Reciprocal epigenetic modification of histone H2B occurs in chromatin during apoptosis in vitro and in vivo.

Histone H2B phosphorylation at Serine 14 (phosS14) has been proposed as an epigenetic marker of apoptotic cells, whereas acetylation at the adjacent Lysine 15 (acK15) is a property of non-dying cells. We investigated the relationship and the potential regulatory mechanisms between these two epigenetic histone modifications and internucleosomal DNA degradation during apoptosis. Using rat primary thymocytes induced to undergo apoptosis with glucocorticoids we found that H2B phosphorylated at Ser14 was associated with soluble, cleaved DNA in apoptotic nuclei. In contrast acK15 was prevalent in non-apoptotic nuclei and scarce in apoptotic nuclei. This switch between K15 acetylation and S14 phosphorylation on H2B was also observed in apoptotic thymocytes from animals treated in vivo with glucocorticoids and in a rat hepatoma cell line (HTC) induced to die by UV-C or Fas ligand. It is interesting to note that the combined use of a histone deacetylase inhibitor and glucocorticoid suppressed both S14 phosphorylation and internucleosomal DNA degradation without inhibiting apoptosis in thymocytes. Using synthetic peptides and a PKC phosphorylation assay system, we show that the deacetylation of K15 was necessary to allow the S14 phosphorylation. These findings suggest that selective chromatin post-translational modifications are associated with DNA degradation during apoptosis.

Beneficial effects of fish oil on glucose metabolism in spontaneously hypertensive rats.

1. Increased interest in fish oil led us to examine their metabolic effects in spontaneously hypertensive rats, which have been reported to have glucose intolerance. 2. Rats were divided into three groups: (i) a control group fed standard rat laboratory chow; (ii) a lard group fed a high-fat diet containing 20% lard; and (iii) a fish oil group fed a high-fat diet containing 20% fish oil for 14 weeks. 3. Systolic blood pressure and fasting blood glucose were markedly increased in the lard group, whereas in the fish oil group they were only transiently increased at the beginning and decreased to levels seen in the control group. 4. Intraperitoneal glucose tolerance test demonstrated that fish oil reversed the impairment of glucose disposal found in the lard group. However, plasma insulin levels were raised transiently at 30 min in the fish oil group compared with the control group. 5. Insulin secretion from pancreatic islets stimulated with glucose in vitro was also enhanced by fish oil. 6. These results lead us to conclude that fish oil improves glucose tolerance by enhancing insulin secretion from pancreatic beta-cells.

Histone H2B phosphorylation in mammalian apoptotic cells. An association with DNA fragmentation.

Histone phosphorylation was investigated in several mammalian cells undergoing apoptosis (human HL-60 and HeLa, mouse FM3A and N18 cells, and rat thymocytes). Among the four nucleosomal core histones (H2A, H2B, H3, and H4), H2B, which is not usually phosphorylated in quiescent or growing cells, was found to be phosphorylated after treatment with various apoptotic inducers. The H2B was phosphorylated around the time when nucleosomal DNA fragmentation was initiated and, like this fragmentation, was completely blocked with Z-Asp-CH(2)-DCB, an inhibitor of ICE or ICE-like caspase. The involved single phosphopeptide of H2B proved to be phosphorylatable in vitro with a protein kinase C, and the site Ser-32 was tentatively identified. Despite typical apoptotic chromatin condensation, the H3 phosphorylation was at a low level, and the sites where phosphorylation did occur did not include any mitosis-specific phosphopeptides. Phosphorylation of H4 was increased, but the other two histone proteins (H1 and H2A) were not appreciably changed. These observations imply that 1) H2B phosphorylation occurs universally in apoptotic cells and is associated with apoptosis-specific nucleosomal DNA fragmentation, 2) chromatin condensation in apoptosis occurs by a different biochemical mechanism from those operating during mitosis or premature chromosome condensation, and 3) this unique phosphorylation of H2B is a useful biochemical hallmark of apoptotic cells.

Identification of a novel phosphorylation site on histone H3 coupled with mitotic chromosome condensation.

Histone H3 (H3) phosphorylation at Ser(10) occurs during mitosis in eukaryotes and was recently shown to play an important role in chromosome condensation in Tetrahymena. When producing monoclonal antibodies that recognize glial fibrillary acidic protein phosphorylation at Thr(7), we obtained some monoclonal antibodies that cross-reacted with early mitotic chromosomes. They reacted with 15-kDa phosphoprotein specifically in mitotic cell lysate. With microsequencing, this phosphoprotein was proved to be H3. Mutational analysis revealed that they recognized H3 Ser(28) phosphorylation. Then we produced a monoclonal antibody, HTA28, using a phosphopeptide corresponding to phosphorylated H3 Ser(28). This antibody specifically recognized the phosphorylation of H3 Ser(28) but not that of glial fibrillary acidic protein Thr(7). Immunocytochemical studies with HTA28 revealed that Ser(28) phosphorylation occurred in chromosomes predominantly during early mitosis and coincided with the initiation of mitotic chromosome condensation. Biochemical analyses using (32)P-labeled mitotic cells also confirmed that H3 is phosphorylated at Ser(28) during early mitosis. In addition, we found that H3 is phosphorylated at Ser(28) as well as Ser(10) when premature chromosome condensation was induced in tsBN2 cells. These observations suggest that H3 phosphorylation at Ser(28), together with Ser(10), is a conserved event and is likely to be involved in mitotic chromosome condensation.

Vanadate triggers the transition from chromosome condensation to decondensation in a mitotic mutant (tsTM13) inactivation of p34cdc2/H1 kinase and dephosphorylation of mitosis-specific histone H3.

At the nonpermissive temperature (39 degrees C), chromosomes remain condensed in a temperature-sensitive cell mutant (tsTM13) arrested in the late stage of mitosis. Highly increased activity of histone H1 kinase, hyperphosphorylation of histone H1, and mitosis-specific histone H3 phosphorylation are maintained, even in telophase. In the present study, the defect of chromosome decondensation in tsTM13 cells was found to be partially normalized by a tyrosine phosphatase inhibitor, vanadate, with induction of chromosome decondensation and the formation of multinucleated cells. In the presence of vanadate, the H1 kinase activity dropped to near normal levels and the amount of the inactive from of p34cdc2 protein phosphorylated at a tyrosine residue was increased. H1 and H3 were also extensively de- phosphorylated, the latter being tightly associated with chromosome decondensation. Serine/threonine-protein phosphatase in late mitosis of the mutant works normally at 39 degrees C. The results indicate that (a) the genetic defect in the mutant may be involved in the control mechanism of the p34cdc2/H1 kinase activity in the late M phase rather than the phosphatase, (b) normalization of the defect of the mutant by vanadate results from inactivation of H1 kinase, and (c) late mitosis-specific events (p34cdc2/H1 kinase inactivation, mitosis-specific dephosphorylation of histone H1 and H3) are closely operating with chromosome decondensation.

Alteration of cell cycle-dependent histone phosphorylations by okadaic acid. Induction of mitosis-specific H3 phosphorylation and chromatin condensation in mammalian interphase cells.

Effects of okadaic acid (OA), a protein phosphatase inhibitor, on chromatin structure and phosphorylation of histones were examined using HeLa and N18 cells. The chromatin condensation in HeLa cells was mild and resemble prometaphase nuclei, while the condensation in N18 cells was extensive and chromatin became a compact body. H2A in HeLa cells was extensively and consistently phosphorylated at the same site throughout the cell cycle, and H3 was demonstrated to be phosphorylated at the mitosis-specific site Ser10. In contrast, H1 phosphorylation was rapidly decreased in most sites within 3 h. The reduction of H1 phosphorylation was accompanied by a quantitative change in the set of H1 phosphopeptides. During the early phase of the OA treatment, H1 phosphorylation was transiently elevated in tandem, whereas H3 phosphorylation reached a maximum somewhat later. The results suggest that mitosis-specific events (cdc2/H1 kinase activation, H1 superphosphorylation, mitosis-specific H3 phosphorylation and chromatin condensation) induced by OA are sequentially associated. The changes appear to reflect a molecular mechanism similar to that operating in normal mitosis.

Cell cycle-dependent suppressive effect of histone H1 on mitosis-specific H3 phosphorylation.

To analyze the mechanism by which histone H3 phosphorylation occurs specifically during mitosis, the effect of H1 on mitosis-specific H3 phosphorylation (Ser-10) was investigated in nucleosomes. H1 interaction with H1-depleted nucleosomes suppressed H3 phosphorylation including Ser-10 by approximately 50%. However, H1 interaction with DNA-free histone octamers failed to suppress H3 phosphorylation. The extent of suppression of H3 phosphorylation in nucleosomes with H1 prepared from synchronized HeLa cells was cell cycle-dependent. Binding with a highly phosphorylated mitotic H1 produced the least suppression of H3 phosphorylation, whereas binding with a lower H1 phosphorylation from G1 phase resulted in the greatest suppression. The results suggest that 1) mitotic H3 phosphorylation is suppressed with a lower level of H1 phosphorylation during interphase and 2) highly phosphorylated H1 during mitosis partially releases the suppression of mitotic H3 phosphorylation.

A temperature-sensitive CHO-K1 cell mutant (tsTM13) defective in chromosome decondensation and spindle deconstruction in M phase.

A temperature-sensitive CHO-K1 cell mutant, tsTM13, exhibited a delayed cell cycle progression from metaphase to telophase at a nonpermissive temperature and was finally arrested from anaphase to telophase. Metaphase chromosomes were overcondensed and chromosome disjunction in anaphase was uncoordinated. In telophase, sister chromatids were segregated and cytokinesis was completed, but chromosome structure remained in a condensed state and the spindle was not deconstructed. The level of phosphorylation of histones H1 and H3 remained high in the later stages of mitosis and the activity of histone H1 kinase was also maintained at a high level. These results strongly suggest that the pleiotropic defects of tsTM13 cells in mitosis are associated with a lack of inactivation of activated histone H1 kinase.

Inactivation of DNA polymerase beta by in vitro phosphorylation with protein kinase C.

The Mr = 38,300 polypeptide of the purified recombinant rat DNA polymerase beta served as an excellent substrate for protein kinase C (PKC) in vitro but not for the catalytic subunit of cAMP-dependent protein kinase. The phosphorylation by PKC resulted in inactivation of DNA polymerase beta activity, and recovery was achieved by dephosphorylation with alkaline phosphatase. Since the phosphorylated DNA polymerase beta was retained with use of a single-stranded DNA-cellulose column, inactivation might occur at a site different from that for the DNA binding. Amino acid sequence analysis of the phosphopeptides revealed that the phosphorylated sites were 2 serine residues at positions 44 and 55 from the NH2 terminus, either or both of which might be involved in the catalytic activity of DNA polymerase beta. Thus, the inactivation of the DNA repair enzyme, DNA polymerase beta, by PKC may be an important process in the modification of DNA metabolism in the nucleus through signal transduction processes.

Mitosis-specific histone H3 phosphorylation in vitro in nucleosome structures.

A mechanism of mitosis-specific enhancement of histone H3 phosphorylation was analyzed in vitro in terms of nucleosome structure. The incorporation of [32P]phosphate into DNA-bound H3 was approximately 5-7 times higher than in DNA-free H3 using the catalytic subunit of cAMP-dependent protein kinase. The two major N-terminal serine sites, including the mitosis-specific site (Ser10) and Ser28, were extensively phosphorylated in the DNA-bound forms. These phosphorylation patterns were identical to those of nucleosomal H3. In contrast, the H3 in DNA-free octamers was very slightly phosphorylated. The major site of H3 phosphorylation in DNA-free H3 was Thr118 in the C-terminus. Results indicate that DNA-binding is essential for the high level of mitosis-specific H3 phosphorylation, and that the nucleosome structure promotes H3 N-terminal phosphorylation in vitro. It also suggests the possibility that H1 prevents H3 phosphorylation during interphase of the cell cycle.

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.

Cell-type-specific expression of mouse DNA polymerase beta-gene is regulated by silencer elements.

RNA blot hybridization analysis revealed that the steady-state level of DNA polymerase beta-mRNA in mouse neuroblastoma N18TG2 cells was approximately five-fold higher than that in NIH/3T3 cells. In order to examine the function of DNA polymerase beta-gene silencers in these two cell lines, we employed a chloramphenicol acetyltransferase (CAT)-transient expression assay using the CAT plasmids containing the silencers linked to various promoter-enhancers. In NIH/3T3 cells, DNA polymerase beta-gene silencers effectively repressed the function of its own promoter and those of several other heterologous promoter-enhancers. In contrast, the silencers only marginally affected the CAT expression directed by DNA polymerase beta-gene promoter and heterologous promoter-enhancers in N18TG2 cells. The extent of the increase of CAT expression by removing silencer elements in NIH/3T3 cells was very similar to the ratio of DNA polymerase beta-mRNA content in N18TG2 cells to that in NIH/3T3 cells. These results indicate that cell-type-specific expression of DNA polymerase beta-gene is primarily controlled by the function of its silencer elements.

Specific site of histone H3 phosphorylation related to the maintenance of premature chromosome condensation. Evidence for catalytically induced interchange of the subunits.

Previously we have found that histone H1 and H3 of tsBN2 cells showing premature chromosome condensation (PCC) at nonpermissive temperature (40.5 degrees C) were phosphorylated extensively as in mitotic cells (Ajiro, K., Nishimoto, T., and Takahashi, T. (1983) J. Biol. Chem. 258, 4534-4538). Under the influence of various chemicals, both the prevention of the PCC induction and the suppression of H3 phosphorylation occurred simultaneously, whereas H1 phosphorylation did not. At the minimum concentration for the inhibition of PCC induction, H1 phosphorylation remained at the control level, but H3 phosphorylation was completely suppressed. Tryptic peptide analysis revealed that the H3 phosphopeptide in PCC was single, and it was observed in the same position as in mitosis. The results suggest that specific site(s) of H3 phosphorylation related to the maintenance of a condensed state of chromatin.

Histone expressions in mouse-rat somatic reconstituted cells.

A critical analysis of histone expression was performed on the four interspecific and the two intraspecific reconstituted cells formed between karyoplast from mouse B16 cells and the cytoplast from rat cells (L6TG.CAPr) or mouse cells (B82.CAPr). All the reconstituted cells had the same pattern of mouse histones and the same amount of mouse-specific H2B. 2 histone as that of mouse nuclear donor cells. A hybrid between B16 and L6TG.CAPr contained both mouse and rat-specific H1b subtypes, whereas no rat-specific H1b was detected in the interspecific reconstituted cells. In both intra- and interspecific reconstituted cells, the proportion of H1b content was lower than that of B16 cells but that of H1 degree was higher, indicating that the mouse H1 patterns from these cells slightly resembled the pattern of slower growing and differentiated cytoplast donor cells. As an effect of the tumor promoter, the H1 pattern tended to revert to that of the nuclear donor cells in agreement with the phenotypic reversion, without any significant change in cell growth.

The induction of chromosome condensation in tsBN2, a temperature-sensitive mutant of BHK21, inhibited by the calmodulin antagonist, W-7.

The induction of premature chromosome condensation (PCC) in tsBN2 cells, a temperature-sensitive (ts) mutant of BHK21/13 which shows PCC at the non-permissive temperature, was almost completely inhibited by 40 microM W-7, an antagonist of calmodulin. The mitotic phosphorylation of histone H1 and H3 was also inhibited by W-7. W-5, a chlorine-deficient analogue of W-7 and which interacts weakly with calmodulin, did not inhibit the induction of PCC, even at a dose of 80 microM. The content of calmodulin in tsBN2 cells was increased by a temperature shift to 40.5 degrees C. All these results suggested that calmodulin is required for the chromosome condensation.

Two distinct types of mitochondrial DNA segregation in mouse-rat hybrid cells. Stochastic segregation and chromosome-dependent segregation.

Two distinct patterns of mitochondrial DNA (mtDNA) segregation were found in different mouse-rat hybrid cell lines. On mouse-rat hybrid cell line, H2, retained complete sets of chromosomes and mtDNAs of both mouse and rat. Even after cultivation for about one year after cloning, the H2 cell population still retained both parental mtDNAs. However, when mtDNAs of H2 subclones were examined, it was found that some individual cells in the H2 cell population contained only mouse or only rat mtDNA, although they still retained complete sets of both kinds of parental chromosomes. This type of mtDNA segregation, named stochastic segregation, is bidirectional and may be caused by the repetition of random sharing of mouse and rat mtDNAs with daughter cells. This segregation occurred spontaneously during long-term cultivation. The second type of mtDNA segregation, named chromosome-dependent segregation, was found in the other mouse-rat hybrid cell lines that segregated either mouse or rat chromosomes. In these hybrid cells, chromosomes and mtDNA of the same species co-segregated. This second type of segregation is unidirectional. The types of mtDNA segregation appear to depend on the stability of the parental chromosomes in the hybrid cells. When both mouse and rat chromosomes retain stably, mtDNA shows stochastic segregation. On the contrary, when either species of chromosomes is segregated from the cells, mtDNA shows chromosome-dependent segregation.

Histone H1 and H3 phosphorylation during premature chromosome condensation in a temperature-sensitive mutant (tsBN2) of baby hamster kidney cells.

The histone phosphorylations of temperature-sensitive mutant cells (tsBN2) were investigated during the induction of premature chromosome condensation (PCC). At the permissive temperature (33.5 degrees C), the histones of the cells were phosphorylated typically as in any other mammalian cell. However, at the nonpermissive temperature (40.5 degrees C), both histone H1 and H3 were phosphorylated extensively as in mitotic cells, and the increase in these phosphorylations throughout S to G2 phase was closely correlated to the frequency of cells showing PCC. The pattern of H1 subtype phosphorylations was quite similar, and the sites of H1 phosphorylation from PCC were the same as those from mitotic cells. Although the degree of phosphorylation was low, H1 and H3 phosphorylations were observed even in G1 phase at the nonpermissive temperature. The effects of metabolic inhibitors on the induction of PCC were parallel in H1 and H3 phosphorylations; actinomycin D failed to inhibit either PCC induction or these phosphorylations, whereas cyclohexamide did, completely inhibiting H3 phosphorylation.