Induction of p53-dependent activation of the human proliferating cell nuclear antigen gene in chromatin by ionizing radiation

A human fibroblast cell line with conditional p53 expression displayed a p53-dependent increase in both the protein and mRNA levels of proliferating cell nuclear antigen (PCNA) after exposure to ionizing radiation (IR). The combination of p53 induction and IR cooperated to activate a transiently expressed human PCNA promoter-reporter gene via a p53-responsive element. Chromatin immunoprecipitation assays with antibodies specific for p53 or p300/CREB-binding protein revealed specific p53-dependent enrichment of PCNA promoter sequences in immunoprecipitates of sheared chromatin prepared from irradiated cells. Maximal and specific association of acetylated histone H4 with the PCNA promoter also depended on p53 induction and exposure to IR. These data demonstrate p53 binding to a target site in the PCNA promoter, recruitment of p300/CREB-binding protein, and localized acetylation of histone H4 in an IR-dependent manner. These molecular events are likely to play a role in mediating activation of PCNA gene expression by p53 during the cellular response to DNA damage. The analyses indicate that the combination of p53 induction and IR activate the PCNA gene via mechanisms similar to that of p21/wild-type p53-activated factor but to a lesser extent. This differential regulation of PCNA and p21/wild-type p53-activated factor may establish the proper ratio of the two proteins to coordinate DNA repair with cell cycle arrest.     

Tissue-specific and differential expression of prothymosin alpha gene during rat development.

We have analyzed the RNA expression of prothymosin alpha (ProT alpha) gene during rat development in several tissues and compared it to that of two proteins related to cell proliferation: proliferating cell nuclear antigen (PCNA)/cyclin and histone H3 (H3). The expression of ProT alpha gene was found to be regulated in a developmental and tissue-specific manner. The mRNA levels of ProT alpha followed a similar time-course in liver, brain, kidney, and testis, being highly increased in the early periods of postnatal development. However, in thymus ProT alpha mRNA showed only moderate changes throughout development. Our findings suggest that ProT alpha participates in developmental processes like cell proliferation and/or differentiation.     

SET8 is degraded via PCNA-coupled CRL4(CDT2) ubiquitylation in S phase and after UV irradiation

The eukaryotic cell cycle is regulated by multiple ubiquitin-mediated events, such as the timely destruction of cyclins and replication licensing factors. The histone H4 methyltransferase SET8 (Pr-Set7) is required for chromosome compaction in mitosis and for maintenance of genome integrity. In this study, we show that SET8 is targeted for degradation during S phase by the CRL4(CDT2) ubiquitin ligase in a proliferating cell nuclear antigen (PCNA)-dependent manner. SET8 degradation requires a conserved degron responsible for its interaction with PCNA and recruitment to chromatin where ubiquitylation occurs. Efficient degradation of SET8 at the onset of S phase is required for the regulation of chromatin compaction status and cell cycle progression. Moreover, the turnover of SET8 is accelerated after ultraviolet irradiation dependent on the CRL4(CDT2) ubiquitin ligase and PCNA. Removal of SET8 supports the modulation of chromatin structure after DNA damage. These results demonstrate a novel regulatory mechanism, linking for the first time the ubiquitin-proteasome system with rapid degradation of a histone methyltransferase to control cell proliferation.     

Expression Levels of the BDNF Gene and Histone Modifications Around Its Promoters in the Ventral Tegmental Area and Locus Ceruleus of Rats During Forced Abstinence from Morphine

Brain-derived neurotrophic factor (BDNF) plays a role in mediating molecular, cellular, and behavioral adaptations underlying drug addiction. Here, we examined the influence of withdrawal from repeated morphine treatment on the expression of BDNF mRNA in the ventral tegmental area (VTA) and locus coeruleus (LC) of the rat brain. We also studied whether alternations in mRNA levels of BDNF in these tissues are associated with histone modifications around promoters II and III of the BDNF gene. Thus, chromatin immunoprecipitation (CHIP) and quantitative (q)-PCR were employed to assess acetylation of histone H3 at K9/K14 and trimethylation of histone H3 at K9. Results of qRT-PCR showed that levels of BDNF mRNA in both VTA and LC were significantly increased 7 days rather than 2 h or 24 h following the last injection of morphine. Consistently, CHIP and qPCR analysis revealed that on day 7 of morphine abstinence, both VTA and LC levels of histone methylation at BDNF promoters II and III of morphine treated rats were significantly lower than control animals. Morphine withdrawal caused only a significant increase in H3 acetylation at the promoter II in the LC. These data demonstrate the involvement of histone H3 methylation in the regulation of gene expression in the VTA and LC of rats during forced abstinence of morphine.     

Elongator complex is critical for cell cycle progression and leaf patterning in Arabidopsis

The mitotic cell cycle in higher eukaryotes is of pivotal importance for organ growth and development. Here, we report that Elongator, an evolutionarily conserved histone acetyltransferase complex, acts as an important regulator of mitotic cell cycle to promote leaf patterning in Arabidopsis. Mutations in genes encoding Elongator subunits resulted in aberrant cell cycle progression, and the altered cell division affects leaf polarity formation. The defective cell cycle progression is caused by aberrant DNA replication and increased DNA damage, which activate the DNA replication checkpoint to arrest the cell cycle. Elongator interacts with proliferating cell nuclear antigen (PCNA) and is required for efficient histone 3 (H3) and H4 acetylation coupled with DNA replication. Levels of chromatin-bound H3K56Ac and H4K5Ac known to associate with replicons during DNA replication were reduced in the mutants of both Elongator and chromatin assembly factor 1 (CAF-1), another protein complex that physically interacts with PCNA for DNA replication-coupled chromatin assembly. Disruptions of CAF-1 also led to severe leaf polarity defects, which indicated that Elongator and CAF-1 act, at least partially, in the same pathway to promote cell cycle progression. Collectively, our results demonstrate that Elongator is an important regulator of mitotic cell cycle, and the Elongator pathway plays critical roles in promoting leaf polarity formation.     

An H3 histone-specific kinase isolated from bovine thymus chromatin.

A substantial portion of the histone phosphorylating activity of bovine thymus chromatin can be isolated by extraction in 0.2 M NaCl. The specificity of this extract for either free histones or washed chromatin substrates was compared. The salt-extracted kinase enzymes favor H2b as the major acceptor when whole free histone is the substrate and H3 when the substrate is intact chromatin. The H3 kinase activity of bovine thymus tissue has been purified free from other detectable histone kinase activities by ammonium sulfate fractionation and is highly specific for H3 histone when assayed either with chromatin or isolated whole histone. The activity is cAMP-independent. Tryptic peptide mapping of the labeled H3 histone reveals a single site of phosphorylation. This site appears to be identical with the major site of metaphase-associated H3 phosphorylation in hepatoma tissue culture cells. No corresponding H3 phosphorylation has been detected in thymus tissue in vivo.     

Tumour suppressor ING1b maintains genomic stability upon replication stress

The lesion bypass pathway, which is regulated by monoubiquitination of proliferating cell nuclear antigen (PCNA), is essential for resolving replication stalling due to DNA lesions. This process is important for preventing genomic instability and cancer development. Previously, it was shown that cells deficient in tumour suppressor p33ING1 (ING1b) are hypersensitive to DNA damaging agents via unknown mechanism. In this study, we demonstrated a novel tumour suppressive function of ING1b in preserving genomic stability upon replication stress through regulating PCNA monoubiquitination. We found that ING1b knockdown cells are more sensitive to UV due to defects in recovering from UV-induced replication blockage, leading to enhanced genomic instability. We revealed that ING1b is required for the E3 ligase Rad18-mediated PCNA monoubiquitination in lesion bypass. Interestingly, ING1b-mediated PCNA monoubiquitination is associated with the regulation of histone H4 acetylation. Results indicate that chromatin remodelling contributes to the stabilization of stalled replication fork and to the regulation of PCNA monoubiquitination during lesion bypass.     

Kinetics of accumulation and depletion of soluble newly synthesized histone in the reciprocal regulation of histone and DNA synthesis

Procedures are presented which permit the identification and analysis of cellular histone that is not bound to chromatin. This histone, called soluble histone, could be distinguished from that bound to chromatin by the state of H4 modification and the lack of H2A ubiquitination. Changes in the levels of newly synthesized soluble histone were analyzed with respect to the balance between histone and DNA synthesis in hamster ovary cells. Pulse-chase protocols suggested that the chase of newly synthesized histone from the soluble fraction into chromatin may have two kinetic components with half-depletion times of about 1 and 40 min. When protein synthesis was inhibited, the pulse-chase kinetics of newly synthesized histone from the solubl fraction into chromatin were not significantly altered from those of the control. However, in contrast to the control, when protein synthesis was inhibited, DNA synthesis was also inhibited with kinetics similar to those of the chase of newly synthesized histone from the soluble fraction. There was a rapid decrease in the rate of DNA synthesis with a half-deceleration time of 1 min down to about 30% of the control rate, followed by a slower decrease with an approximate half-deceleration time of 40 min. When DNA synthesis was inhibited, newly synthesized histone accumulated in the soluble fraction, but H2A and H2B continued to complex with chromatin at a significant rate. Soluble histone in G1 cells showed the same differential partitioning of H4/H3 and H2A/H2B between the soluble and chromatin-bound fractions as was found in cycling cells with inhibited DNA synthesis. These results support a unified model of reciprocal regulatory mechanisms between histone and DNA synthesis in the assembly of chromatin.     

Molecular cloning and differential expression of somatic and testis-specific H2B histone genes during rat spermatogenesis

We have cloned cDNA of a testis-specific histone, TH2B (a variant of H2B), and rat somatic H2B gene to investigate regulation of testis-specific histone genes during rat spermatogenesis. The amino acid sequences deduced from DNA sequences show extensive sequence divergence in the N-terminal third of the two histones. The rest is highly conserved. One cysteine residue was found in TH2B. No cysteine is present in somatic histones except in H3 histone. We investigated the expression of TH2B and H2B genes using the regions of sequence divergence as hybridization probes. The TH2B gene is expressed only in the testis, and the expression of this gene is detected 14 days after birth, reaching a maximum at Day 20. The level of H2B mRNA shows a reciprocal pattern. This contrasting pattern can be explained by the gradually changing proportion of spermatogonia and spermatocytes with testicular maturation. In situ cytohybridization studies show that H2B gene is expressed primarily in proliferating spermatogonia and preleptotene spermatocytes, whereas TH2B gene is expressed exclusively in pachytene spermatocytes which first appear in testis about 14 days after birth. H2B and TH2B genes appear to be ideal markers for the study of proliferation and differentiation events in spermatogenesis and their regulatory mechanisms.     

The role of H2B histones from the sea urchin sperm in the formation of supranucleosome structures

The structural role of histone H2B from sea urchin sperm (H2Bsp) has been examined in experiments on reconstitution of chromatin from DNA and core histones taken in three variants: (1) four core histones from sea urchin sperm; (2) four core histones from calf thymus; (3) (H3, H4, H2A) from calf thymus and H2Bsp. It is shown that H2Bsp when present in reconstituted chromatin induces its aggregation. Fidelity of the reconstitution of nucleosomes has been tested using DNase I probe, one- and two-dimensional electrophoresis and electron microscopy. The reconstitutes that contain H2Bsp appear under electron microscope mainly as regular closely spaced large granules, about 450 A in diameter, which are very similar to the granules found in "native" sea urchin sperm chromatin. The reconstitutes formed by four core histones from calf thymus appear as randomly arranged particles, about 100 A in diameter. We conclude that histone H2Bsp participates in interactions between nucleosomes and is involved in the formation of the condensed supranucleosomal structure in sea urchin sperm chromatin.     

Histones synthesized at different stages of myogenesis are differentially degraded in myotube cells

We recently reported that cultures of terminally differentiating myotube cells synthesize histones in reduced but significant amounts in comparison with proliferating myoblasts (Wunsch et al., 1987, Dev. Biol., 119: 85-93). In this study, the stability of myotube histone has been determined, comparing the degradation of de novo-synthesized histones in nascent (day 3) and maturing (day 4) myotubes with histones in the same cells that had been previously made during myoblast proliferation (day 1). Histones synthesized in proliferating myoblasts and myotubes were pulse-labeled with 3H-lysine and chased up to seven days, followed by determinations of radioactivity remaining in histone bands using fluorography of one- and two-dimensional polyacrylamide gels. Considered in aggregate, core histones synthesized de novo in nascent (day 3) myotubes were degraded most rapidly, followed by myotube histones that had been previously made during the proliferative phase (day 1) of myogenesis. De novo-synthesized histones in maturing (day 4) myotubes were relatively stable. Individual histone classes were degraded in the following order of increasing half-life, regardless of the differentiative stage at which they were synthesized: H2A.Z, H2A, H2B, H3(.2, day 1; .3, days 3 and 4), H4.     

Human ING1 proteins differentially regulate histone acetylation

ING1 proteins are nuclear, growth inhibitory, and regulate apoptosis in different experimental systems. Here we show that similar to their yeast homologs, human ING1 proteins interact with proteins associated with histone acetyltransferase (HAT) activity, such as TRRAP, PCAF, CBP, and p300. Human ING1 immunocomplexes contain HAT activity, and overexpression of p33(ING1b), but not of p47(ING1a), induces hyperacetylation of histones H3 and H4, in vitro and in vivo at the single cell level. p47(ING1a) inhibits histone acetylation in vitro and in vivo and binds the histone deacetylase HDAC1. Finally, we present evidence indicating that p33(ING1b) affects the degree of physical association between proliferating cell nuclear antigen (PCNA) and p300, an association that has been proposed to link DNA repair to chromatin remodeling. Together with the finding that human ING1 proteins bind PCNA in a DNA damage-dependent manner, these data suggest that ING1 proteins provide a direct linkage between DNA repair, apoptosis, and chromatin remodeling via multiple HAT.ING1.PCNA protein complexes.     

Histone Acetylation Regulation in Sleep Deprivation-Induced Spatial Memory Impairment

Sleep disorders negatively affect cognition and health. Recent evidence has indicated that chromatin remodeling via histone acetylation regulates cognitive function. This study aimed to investigate the possible roles of histone acetylation in sleep deprivation (SD)-induced cognitive impairment. Results of the Morris water maze test showed that 3 days of SD can cause spatial memory impairment in Wistar rats. SD can also decrease histone acetylation levels, increase histone deacetylase 2 (HDAC2) expression, and decrease histone acetyltransferase (CBP) expression. Furthermore, SD can reduce H3 and H4 acetylation levels in the promoters of the brain-derived neurotrophic factor (Bdnf) gene and thus significantly downregulate BDNF expression and impair the activity of key BDNF signaling pathways (pCaMKII, pErk2, and pCREB). However, treatment with the HDAC inhibitor trichostatin A attenuated all the negative effects induced by SD. Therefore, BDNF and its histone acetylation regulation may play important roles in SD-induced spatial memory impairment, whereas HDAC inhibition possibly confers protection against SD-induced impairment in spatial memory and hippocampal functions.