Post-translational changes of chromosomal proteins in rat cerebellum during postnatal development

Acetylation, phosphorylation and methylation of nuclear proteins in rat cerebellum at 10 and 30 days of age were investigated in vitro. Isolated nuclei were incubated in the presence of [1-14C]acetyl CoA, S-adenosyl [methyl-3H]methionine and [gamma-32P]ATP and then separated into histones and non histone proteins (NHP), which were further fractionated by polyacrylamide gel electrophoresis. The results obtained indicate that acetylation, phosphorylation and methylation of both basic and acidic proteins decrease from 10 to 30 days of age. Electrophoretic analysis of histones shows that the decrease mainly concerns H1, H3, and H2b fractions. The H3 fraction is always more labeled than the other fractions and shows the major changes during postnatal development. Phosphorylation of H2a and H4 fractions increases from 10 to 30 days of age, whereas acetylation and methylation of these fractions do not show significant changes from 10 to 30 days. The densitometric and radioactive patterns of NHP show considerable changes between 10 and 30 days, especially in the high molecular weight region. The incorporation of 14C-acetyl and 3H-methyl groups and of 32P phosphate appears to be generalized throughout the molecular weight range and decreases from 10 to 30 days of age. The methylation of an as yet unidentified protein with a molecular weight of approximately 110,000 daltons occurred at both ages.     

Acetylation and Phosphorylation of Histones and Nonhistone Chromosomal Proteins in Neuronal and Glial Nuclei Purified from Cerebral Hemispheres of Developing Rat Brain

The processes of acetylation and phosphorylation of histones and nonhistone proteins (NHPs) in neuronal and glial nuclei purified from cerebral hemispheres of rats at 1, 10, and 30 days of age were investigated. Purified neuronal and glial nuclei were incubated in the presence of [3H]acetyl-CoA and of [gamma-32P]ATP. Histones and NHPs were extracted and fractionated by gel electrophoresis. Densitometric and radioactive patterns were obtained. The results showed an increase of acetylation and phosphorylation from 1 to 10 and 30 days of age in both neuronal and glial nuclei in almost all histone and NHP fractions. Among the histones, the H3 fraction was always more labeled than the other fractions and showed the most remarkable differences during postnatal development. In the NHP fractions, the increase in acetylation from 1 to 10 and 30 days of age was more evident in the low-molecular-weight region of neuronal nuclei than in the corresponding fraction of glial nuclei. The appearance of highly phosphorylated proteins (70,000-90,000 daltons)--absent at 1 day, appearing at 10 days, and more evident at 30 days of age--was observed in both neuronal and glial nuclei.     

Methylation of Chromosomal Proteins in Neuronal and Glial Nuclei Purified from Cerebral Hemispheres of Rat During Postnatal Development

The process of methylation of chromosomal proteins [histones and nonhistone proteins (NHP)] in neuronal and glial cell nuclei obtained from cerebral hemispheres of rats at 1, 10, and 30 days of age was investigated. Purified neuronal and glial nuclei were incubated in the presence of S-adenosyl[methyl-3H]methionine. Histone and NHPs were extracted and fractionated by polyacrylamide gel electrophoresis. The results obtained indicate remarkable differences in the process of methylation of histones and NHPs between neuronal and glial nuclei, especially during the first period of postnatal development. In both nuclear populations the histone fraction H3 was labeled to a greater degree than the other fractions and showed the major changes during postnatal development. The densitometric and radioactive patterns of NHPs show considerable changes in the two nuclear populations at the various ages examined. The main difference between neuronal and glial nuclei consists in the intense methylation of proteins with a molecular weight of approximately 100,000, which are present in neuronal nuclei and virtually absent in glial ones. The results obtained may be correlated with the different chromatin structures of neuronal and glial nuclei and with the patterns of maturation and differentiation of neuronal and glial cells during postnatal development.     

Lysine methylation: beyond histones

Posttranslational modifications (PTMs) of histone proteins, such as acetylation, methylation, phosphorylation, and ubiquitylation, play essential roles in regulating chromatin dynamics. Combinations of different modifications on the histone proteins, termed 'histone code' in many cases, extend the information potential of the genetic code by regulating DNA at the epigenetic level. Many PTMs occur on non-histone proteins as well as histones, regulating protein-protein interactions, stability, localization, and/or enzymatic activities of proteins involved in diverse cellular processes. Although protein phosphorylation, ubiquitylation, and acetylation have been extensively studied, only a few proteins other than histones have been reported that can be modified by lysine methylation. This review summarizes the current progress on lysine methylation of non-histone proteins, and we propose that lysine methylation, like phosphorylation and acetylation, is a common PTM that regulates proteins in diverse cellular processes.     

Effect of biotin on phosphorylation,acetylation, methylation of rat liver histones

Biotin deficient rat liver histones showed decreased phosphorylation and methylation, and increased acetylation rates as compared to normal rat liver histones: these alterations may be related to the observed lower stability of the interactions between histones and DNA. The modifications of the metabolic process might be the consequence of an alteration of the synthesis of the enzymes involved in histone phosphorylation, acetylation and methylation mechanisms and are presumably related to a biotin effect upon the synthesis of RNA and proteins.     

Chromosomal histone modification patterns--from conservation to diversity

The organization of DNA into chromatin regulates expression and maintenance (replication, repair, recombination, segregation) of genetic information in a dynamic manner. The N-terminal tails of the nucleosomal core histones are subjected to post-translational modifications such as acetylation, methylation, phosphorylation, ubiquitination, glycosylation, ADP-ribosylation, carbonylation and sumoylation. These modifications, together with DNA methylation, control the folding of the nucleosomal array into higher order structures and mediate signalling for cellular processes. Although histones and their modifications are highly conserved, recent data show that chromosomal distribution of individual modifications (acetylation, methylation, phosphorylation) can differ along the cell cycle as well as among and between groups of eukaryotes. This implies the possibility of evolutionary divergence in reading the "histone code".     

Effect of norepinephrine on chromatin protein phosphorylation in C-6 glioma cells

Rat C₆ glioma cultures were exposed to labelled sodium phosphate after treatment with NE with or without propanolol. Histones and non-histone proteins (NHP) were extracted from chromatin and there was no significant change in the specific activity of the total pool of histones and NHP between control and other two groups. However, after electrophoretic separation F₂a₂ histone showed a 60% increase while F₂b and F₃ histones exhibited a 40% decrease in phosphorylation in response to NE. There was no significant change in the gel pattern of NHP from different groups on SDS-PAGE. However, the 30k dalton NHP showed an increase in phosphorylation in response to NE and this increment was blocked by propanolol. The possible role of β-receptors on nuclear protein phosphorylation and genomic expression is discussed.     

Phosphorylation of non-histone proteins associated with mitosis in HeLa cells

Our previous studies indicated that certain non-histone proteins (NHP) extractable with 0.2 M NaCl from mitotic HeLa cells induce germinal vesicle breakdown and chromosome condensation in Xenopus laevis oocytes. Since the maturation-promoting activity of the mitotic proteins is stabilized by phosphatase inhibitors, we decided to examine whether phosphorylation of NHP plays a role in the condensation of chromosomes during mitosis. HeLa cells, synchronized in S phase, were labeled with ³²P at the end of S phase, and the cells subsequently collected while they were in G2, mitosis, or G1. Cytoplasmic, nuclear, or chromosomal proteins were extracted and separated by gel electrophoresis. The labeled protein bands were detected by radioautography. The results indicated an 8–10-fold increase in the phosphorylation of NHP from mid-G2 to mitosis, followed by a similar-size decrease as the cells divided and entered G1. The NHP phosphorylation rate increased progressively during G2 traverse and reached a peak in mitosis. Radioautography of the separated NHP revealed eight prominent, extensively phosphorylated protein bands with molecular masses ranging from 27.5 to 100 kD. These NHP were rapidly dephosphorylated during M-G1 transition. Phosphorylation—dephosphorylation of NHP appeared to be a dynamic process, with the equilibrium shifting to phosphorylation during G2-M and dephosphorylation during M-G1 transitions. These results suggest that besides histone H1 phosphorylation, phosphorylation of this subset of NHP may also play a part in mitosis.     

Chromosome remodeling related to hepatitis B virus replication in HepG2 cells

Hepatitis B Virus (HBV) covalently closed circular DNA (cccDNA) is the main replicative intermediate of HBV and is organized into minichromosomes by the interaction with histone and nonhistone proteins. The remodeling of HBV minichromosomes such as post-translational modifications of histone proteins plays an important role in regulating HBV replication. To determine whether other remodeling occurs in addition to acetylation of cccDNA-bound H3 histones in the presence of HBV replication, a cell culture replication model of HBV was used to assess the dynamic status of acetylation, phosphorylation, and methylation of cccDNA-bound H3 histones at various times after transient transfection of linear HBV DNA into human hepatoma, HepG2 cells. H3 histones bound to cccDNA were found to be phosphorylated, mono-methylated, and acetylated in HepG2 cells containing replicating HBV. The acetylation and methylation status of H3 histones bound to cccDNA paralleled HBV replication. Our results demonstrate that phosphorylation and methylation occur in the remodeling of HBV minichromosomes during HBV replication. The modifications of cccDNA-bound H3 histones were associated with the level of HBV replication. These findings suggest that alterations in the extent of minichromosome remodeling might be a potential target to inhibit HBV replication in the development of effective novel antiviral agents.     

Molecular implementation and physiological roles for histone H3 lysine 4 (H3K4) methylation

Chromosomal surfaces are ornamented with a variety of post-translational modifications of histones, which are required for the regulation of many of the DNA-templated processes. Such histone modifications include acetylation, sumoylation, phosphorylation, ubiquitination, and methylation. Histone modifications can either function by disrupting chromosomal contacts or by regulating non-histone protein interactions with chromatin. In this review, recent findings will be discussed regarding the regulation of the implementation and physiological significance for one such histone modification, histone H3 lysine 4 (H3K4) methylation by the yeast COMPASS and mammalian COMPASS-like complexes.     

有丝分裂期间蛋白质的翻译后修饰及其功能

有丝分裂期间蛋白质的翻译后修饰对于有丝分裂顺利完成以及细胞功能发挥具有重要的调控作用。常见的修饰类型包括磷酸化修饰、糖基化修饰、SUMO化修饰、乙酰化修饰、甲基化修饰。这些翻译后修饰可以维持染色体结构、促进后期染色体分离、协助末期核膜重新形成。本文对有丝分裂过程中相关蛋白质翻译后修饰的最新类型和功能进行了系统总结,以期能为肿瘤基础研究提供新的方向。     

Switching on Chromatin: Mechanistic Role of Histone H4-K16 Acetylation

DNA in eukaryotic organisms does not exist free in cells, but instead is present as chromatin, a complex assembly of DNA, histone proteins, and chromatin-associated proteins. Chromatin exhibits a complex hierarchy of structures, but in its simplest form it is composed of long linear arrays of nucleosomes. Nucleosomes contain 147 base pairs of DNA wrapped around a histone octamer, consisting of two copies each of histones H2A, H2B, H3 and H4, where 15-38 amino terminal residues of each histone protein extends past the DNA gyres to form histone “tails” 1. Chromatin provides a versatile regulatory platform for nearly all cellular processes that involve DNA, and improper chromatin regulation results in a wide range of diseases, including various cancers and congenital defects. One major way that chromatin regulates DNA utilization is through a wide range of post-translational modification of histones, including serine and threonine phosphorylation, lysine acetylation, methylation, ubiquitination, and sumoylation, and arginine methylation 2. Histone H4 K16 acetylation is a modification that occurs on the H4 histone tail and is one of the most frequent of the known histone modifications. We have demonstrated that this mark both disrupts formation of higher-order chromatin structure and changes the functional interaction of chromatin-associated proteins 3. Our results suggest a dual mechanism by which H4 K16 acetylation can ultimately facilitate genomic functions.     

Histone methylation. Its occurrence in different cell types and relation to histone H4 metabolism in developing trout testis.

Histone methylation in developing trout testis has been observed in the diploid stem cells and primary spermatocytes, which actively synthesize DNA and histones. In spermatids, histone methylation is minimal and so probably plays no role in the replacement of histones by protamine which is characteristic of this cell type. No turnover of histone methyl groups could be detected over several hours, so that unlike acetylation or phosphorylation of histones, methylation in this tissue appears to be a stable, irreversible modification. When histone H4, labeled with [14C]methyl groups, is separated on starch gels into acetylated and phosphorylated derivatives, [14C]methyl label does not appear in positions characteristic of newly synthesized histone H4, i.e. the highly acetylated (di-, tri-, and tetra-acetylated), unphosphorylated species. [14C]Methyl label appears rather in the unphosphorylated, and unacetylated or monoacetylated species, shifting with time to the monophosphorylated form of histone H4. These data suggest a temporal sequence of events for histone H4: synthesis, then acetylation and deacetylation, followed by methylation and phosphorylation. Occurring late after histone synthesis and assembly into chromatin, histone methylation might then be necessary for histone interactions with other molecules (e.g. histone phosphokinase) prior to mitosis.     

Characterization of post-translational modifications of the linker histones H1 and H5 from chicken erythrocytes using mass spectrometry

Histone linker proteins H1 and H5 were purified from chicken erythrocyte cell nuclei under nondenaturing conditions. The purified linker histones were analyzed using in-solution enzymatic digestions followed by nanoflow reverse-phase high-performance liquid chromatography tandem mass spectrometry. We have identified all six major isoforms of the chicken histone H1 (H101, H102, H103, H110, H11R and H11L) and, in addition, the specialist avian isoform H5. In all the histone variants, both the acetylated and nonacetylated N (alpha)-terminal peptides were identified. Mass spectrometry analysis also enabled the identification of a wide range of post-translational modifications including acetylation, methylation, phosphorylation and deamidation. Furthermore, a number of amino acids were identified that were modified with both acetylation and methylation. These results highlight the extensive modifications that are present on the linker histone proteins, indicating that, similar to the core histones, post-translational modifications of the linker histones may play a role in chromatin remodelling and gene regulation.