Isolation and sequence analysis of sea urchin (Lytechinus pictus) histone H4 messenger RNA

Histone messenger RNAs isolated from early blastula stage Lytechinus pictus sea urchin embryos have been separated into discrete RNA bands on polyacrylamide gels. The most rapidly migrating of these molecules, the putative histone H4 mRNA, has been digested with T₁ ribonuclease to generate oligonucleotides for nucleotide sequence analysis. Many of these sequences are colinear with the highly conserved amino acid sequence of histone H4 protein as determined for both cows and peas.Histone H4 messenger RNA hybridizes in conditions of DNA excess to sea urchin DNA which is repeated approximately 470-fold. Despite this level of repetition the nucleotide sequence of the H4 messenger RNA reflects little evolutionary divergence within the H4 genes of L. pictus as judged by the stoichiometric yield of T₁ oligonucleotides and the hybridization and thermal stability of histone H4 mRNA-DNA hybrids.     

Quantitation of the accumulation of histone messenger RNA during oogenesis in Xenopus leavis

The accumulation of messenger RNA coding for histone H3 in oogenesis of Xenopus laevis was studied by quantitative hybridization techniques, using a cloned genomic DNA fragment as a probe. This probe was isolated from cloned Xenopus histone DNA and contains most of the H3 coding sequences. Histone H3 mRNA accumulation was found to be completed before the maximum lampbrush stage. Hybridization of RNA blots with DNA probes containing genes for histones H2A, H2B, and H4 suggests the same accumulation pattern for the mRNAs coding for these histones as for histone H3 mRNA. The amount of H3 mRNA in the mature oocyte was established to be 130 ± 68 pg, i.e., about 5 × 10⁸ copies.     

Yeast CAF-1 assembles histone (H3-H4)2 tetramers prior to DNA deposition

Following acetylation, newly synthesized H3-H4 is directly transferred from the histone chaperone anti-silencing factor 1 (Asf1) to chromatin assembly factor 1 (CAF-1), another histone chaperone that is critical for the deposition of H3-H4 onto replicating DNA. However, it is unknown how CAF-1 binds and delivers H3-H4 to the DNA. Here, we show that CAF-1 binds recombinant H3-H4 with 10- to 20-fold higher affinity than H2A-H2B in vitro, and H3K56Ac increases the binding affinity of CAF-1 toward H3-H4 2-fold. These results provide a quantitative thermodynamic explanation for the specific H3-H4 histone chaperone activity of CAF-1. Surprisingly, H3-H4 exists as a dimer rather than as a canonical tetramer at mid-to-low nanomolar concentrations. A single CAF-1 molecule binds a cross-linked (H3-H4)₂ tetramer, or two H3-H4 dimers that contain mutations at the (H3-H4)₂ tetramerization interface. These results suggest that CAF-1 binds to two H3-H4 dimers in a manner that promotes formation of a (H3-H4)₂ tetramer. Consistent with this idea, we confirm that CAF-1 synchronously binds two H3-H4 dimers derived from two different histone genes in vivo. Together, the data illustrate a clear mechanism for CAF-1-associated H3-H4 chaperone activity in the context of de novo nucleosome (re)assembly following DNA replication.     

Potent activity against K562 cells by polyamide–seco-CBI conjugates targeting histone H4 genes

We designed and synthesized conjugates between pyrrole–imidazole polyamides and seco-CBI that alkylate within the coding regions of the histone H4 genes. DNA alkylating activity on the histone H4 fragment and cellular effects against K562 chronic myelogenous leukemia cells were investigated. One of the conjugates, 5-CBI, showed strong DNA alkylation activity and good sequence specificity on a histone H4 gene fragment. K562 cells treated with 5-CBI down-regulated the histone H4 gene and induced apoptosis efficiently. Global gene expression data revealed that a number of histone H4 genes were down-regulated by 5-CBI treatment. These results suggest that sequence-specific DNA alkylating agents may have the potential of targeting specific genes for cancer chemotherapy.     

The Rtt109 histone acetyltransferase facilitates error-free replication to prevent CAG/CTG repeat contractions

Lysine 56 is acetylated on newly synthesized histone H3 in yeast, Drosophila and mammalian cells. All of the proteins involved in histone H3 lysine 56 (H3K56) acetylation are important for maintaining genome integrity. These include Rtt109, a histone acetyltransferase, responsible for acetylating H3K56, Asf1, a histone H3/H4 chaperone, and Hst3 and Hst4, histone deacetylases which remove the acetyl group from H3K56. Here we demonstrate a new role for Rtt109 and H3K56 acetylation in maintaining repetitive DNA sequences in Saccharomyces cerevisiae. We found that cells lacking RTT109 had a high level of CAG/CTG repeat contractions and a twofold increase in breakage at CAG/CTG repeats. In addition, repeat contractions were significantly increased in cells lacking ASF1 and in an hst3Δhst4Δ double mutant. Because the Rtt107/Rtt101 complex was previously shown to be recruited to stalled replication forks in an Rtt109-dependent manner, we tested whether this complex was involved. However, contractions in rtt109Δ cells were not due to an inability to recruit the Rtt107/Rtt101 complex to repeats, as absence of these proteins had no effect on repeat stability. On the other hand, Dnl4 and Rad51-dependent pathways did play a role in creating some of the repeat contractions in rtt109Δ cells. Our results show that H3K56 acetylation by Rtt109 is important for stabilizing DNA repeats, likely by facilitating proper nucleosome assembly at the replication fork to prevent DNA structure formation and subsequent slippage events or fork breakage.     

Hypoxia causes epigenetic gene regulation in macrophages by attenuating Jumonji histone demethylase activity

Epigenetic processes elicit changes in gene expression by modifying DNA bases or histone side chains without altering DNA sequences. Recently discovered Jumonji histone demethylases (JHDMs) affect gene expression by demethylating lysine residues of histone tails. JHDMs belong to a family of dioxygenases and share similarities with prolyl hydroxylases (PHDs). Therefore, we investigated the influence of hypoxia in macrophages on histone methylation. All JHDM family members JMJD1A–C and JMJD2A–D are expressed in macrophages. Thus, we analyzed the methylation status of histone H3 residues not only under hypoxia but also after treatment with the dioxygenase-inhibitors DMOG, NO and ROS. Western analysis revealed increased methylations in H3K9me2/me3 and H3K36me3 at pO₂ below 3%, DMOG, NO and ROS treatment. Chromatin immunoprecipitation (ChIP) assays demonstrated increased repressive marks H3K9me2 and H3K9me3 in specific promoter regions of the chemokine Ccl2 and the chemokine receptors Ccr1 and Ccr5, which correlated with a downregulation of their mRNA expression under hypoxic conditions. In contrasts, the hypoxia-inducible factor (HIF) target gene adrenomedullin (ADM) mRNA was upregulated and no increase in its histone modification was observed. We suggest that hypoxia and a concomitant loss of JHDM activity increases H3K9 methylation and decreases chemokine expression.     

Reduction of histone cytotoxicity by the Alzheimer β-amyloid peptide precursor

In a search for Alzheimer β-amyloid peptide precursor ligands, Potempska et al. (Arch. Biochem. Biophys. (1993) 304, 448) found that histones bind with high affinity and specificity to the secreted precursor. Because exogenous histones can be cytotoxic, we compared the effects of histones on the viability of cells which produce little β-amyloid peptide precursor (U-937) to those on cells that produce twenty times as much precursor (COS-7). Addition of purified histones caused necrosis of U-937 cells (histone H4, LD₅₀=1.5 μM). Extracellular Aβ precursor in the submicromolar range prevented histone-induced U-937 cell necrosis. Cell-surface precursor also reduced histone toxicity: COS-7 cells were less sensitive to the toxic effects of histone H4 (LD₅₀=5.4 μM). COS-7 cells in which the expression of an APP mRNA-directed ribozyme reduced the synthesis of the protein by up to 80% were more sensitive to histone H4 (LD₅₀=3.2 μM) than cells that expressed the vector alone. Histone H4 binds to cell-associated Aβ precursor. Cells expressing the Aβ precursor-directed ribozyme bound less ¹²⁵I-labeled histone H4 than those expressing the vector alone. In the limited extracellular space of tissues in vivo, both secreted and cell-surface Aβ precursor protein may play significant roles in trapping chromatin or histones and removing them from the extracellular milieu.     

Uncoordinate synthesis of histone H1 in cells arrested in the G1 phase

It has been known for several years that DNA replication and histone synthesis occur concomitantly in cultured mammalian cells. Normally all five classes of histones are synthesized coordinately. However, mouse myeloma cells, synchronized by starvation for isoleucine, synthesize increased amounts of histone H1 relative to the four nucleosomal core histones. This unscheduled synthesis of histone H1 is reduced within 1 h after refeeding isoleucine, and is not a normal component of G1. The synthesis of H1 increases coordinately again with other histones during the S phase. The DNA synthesis inhibitors, cytosine arabinoside and hydroxyurea, block all histone synthesis in S-phase cells. The levels of histone H1 mRNA, relative to the other histone mRNAs, is increased in isoeleucine-starved cells and decreases rapidly after refeeding isoleucine. The increased incorporation of histone H1 is at least partially due to the low isoleucine content of histone H1. Starvation of cells for lysine resulted in a decrease in H1 synthesis relative to core histones. Again the ratio was altered on refeeding the amino acid. 3T3 cells starved for serum also incorporated only H1 histones into chromatin. The ratio of different H1 proteins also changed. The synthesis of the H1⁰ protein was predominant in G₀ cells, and reduced in S-phase cells. These data indicate the metabolism of H1 is independent of the other histones when cell growth is arrested.     

Methylation of histone H3 at lysine 4 and expression of the maltase-glucoamylase gene are reduced by dietary resistant starch

Methylated histone H3 at lysine 4 (K4) is associated with euchromatin and is involved in the transactivation of genes. However, it is unknown whether histone methylation is involved with changes in gene expression induced by nutrients. In this study, we examined whether methylations of histone H3 at K4 on maltase-glucoamylase (Mgam), which is responsible for the digestion of starch in the small intestine, as well as Mgam expression were altered by feeding rats an indigestible starch (resistant starch, RS). The mRNA and protein levels and the activities of MGAM were reduced in rats fed an RS diet compared with those fed a regular starch diet. Furthermore, we found that decreases in di- and tri-methylation of histone H3 at K4, as well as reduced acetylation of histones H3 and H4 on the Mgam gene were associated with a reduction of Mgam gene expression. These results suggest that the reductions of jejunal MGAM levels and activities caused by the RS diet are regulated at the mRNA level through a decrease in methylation of histone H3 at K4 and reduced acetylation of histones H3 and H4 on the Mgam gene.     

Differential epigenetic regulation of BDNF and NT-3 genes by trichostatin A and 5-aza-2′-deoxycytidine in Neuro-2a cells

To understand epigenetic regulation of neurotrophins in Neuro-2a mouse neuroblastoma cells, we investigated the alteration of CpG methylation of brain-derived neurotrophic factor (BDNF) promoter I and neurotrophin-3 (NT-3) promoter IB and that of histone modification in Neuro-2a cells. Bisulfite genomic sequencing showed that the CpG sites of BDNF promoter I were methylated in non-treated Neuro-2a cells and demethylated following 5-aza-2′-deoxycytidine (5-aza-dC) treatment. In contrast, methylation status of the NT-3 promoter IB did not change by 5-aza-dC treatment in Neuro-2a cells. Furthermore, we demonstrated that BDNF exon I-IX mRNA was induced by trichostatin A (TSA) treatment. However, NT-3 exon IB-II mRNA was not induced by TSA treatment. Chromatin immunoprecipitation assays showed that the levels of acetylated histones H3 and H4 on BDNF promoter I were increased by TSA. These results demonstrate that DNA methylation and/or histone modification regulate BDNF gene expression, but do not regulate NT-3 gene expression in Neuro-2a cells.     

PGE2 induces interleukin-8 derepression in human astrocytoma through coordinated DNA demethylation and histone hyperacetylation

We have recently reported that in astrocytoma cells the expression of interleukin-8 (IL-8) is upregulated by prostaglandin E2 (PGE2). Unfortunately, the exact mechanism by which this happens has not been clarified yet. Here, we have investigated whether IL-8 activation by PGE2 involves changes in DNA methylation and/or histone modifications in 46 astrocytoma specimens, two astrocytoma cell lines and normal astrocytic cells. The DNA methylation status of the IL-8 promoter was analyzed by bisulphite sequencing and by methylation DNA immunoprecipitation analysis. The involvement of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs), as well as histone acetylation levels, was assayed by chromatin immunoprecipitation. IL-8 expression at promoter, mRNA and protein level was explored by transfection, real-time PCR and enzyme immunoassay experiments in cells untreated or treated with PGE2, 5-aza-2'-deoxycytidine (5-aza-dC) and HDAC inhibitors, alone or in combination. EMSA was performed with crude cell extracts or recombinant protein. We observed that PGE2 induced IL-8 activation through: (1) demethylation of the single CpG site 5 located at position -83 within the binding region for CEBP-β in the IL-8 promoter; (2) C/EBP-β and p300 co-activator recruitment; (3) H3 acetylation enhancement and (4) reduction in DNMT1, DNMT3a, HDAC2 and HDAC3 association to CpG site 5 region. Treatment with 5-aza-dC or HDAC inhibitors of class I HDACs strengthened either basal or PGE2-mediated IL-8 expression. These findings have elucidated an orchestrated mechanism triggered by PGE2 whereby concurrent association of site-specific demethylation and histone H3 hyperacetylation resulted in derepression of IL-8 gene expression in human astrocytoma.     

Inhibition of histone deacetylases by Trichostatin A leads to a HoxB4-independent increase of hematopoietic progenitor/stem cell frequencies as a result of selective survival

BackgroundDNA and chromatin modifications are critical mediators in the establishment and maintenance of cell type-specific gene expression patterns that constitute cellular identities. One type of modification, the acetylation and deacetylation of histones, occurs reversibly on lysine ε-NH₃⁺ groups of core histones via histone acetyl transferases (HAT) and histone deacetylases (HDAC). Hyperacetylated histones are associated with active chromatin domains, whereas hypoacetylated histones are enriched in non-transcribed loci.MethodsWe analyzed global histone H4 acetylation and HDAC activity levels in mature lineage marker-positive (Lin⁺) and progenitor lineage marker-negative (Lin^?) hematopoietic cells from murine bone marrow (BM). In addition, we studied the effects of HDAC inhibition on hematopoietic progenitor/stem cell (HPSC) frequencies, cell survival, differentiation and HoxB4 dependence.ResultsWe observed that Lin^? and Lin⁺ cells do not differ in global histone H4 acetylation but in HDAC activity levels. Further, we saw that augmented histone acetylation achieved by transient Trichostatin A (TSA) treatment increased the frequency of cells with HPSC immunophenotype and function in the heterogeneous pool of BM cells. Induction of histone hyperacetylation in differentiated BM cells was detrimental, as evidenced by preferential death of mature BM cells upon HDAC inhibition. Finally, TSA treatment of BM cells from HoxB4^?/? mice revealed that the HDAC inhibitor-mediated increase in HPSC frequencies was independent of HoxB4.ConclusionsOverall, these data indicate the potential of chromatin modifications for the regulation of HPSC. Chromatin-modifying agents may provide potential strategies for ex vivo expansion of HPSC.     

Alterations in amounts and covalent modifications of low-molecular-weight chromosomal proteins in Chinese hamster ovary cells during polyamine depletion

The effect of polyamine depletion on phosphorylation and ADP-ribosylation of low-M_r chromosomal proteins was studied in intact, mutant Chinese hamster ovary cells (CHO-P22) devoid of ornithine decarboxylase activity. When starved of polyamines for 6 days, severe polyamine deficiency develops and the cells gradually stop growing. The rate of DNA synthesis was retarded to 16% of the control value and to 29% in density-inhibited cells. The synthesis of high-mobility-group (HMG) proteins was decreased by 65% in polyamine-depleted cells and by 40% in density-inhibited cells. The synthesis of core histones was decreased by 40% both in polyamine-depleted and density-inhibited cells. In polyamine-depleted cells the molar ratio of the higher-M_r HMG proteins (HMG 1 + 2) to the lower-M_r HMG proteins (HMG 14 + P) was about one-half of that found in cells grown in the presence of putrescine or in density-inhibited cells. In contrast to HMG proteins, no major differences were found in the content of core histones in these cell populations. In the perchloric acid-soluble fraction of nuclear proteins, ³²P was incorporated mainly into histone H1, HMG P and a protein migrating more slowly than HMG 1 (protein P1). Specific changes in the ³²P-labeling and migration of a number of protein bands, including histone H1, was observed in polyamine-depleted cells as compared to cells grown in the presence of putrescine or to density-inhibited cells. ADP-ribosylation experiments using [³H]adenosine showed a different pattern of label distribution; the higher-M_r HMG proteins from polyamine-depleted cells contained about one-half the amount of label found in the proteins from control cells. The lower-M_r HMG proteins and histone H1 were the preferentially labeled proteins in polyamine-depleted cells. Labeling of core histones with [³²P]orthophosphate or [³H]adenosine did not differ markedly in the two cell populations. The results obtained using intact polyamine auxotrophic cells indicated that polyamine depletion is connected with more severe alterations in amounts and covalent modifications (phosphorylation and ADP-ribosylation) of HMG chromosomal proteins and histone H1 than core histones.