Histone H4 acetylation and DNA methylation dynamics during pollen development

Summary The first pollen mitosis results in generative and vegetative cells which are characterised by a striking difference in their chromatin structure. In this study, histone H4 acetylation and DNA methylation have been analysed during pollen development inLilium longiflorum. Indirect immunofluorescence procedures followed by epifluorescence and laser scanning microscopy enabled a relative quantification of H4 acetylation and DNA methylation in microspores, immature binucleate pollen, mature pollen, and pollen tubes. The results show that histone H4 of the vegetative nucleus, in spite of its decondensed chromatin structure, is strongly hypoacetylated at lysine positions 5 and 8 in comparison with both the original microspore nucleus and the generative-cell nucleus. These H4 terminal lysines in the vegetative nucleus are, however, progressively acetylated during the following pollen tube growth. The DNA methylation analysis inversely correlates with the histone acetylation data. The vegetative nucleus in mature pollen grains is heavily methylated, but a dramatic nonreplicative demethylation occurs during the pollen tube development. Changes neither in H4 acetylation nor in DNA methylation have been found during development of the generative nucleus. The results obtained indicate that the vegetative nucleus enters the quiescent state (accompanied by DNA hypermethylation and H4 underacetylation) during the maturation of pollen grain which enables pollen grains a long-term survival without external source of nutrients until they reach the stigma.     

A fine balance: epigenetic control of cellular quiescence by the tumor suppressor PRDM2/RIZ at a bivalent domain in the cyclin a gene

Adult stem cell quiescence is critical to ensure regeneration while minimizing tumorigenesis. Epigenetic regulation contributes to cell cycle control and differentiation, but few regulators of the chromatin state in quiescent cells are known. Here we report that the tumor suppressor PRDM2/RIZ, an H3K9 methyltransferase, is enriched in quiescent muscle stem cells in vivo and controls reversible quiescence in cultured myoblasts. We find that PRDM2 associates with >4400 promoters in G₀ myoblasts, 55% of which are also marked with H3K9me2 and enriched for myogenic, cell cycle and developmental regulators. Knockdown of PRDM2 alters histone methylation at key promoters such as Myogenin and CyclinA2 (CCNA2), and subverts the quiescence program via global de-repression of myogenesis, and hyper-repression of the cell cycle. Further, PRDM2 acts upstream of the repressive PRC2 complex in G₀. We identify a novel G₀-specific bivalent chromatin domain in the CCNA2 locus. PRDM2 protein interacts with the PRC2 protein EZH2 and regulates its association with the bivalent domain in the CCNA2 gene. Our results suggest that induction of PRDM2 in G₀ ensures that two antagonistic programs—myogenesis and the cell cycle—while stalled, are poised for reactivation. Together, these results indicate that epigenetic regulation by PRDM2 preserves key functions of the quiescent state, with implications for stem cell self-renewal.     

H4K20 methylation regulates quiescence and chromatin compaction

The transition between proliferation and quiescence is frequently associated with changes in gene expression, extent of chromatin compaction, and histone modifications, but whether changes in chromatin state actually regulate cell cycle exit with quiescence is unclear. We find that primary human fibroblasts induced into quiescence exhibit tighter chromatin compaction. Mass spectrometry analysis of histone modifications reveals that H4K20me2 and H4K20me3 increase in quiescence and other histone modifications are present at similar levels in proliferating and quiescent cells. Analysis of cells in S, G₂/M, and G₁ phases shows that H4K20me1 increases after S phase and is converted to H4K20me2 and H4K20me3 in quiescence. Knockdown of the enzyme that creates H4K20me3 results in an increased fraction of cells in S phase, a defect in exiting the cell cycle, and decreased chromatin compaction. Overexpression of Suv4-20h1, the enzyme that creates H4K20me2 from H4K20me1, results in G₂ arrest, consistent with a role for H4K20me1 in mitosis. The results suggest that the same lysine on H4K20 may, in its different methylation states, facilitate mitotic functions in M phase and promote chromatin compaction and cell cycle exit in quiescent cells.     

A Highlights from MBoC Selection: Trehalose Is a Key Determinant of the Quiescent Metabolic State That Fuels Cell Cycle Progression upon Return to Growth

When conditions are unfavorable, virtually all living cells have the capability of entering a resting state termed quiescence or G0. Many aspects of the quiescence program as well as the mechanisms governing the entry and exit from quiescence remain poorly understood. Previous studies using the budding yeast Saccharomyces cerevisiae have shown that upon entry into stationary phase, a quiescent cell population emerges that is heavier in density than nonquiescent cells. Here, we show that total intracellular trehalose and glycogen content exhibits substantial correlation with the density of individual cells both in stationary phase batch cultures and during continuous growth. During prolonged quiescence, trehalose stores are often maintained in favor over glycogen, perhaps to fulfill its numerous stress-protectant functions. Immediately upon exit from quiescence, cells preferentially metabolize trehalose over other fuel sources. Moreover, cells lacking trehalose initiate growth more slowly and frequently exhibit poor survivability. Together, our results support the view that trehalose, which is more stable than other carbohydrates, provides an enduring source of energy that helps drive cell cycle progression upon return to growth.     

组蛋白去乙酰化酶抑制剂的研究进展

核小体是真核生物染色质的基本单位,通过对组蛋白核心的N-端的乙酰化、甲基化、磷酸化、遍在蛋白化的修饰作用而影响细胞的功能。组蛋白乙酰化酶(histone acetylase HAT)及组蛋白去乙酰化酶(Histone Deacetylases HDAC)之间的动态平衡控制着染色质的结构和基因表达。当组蛋白去乙酰化水平增加,乙酰化水平相对降低,即会导致正常的细胞周期与代谢行为的改变而诱发肿瘤,及神经退行性变。组蛋白去乙酰化酶抑制剂(Histone Deacetylases-inhibitor HDACi)目前是国内外研究的热点。其中,曲古霉素A(Trichostatin A TSA),是最早发现的天然组蛋白去乙酰化酶抑制剂;伏立诺他(Suberoylanilide Hydroxamic Acid SAHA)已经美国FDA批准用于治疗皮肤T细胞淋巴瘤。本文就HDACi分类及其功能出发综述HDACi的作用机制及研究进展。     

Dynamic Acetylation of All Lysine 4–Methylated Histone H3 in the Mouse Nucleus: Analysis at c-fos and c-jun

A major focus of current research into gene induction relates to chromatin and nucleosomal regulation, especially the significance of multiple histone modifications such as phosphorylation, acetylation, and methylation during this process. We have discovered a novel physiological characteristic of all lysine 4 (K4)–methylated histone H3 in the mouse nucleus, distinguishing it from lysine 9–methylated H3. K4-methylated histone H3 is subject to continuous dynamic turnover of acetylation, whereas lysine 9–methylated H3 is not. We have previously reported dynamic histone H3 phosphorylation and acetylation as a key characteristic of the inducible proto-oncogenes c-fos and c-jun. We show here that dynamically acetylated histone H3 at these genes is also K4-methylated. Although all three modifications are proven to co-exist on the same nucleosome at these genes, phosphorylation and acetylation appear transiently during gene induction, whereas K4 methylation remains detectable throughout this process. Finally, we address the functional significance of the turnover of histone acetylation on the process of gene induction. We find that inhibition of turnover, despite causing enhanced histone acetylation at these genes, produces immediate inhibition of gene induction. These data show that all K4-methylated histone H3 is subject to the continuous action of HATs and HDACs, and indicates that at c-fos and c-jun, contrary to the predominant model, turnover and not stably enhanced acetylation is relevant for efficient gene induction.     

关于组蛋白甲基化的研究

主要阐述了组蛋白甲基转移酶的类型,组蛋白H3中第9位赖氨酸甲基化与异染色质的形成、常染色体中基因表达的调控,以及与DNA甲基化之间的关系,说明了组蛋白甲基化与组蛋白乙酰化、磷酸化的相互关系, 指出组蛋白甲基化对维持细胞各种状态的平衡起到极其重要的作用。 Abstract： The types of histone methyltransferases, the relationship between methylation of Lysine 9 of H3 and the formation of heterochromatin, gene regulation in euchromatin, and that with DNA methylation, were mainly introduced. The interrelation between histone methylation and histone acetylation/phosphorylation was summarized. It is showed that histone methylation plays a very important role in maintaining the balance state of cell. The future research tendency of histone methylation was fantanstic.     

Histone acetyltransferase activity during the cell cycle

Histone acetyltransferase activity was measured in isolated nuclei during the synchronous cell cycle of the myxomycete Physarum polycephalum. Nuclei were incubated with [14C]acetyl-coenzyme A and an excess of exogenous calf thymus histones. The activity is periodic during the cell cycle; it rises during the S-phase to reach a maximum in the early G2-period with a decline in mid and late G2. Comparison of the pattern of enzyme activity with the in vivo acetylation of histones during the cell cycle reveals that the enzyme activity does not wholly determine the acetylation state, indicating that other factors, including possibly the structural state of chromatin, are responsible for the observed cell cycle pattern of in vivo histone acetylation.     

Histone deacetylase inhibitors decrease reelin promoter methylation in vitro

We investigated the effects of agents that induce reelin mRNA expression in vitro on the methylation status of the human reelin promoter in neural progenitor cells (NT2). NT2 cells were treated with the histone deacetylase inhibitors, trichostatin A (TSA) and valproic acid (VPA), and the methylation inhibitor aza-2'-deoxycytidine (AZA) for various times. All three drugs reduced the methylation profile of the reelin promoter relative to untreated cells. The acetylation status of histones H3 and H4 increased following treatment with VPA and TSA at times as short as 15 min following treatment; a result consistent with the reported mode of action of these drugs. Chromatin immunoprecipitation experiments showed that these changes were accompanied by changes occurring at the level of the reelin promoter as well. Interestingly, AZA decreased reelin promoter methylation without concomittantly increasing histone acetylation. In fact, after prolonged treatments with AZA, the acetylation status of histones H3 and H4 decreased relative to untreated cells. We also observed a trend towards reduced methylated H3 after 18 h treatment with TSA and VPA. Our data indicate that while TSA and VPA act to increase histone acetylation and reduce promoter methylation, AZA acts only to decrease the amount of reelin promoter methylation.     

Histone h3 lysine 4 methylation marks postreplicative human cytomegalovirus chromatin

In the nuclei of permissive cells, human cytomegalovirus genomes form nucleosomal structures initially resembling heterochromatin but gradually switching to a euchromatin-like state. This switch is characterized by a decrease in histone H3 K9 methylation and a marked increase in H3 tail acetylation and H3 K4 methylation across the viral genome. We used ganciclovir and a mutant virus encoding a reversibly destabilized DNA polymerase to examine the impact of DNA replication on histone modification dynamics at the viral chromatin. The changes in H3 tail acetylation and H3 K9 methylation proceeded in a DNA replication-independent fashion. In contrast, the increase in H3 K4 methylation proved to depend widely on viral DNA synthesis. Consistently, labeling of nascent DNA using "click chemistry" revealed preferential incorporation of methylated H3 K4 into viral (but not cellular) chromatin during or following DNA replication. This study demonstrates largely selective epigenetic tagging of postreplicative human cytomegalovirus chromatin.     

Histone deacetylase inhibitors: anticancer compounds

The reversible acetylation of proteins is mediated by histone acetyltransferases which acetylate proteins and histone deacetylases that remove the acetyl groups. High levels of histone acetylation are correlated with active genes, while hypoacetylation of histones corresponds with gene repression. Importantly, acetylation also occurs on non-histone proteins and this can affect the activity and stability of these proteins. Aberrant epigenetic changes are a common hallmark of tumors and imbalances in the activities of deacetylases have been associated with cancers. Accordingly, inhibitors to the histone deacetylases are in clinical trials for the treatment of several cancer types. These drugs mediate a number of molecular changes and in turn can induce cell cycle arrest, apoptosis or differentiation of cancer cells while displaying limited toxicity in normal cells.     

Differential Histone Acetylation in Alfalfa (Medicago sativa) Due to Growth in NaCl : Responses in Salt Stressed and Salt Tolerant Callus Cultures

The steady state distribution of histone variant proteins and their modifications by acetylation were characterized in wild type and salinity stress adapted alfalfa (Medicago sativa). Isotopic labeling detected dynamic acetylation at four sites in the histone H3 variants and five sites in histones H4 and H2B. Histone variant H3.2 was the most highly acetylated histone with 25% higher steady state acetylation and a two- to threefold higher acetylation labeling than histone H3.1. Histone phosphorylation was limited to histone variants H1.A, H1.B, and H1.C and to histone H2A.3, which was also acetylated. Histone variant composition was unaffected by cellular exposure to NaCl. Histone acetylation was qualitatively similar in salt-tolerant and salt-sensitive cells under normal growth conditions. However, short term salt stress in salt sensitive cells or continued growth at 1% NaCl in salt tolerant cells led to major increases in the multiacetylated forms of histone H4 and the two variants of histone H3. These changes were more pronounced in the diploid than in the tetraploid alfalfa strains. The increase in multiacetylation of core histones serves as an in vivo reporter suggesting an altered intranuclear ionic environment in the presence of salt. It may also represent an adaptive response in chromatin structure to permit chromatin function in a more saline intranuclear environment.     

Histone Deacetylase 2 (HDAC2) Regulates Chromosome Segregation and Kinetochore Function via H4K16 Deacetylation during Oocyte Maturation in Mouse

Changes in histone acetylation occur during oocyte development and maturation, but the role of specific histone deacetylases in these processes is poorly defined. We report here that mice harboring Hdac1 ^−/+/Hdac2 ^−/− or Hdac2 ^−/− oocytes are infertile or sub-fertile, respectively. Depleting maternal HDAC2 results in hyperacetylation of H4K16 as determined by immunocytochemistry—normal deacetylation of other lysine residues of histone H3 or H4 is observed—and defective chromosome condensation and segregation during oocyte maturation occurs in a sub-population of oocytes. The resulting increased incidence of aneuploidy likely accounts for the observed sub-fertility of mice harboring Hdac2 ^−/− oocytes. The infertility of mice harboring Hdac1 ^−/+/Hdac2 ^−/−oocytes is attributed to failure of those few eggs that properly mature to metaphase II to initiate DNA replication following fertilization. The increased amount of acetylated H4K16 likely impairs kinetochore function in oocytes lacking HDAC2 because kinetochores in mutant oocytes are less able to form cold-stable microtubule attachments and less CENP-A is located at the centromere. These results implicate HDAC2 as the major HDAC that regulates global histone acetylation during oocyte development and, furthermore, suggest HDAC2 is largely responsible for the deacetylation of H4K16 during maturation. In addition, the results provide additional support that histone deacetylation that occurs during oocyte maturation is critical for proper chromosome segregation.