretreated with control IgG. We found that the blockade of histone H1 modulated DCs toward tolerogenic status, decreased the cytotoxicity of LAK and NK cells, and induced CD4 +CD25 + T-cells. These results suggest that the use of anti-histone H1 Abs might be a useful strategy for the development of a form of immunosuppression.

Binding properties of anti-histone H1 antibodies suggest that nuclei of necrotic cells rather than cells undergoing apoptosis are engulfed in the LE cell phenomenon

Binding properties of anti-histone H1 antibodies suggest that nuclei of necrotic cells rather than cells undergoing apoptosis are engulfed in the LE cell phenomenon

Characterization of immunosuppressive factors expressed in serum by rat tolerogenic liver transplantation

Background In a rat tolerogenic model of orthotopic liver transplantation (OLT), recipient serum after OLT (post-OLT serum) possesses strong immunosuppressive activity. This study aimed to identify immunosuppressive factors present in early post-OLT serum. Methods Immunosuppressive activity was evaluated in vitro by inhibition of the mixed-lymphocyte reaction (MLR). Autoantigens recognized by MLR-inhibitory IgG were identified by the internal protein sequencing. Results Recipient post-OLT serum inhibited MLR, and OLT-inducible IgG was the major immunosuppressive factor. IgG from post-OLT sera (2 to 3 weeks) specifically reacted to 31; 34; and 73-kd autoantigens on spleen cells. The internal sequences of the 31- and 34-kd antigens coincided completely with those of histone H1 molecules. Immunodepletion of anti-histone H1 antibodies (Abs) from early post-OLT serum abolished the MLR-inhibitory activity. Furthermore, rabbit polyclonal Ab-directed histone H1 not only significantly suppressed rat and human MLR but also prolonged survival of heart allografts. Flow-cytometric analysis revealed that some live PVG splenocytes were stained with antihistone H1 Abs, and that these positive cells increased on Con A stimulation. Western blot analysis indicated that several cross-reactive antigens against anti-histone H1 Abs were found in their membrane fraction. Conclusions In this study we provide evidence that autoreactive Abs, against histone H1 are a major OLT-induced graft survival factor, and may play at least a part in overcoming the acute rejection phase to establish solid allograft tolerance.

Cytosolic Histone H1 Releasing under Different Apoptotic Stimuli in Chronic Lymphocytic Leukemia (CLL).

Recently, it has been demonstrated that nuclear histone H1 could be released into cytoplasm when apoptosis is induced by DNA double-strand breaks, this process being dependent of p53 functional status. In addition, cytosolic histone H1.2 was shown to induce cytochrome C release in a Bak-dependent manner. The aims of this study were: 1) to analyze the presence of histone H1.2 in the cytosol of peripheral blood purified CLL cells during apoptosis induced by different chemotherapeutic drugs or ionizing radiation in CLL cells in culture; 2) to correlate the presence of cytosolic histone H1 with the p53 functional status. FISH analysis was used to select samples with (n=2) or without (n=4) p53 deletion. Peripheral blood purified CLL cells were cultured for 24 hours with no treatment, after irradiation (25 Gy), in the presence of fludarabine and mitoxantrone (FM) (1mg/ml and 0.5 mg/ml respectively), or with etoposide (5.8 mg/ml). Cell viability and analysis of apoptosis were performed by annexin V/PI staining and FACscan analysis. Cytosolic and nuclear fractions were separated with the Nuclear/Cytosol Fractionation kit (Biovision). The presence of histone H1 in the cytosolic fraction was assessed by Western Blott using the anti-histone H1 antibody (Upstate). Nuclear contamination of this fraction was detected by using anti-ribonucleoprotein (hnRNP) antibody (Abcam). In p53 wild-type CLL cases, increased apoptosis was observed under all stimuli, being the FM combination the most effective. In contrast, CLL cases with p53 deletion displayed a lower or no response to the different treatments. In p53 wild-type CLL cases, histone H1 was only observed in cytosolic fractions at 24 hours when irradiation or etoposide treatment was applied, but not with FM treatment. Interestingly, cytosolic histone H1 was also present in the control cells at 24h of culture, indicating that H1 was also involved in the spontaneous apoptosis observed due to the microambient deprivation of CLL cells in culture. This histone H1 traffic was also confirmed using immunofluorescence analysis. Conversely, in p53 deleted CLL cases histone H1 was not detected into cytosolic fractions with any treatment. In conclusion, cytosolic histone H1 appeared in CLL cases in a p53 dependent manner when DNA double-strand breaks were induced. These results suggest that histone H1 plays an important role in the p53-dependent cytosol apoptotic signaling.

Understanding global changes in histone H1 phosphorylation using mass spectrometry

Histone post-translational modifications have been implicated in a variety of biological processes such as gene expression, DNA replication, and chromatin assembly. The modifications include methylation, acetylation, phosphorylation, ubiquitination, glycosylation, and ADP-ribosylation. For several years, we have been investigating the role of histone H1 phosphorylation in transcription using the hormone inducible mouse mammary tumor virus (MMTV) promoter. When mouse cells were exposed to prolonged treatment with dexamethasone, a significant decrease in the level of histone H1 phosphorylation was observed. Traditionally, Western analyses with anti-histone H1 and phospho-specific H1 antibodies were performed to observe changes in phosphorylation levels of the bulk H1 histones. More recently, we have applied electrospray ionization mass spectrometry to the analysis of histone H1 isoforms. Utilizing this approach, we have investigated the phosphorylation state of the specific H1 isoforms before and after prolonged treatment with dexamethasone. Specifically, we could determine that the relative phosphorylation levels of the histone H1.3, H1.4, and H1.5 isoforms decrease after prolonged hormone exposure. Recent advancements in mass spectrometry have proven invaluable toward the analysis of post-translational modifications on proteins. The continued developments in the area of mass spectrometry should provide new insights into not only the function of proteins but also into the basic regulatory mechanisms that control cellular functions.

Comparative analysis of anti-histone and anti-chromatin antibody specificity in lupus erythematosus cell-positive and -negative sera and their relation to disease activity

Anti-histone and anti-chromatin antibody responses play a central role in the autoimmune response of systemic lupus erythematosus (SLE). Furthermore, anti-histone H1 antibodies are essential for the formation of the lupus erythematosus cell (LEC) phenomenon. In this study, the binding properties of LEC+ and LEC- SLE sera to chromatin-associated nuclear antigens (histones H1, H2A, H2B, H3, H4; complexes of H2A-H2B, [H2A-H2B]-DNA, H1-DNA; total and H1-stripped chromatin; native and denatured DNA) were investigated. In addition, sera from patients with drug-induced lupus (by procainamide, hydralazine, or quinidine), as well as from patients with rheumatoid arthritis and osteoarthritis, were assessed. Enzyme-linked immunosorbent assay was used to detect specific antibody binding. Mirroring the important role of histone H1 in the formation of LE cells, anti-histone H1 reactivity was 8-fold higher in LEC+ sera than in LEC- sera. In addition, reactivities to most of the other antigens tested, i.e., other histones and histone-DNA complexes as well as chromatin and DNA, were significantly higher in LEC+ sera than in LEC- sera. All but 1 serum sample from the patients with drug-induced lupus were negative for LE cell formation as well as for anti-histone H1 reactivity, but displayed high antibody reactivities to histone-DNA complexes, including chromatin. Sera from patients with rheumatoid arthritis and osteoarthritis did not show significant binding to these antigens. When comparing the clinical features of LEC+ and LEC-SLE patients, severe organ involvement, including nephritis and central nervous system involvement, was common in the LEC+ group, but rare in the LEC- group. A positive LE cell phenomenon not only correlated with the presence of high anti-histone H1 antibody levels in SLE, but also indicated serologically and clinically active disease with major organ involvement.

Identification and characterization of a calreticulin-binding nuclear protein as histone (H1), an autoantigen in systemic lupus erythematosus.

Our objective was to identify nuclear calreticulin-binding protein(s) and investigate whether there is a correlation between presence of autoantibodies against calreticulin and calreticulin-binding protein(s) in the sera of patients suffering from systemic lupus erythematosus (SLE). The ligand overlay procedure using digoxigenin-labelled calreticulin was used to identify a calreticulin-binding protein in the nuclear fraction of bovine brain. Fractionation of the nuclear components was used to localize the major positive calreticulin-binding protein. The protein was partially purified using hydroxylapatitie chromatography and subjected to NH2-amino acid sequence analysis. Immunoblots using the sera of SLE patients were then carried out on calreticulin and the calreticulin-binding protein. The calreticulin-binding protein present in the nucleoplasm was identified as histone H1. Approximately 62% (26/42) patients with SLE had IgG antibodies directed against H1 whereas the sera of healthy individuals did not react with the antigen; 36% of patients with SLE had both anti-calreticulin and anti-histone H1 antibodies. Phosphorylation of the latter protein did not alter its immunoreactivity. These findings demonstrate that the concomitant presence of autoantibodies directed against both calreticulin and histone H1 occurs frequently in patients with SLE and may help shed some light on the mechanisms which bring about the autoimmune response.

Developmental changes in DNA methylation of the two tobacco pollen nuclei during maturation.

Changes in DNA methylation during tobacco pollen development have been studied by confocal fluorescence microscopy using a monoclonal anti-5-methylcytosine (anti-m5C) antibody and a polyclonal anti-histone H1 (anti-histone) antibody as an internal standard. The specificity of the anti-m5C antibody was demonstrated by a titration series against both single-stranded DNA and double-stranded DNA substrates in either the methylated or unmethylated forms. The antibody was found to show similar kinetics against both double- and single-stranded DNA, and the fluorescence was proportional to the amount of DNA used. No signal was observed with unmethylated substrates. The extent of methylation of the two pollen nuclei remained approximately constant after the mitotic division that gave rise to the vegetative and generative nuclei. However, during the subsequent development of the pollen, the staining of the generative nucleus decreased until it reached a normalized value of (1)/(5) of that of the vegetative nucleus. The use of a confocal microscope makes these data independent of possible focusing artefacts. The anti-histone antibody was used as a control to show that, while the antibody staining directed against 5-methylcytosine changed dramatically during pollen maturation, the histone signal did not. We observed the existence of structural dimorphism amongst tobacco pollen grains, the majority having three pollen apertures and the rest with four. However, the methylation changes observed occurred to the same extent in both subclasses.

Ontogeny of histone H1 in guinea pig renal cortex. 258

Histone H1 has been implicated in transcriptional regulation, in some instances by inhibiting thyroid receptorDNA interactions. We have shown a postnatal increase in renal cortical Na,KATPase abundance and activity in guinea pig and demonstrated a potential role for thyroid hormones in mediating this increase. Therefore, we wish to understand the ontogeny of H1 histones in the kidney around the time of birth. SDS polyacrylamide gel electrophoresis(SDSPAGE) of renal cortical homogenates revealed a 32 kDa protein band greatly more abundant in renal cortex from nearterm guinea pig fetuses (> 60 days gestation; term is 68 days) than from postnatal cortex. An 11amino acid fragment from this band was sequenced and found to be from a histone H1. Immunoblots of renal cortical homogenates from fetal (F), newborn (NB) and adult guinea pigs (n=6/group) were probed with an antihistone H1 antibody as well as antiserum to guinea pig outer medullary Na,KATPaseαβheterodimer. The abundances of these proteins were inversely related, with histone H1/Na,KATPase declining with maturation (F 1.9±0.5; NB 0.7±0.1; adults 0.02±0.01). This decline was largely accounted for simply by postnatal tubular cell hypertrophy. We isolated nuclei from renal cortex of the 3 groups, measured total histone H1 in silverstained SDSPAGE gels of nuclei and noted, however, that there was still a maturational decline in histone H1 abundance; F had 32±13% greater total histone H1 than NB and 26±12% greater than adults, while the abundance of the nucleosome core histones (H2H4) in these gels did not change with maturation (n=23/group). We also measured histone H1 isoform abundances in nuclei preps from the 3 groups. Similar to prior studies of the postnatal development of the mouse and rat kidneys, we noted a maturational decline in the ratio of histone H1A/histone H1° abundances in guinea pig renal cortical nuclei, (F 4.6±0.2; NB 2.6±0.3; adults 2.1±0.2, n=23/group). We conclude that there are maturational decreases in total H1 histone and shifts in histone H1 isoforms. We speculate that these changes may influence the maturation of renal tubular function. Glucocorticoid and thyroid receptors and response elements are known to interact with histone H1, and so the abundance of histone H1 isoforms may be a critical determinant of how the developing kidney responds to hormonal stimuli.

Rabbit anti-chromatin antibodies recognize similar epitopes on a histone H1 molecule as lupus autoantibodies

Autoantibodies in systemic lupus erythematosus (SLE) are believed to be produced through an antigen-driven mechanism, i.e., autosensitization with autoantigens. If this is the case, it is conceivable that antigenic determinants reactive with SLE autoantibodies and induced antibodies raised by immunization are quite similar. To examine this issue, rabbits were immunized with purified histone H1 or chromatin. Although immunization with purified histone H1 did not produce detectable amounts of anti-histone H1 antibodies, immunization with chromatin mounted relatively good antibody responses against not only all histone subunits but also ssDNA. Epitopes on a histone H1 molecule reactive with SLE autoantibodies and rabbit antisera were determined using deletion mutants of a histone Hl molecule. At least two epitopes reactive with induced antibodies were found on a histone H1 and these were closely related to or the same as major autoepitopes in SLE. These data might indicate that B cells producing anti-histone Hl autoantibodies are quite similar to those induced by immunization and provide additional evidence that autosensitization with an autoantigen might be operative and a particulate antigen chromatin could be one of the immunogens for the production of anti-nuclear antibodies of various kinds in SLE.