Cellular memory and the histone code

The histone tails on the nucleosome surface are subject to enzyme-catalyzed modifications that may, singly or in combination, form a code specifying patterns of gene expression. Recent papers provide insights into how a combinatorial code might be set and read. They show how modification of one residue can influence that of another, even when they are located on different histones, and how modifications at specific genomic locations might be perpetuated on newly assembled chromatin.     

Histone modifications: signalling receptors and potential elements of a heritable epigenetic code

The genetic code epitomises simplicity, near universality and absolute predictive power. By contrast, epigenetic information, in the form of histone modifications, is characterised by complexity, diversity and an overall tendency to respond to changes in genomic function rather than to predict them. Perhaps the transient changes in histone modifications involved in intranuclear signalling and ongoing chromatin functions mask stable, predictive modifications that lie beneath. The current rapid progress in unravelling the diversity and complexity of epigenetic information might eventually reveal an underlying histone or epigenetic code. But whether it does or not, it will certainly provide unprecedented opportunities, both for understanding how the genome responds to environmental and metabolic change and for manipulating its activities for experimental and therapeutic benefit.     

Identification of lysine 37 of histone H2B as a novel site of methylation

Recent technological advancements have allowed for highly-sophisticated mass spectrometry-based studies of the histone code, which predicts that combinations of post-translational modifications (PTMs) on histone proteins result in defined biological outcomes mediated by effector proteins that recognize such marks. While significant progress has been made in the identification and characterization of histone PTMs, a full appreciation of the complexity of the histone code will require a complete understanding of all the modifications that putatively contribute to it. Here, using the top-down mass spectrometry approach for identifying PTMs on full-length histones, we report that lysine 37 of histone H2B is dimethylated in the budding yeast Saccharomyces cerevisiae. By generating a modification-specific antibody and yeast strains that harbor mutations in the putative site of methylation, we provide evidence that this mark exist in vivo. Importantly, we show that this lysine residue is highly conserved through evolution, and provide evidence that this methylation event also occurs in higher eukaryotes. By identifying a novel site of histone methylation, this study adds to our overall understanding of the complex number of histone modifications that contribute to chromatin function.     

Functional consequences of histone modifications

Covalent modifications of the histone proteins have well-known roles in gene expression. Experiments reported during the past year have extended this paradigm to include roles for histone acetylation and phosphorylation in DNA double-strand break repair. In addition, new results now provide a definitive example of an acetylation histone code, whereas others reveal the workings of a charge patch mechanism. Finally, exciting research has identified new modifications, complex modification cascades, and functional links to DNA methylation and RNA interference pathways.     

Local stability and evolution of the genetic code

The standard genetic code is known to be robust to translation errors and point mutations. We studied how small modifications of the standard code affect its robustness. The robustness was assessed in terms of a proper stability function, the negative variations of which correspond to a more robust code. The fraction of more robust codes obtained under small modifications appeared to be unexpectedly high, about 0.1-0.4 depending on the choice of stability function and code modifications, yet significantly lower than the corresponding fraction in the random codes (about a half). In this sense the standard code ought to be considered distinctly non-random in accordance with previous observations. The distribution of the negative variations of stability function revealed very abrupt drop beyond one standard deviation, much sharper than for Gaussian distribution or for the random codes with the same number of codons in the sets coding for amino acids or stop-codons. This behavior holds for both the standard code as a whole and its binary NRN-NYN, NWN-NSN, and NMN-NKN blocks. Previously, it has been proved that such binary block structure is necessary for the robustness of a code and is inherent to the standard genetic code. The modifications of the standard code corresponding to more robust coding may be related to the different variants of the code. These effects may also contribute to the rates of replacements of amino acids. The observed features demonstrate the joint impact of random factors and natural selection during evolution of the genetic code.     

Histone-modifying enzymes: encrypting an enigmatic epigenetic code

Histone-modifying enzymes catalyze a diverse array of post-translational modifications of core and linker histones within chromatin. These modifications govern a multitude of genomic functions, particularly gene expression, and are believed to constitute an epigenetic code. Histone-modifying enzymes inscribe this code by catalyzing site-selective modifications, which are subsequently interpreted by effector proteins that recognize specific covalent marks. The substrate specificity of these enzymes is of fundamental biological importance because it underpins this epigenetic code. Recently, the structural basis of this specificity has been examined with regards to recently determined structures of GCN5 acetyltransferases and SET domain methyltransferases in complex with their cognate histone substrates.     

组蛋白翻译后修饰技术研究进展

翻译后修饰调控着真核生物大部分蛋白质的活性,这些修饰的解读对研究生物功能是必不可少的。组蛋白翻译后修饰是蛋白质翻译后修饰中研究的较好一类小分子碱性蛋白,易被各种生物大分子修饰,尤其易发生在N-末端的尾部。不同组合式修饰构成了"组蛋白密码",在细胞的发育、生长、分化和动态平衡中,组蛋白密码影响着染色体的结构状态,进而调控基因的表达状态。组蛋白翻译后修饰的研究可作为一种模式来解析蛋白质复杂的修饰状态及研究其分子功能。翻译后修饰分析技术的发展对组蛋白密码的解析是至关重要的。重点讨论组蛋白修饰分析技术的发展和应用。     

Do protein motifs read the histone code?

The existence of different patterns of chemical modifications (acetylation, methylation, phosphorylation, ubiquitination and ADP-ribosylation) of the histone tails led, some years ago, to the histone code hypothesis. According to this hypothesis, these modifications would provide binding sites for proteins that can change the chromatin state to either active or repressed. Interestingly, some protein domains present in histone-modifying enzymes are known to interact with these covalent marks in the histone tails. This was first shown for the bromodomain, which was found to interact selectively with acetylated lysines at the histone tails. More recently, it has been described that the chromodomain can be targeted to methylation marks in histone N-terminal domains. Finally, the interaction between the SANT domain and histones is also well documented. Overall, experimental evidence suggests that these domains could be involved in the recruitment of histone-modifying enzymes to discrete chromosomal locations, and/or in the regulation their enzymatic activity. Within this context, we review the distribution of bromodomains, chromodomains and SANT domains among chromatin-modifying enzymes and discuss how they can contribute to the translation of the histone code.     

The Tubulin Code

Microtubules create diverse arrays with specific cellular functions such as the mitotic spindle, cilia, and bundles inside neurons. How microtubules are regulated to enable specific functions is not well understood. Recent work has shown that posttranslational modifications of the tubulin building blocks mark subpopulations of microtubules and regulate downstream microtubule-based functions. In this way, the tubulin modifications generate a “code” that can be read by microtubule-associated proteins in a manner analogous to how the histone code directs diverse chromatin functions. Here we review recent progress in understanding how the tubulin code is generated, maintained, and read by microtubule effectors.     

Unassigned Codons, Nonsense Suppression, and Anticodon Modifications in the Evolution of the Genetic Code

The origin of the genetic code is a central open problem regarding the early evolution of life. Here, we consider two undeveloped but important aspects of possible scenarios for the evolutionary pathway of the translation machinery: the role of unassigned codons in early stages of the code and the incorporation of tRNA anticodon modifications. As the first codons started to encode amino acids, the translation machinery likely was faced with a large number of unassigned codons. Current molecular scenarios for the evolution of the code usually assume the very rapid assignment of all codons before all 20 amino acids became encoded. We show that the phenomenon of nonsense suppression as observed in current organisms allows for a scenario in which many unassigned codons persisted throughout most of the evolutionary development of the code. In addition, we demonstrate that incorporation of anticodon modifications at a late stage is feasible. The wobble rules allow a set of 20 tRNAs fully lacking anticodon modifications to encode all 20 canonical amino acids. These observations have implications for the biochemical plausibility of early stages in the evolution of the genetic code predating tRNA anticodon modifications and allow for effective translation by a relatively small and simple early tRNA set.     

Writing and Reading the Tubulin Code

Microtubules give rise to intracellular structures with diverse morphologies and dynamics that are crucial for cell division, motility, and differentiation. They are decorated with abundant and chemically diverse posttranslational modifications that modulate their stability and interactions with cellular regulators. These modifications are important for the biogenesis and maintenance of complex microtubule arrays such as those found in spindles, cilia, neuronal processes, and platelets. Here we discuss the nature and subcellular distribution of these posttranslational marks whose patterns have been proposed to constitute a tubulin code that is interpreted by cellular effectors. We review the enzymes responsible for writing the tubulin code, explore their functional consequences, and identify outstanding challenges in deciphering the tubulin code.     

Modifications of human histone H3 variants during mitosis

Phosphorylation of histone H3 is a hallmark event in mitosis and is associated with chromosome condensation. Here, we use a combination of immobilized metal affinity chromatography and tandem mass spectrometry to characterize post-translational modifications associated with phosphorylation on the N-terminal tails of histone H3 variants purified from mitotically arrested HeLa cells. Modifications observed in vivo on lysine residues adjacent to phosphorylated Ser and Thr provide support for the existence of the "methyl/phos", binary-switch hypothesis [Fischle, W., Wang, Y., and Allis, C. D. (2003) Nature 425, 475-479]. ELISA with antibodies selective for H3 at Ser10, Ser28, and Thr3 show reduced activity when adjacent Lys residues are modified. When used together, mass spectrometry and immunoassay methods provide a powerful approach for elucidation of the histone code and identification of histone post-translational modifications that occur during mitosis and other specific cellular events.     

Rewiring translation - Genetic code expansion and its applications

With few minor variations, the genetic code is universal to all forms of life on our planet. It is difficult to imagine that one day organisms might exist that use an entirely different code to translate the information of their genome. Recent developments in the field of synthetic biology, however, have opened the gate to their creation. The genetic code of several organisms has been expanded by the heterologous expression of evolved aminoacyl-tRNA synthetase/tRNA(CUA) pairs that mediate the incorporation of unnatural amino acids in response to amber codons. These UAAs introduce exciting new features into proteins, such as spectroscopic probes, UV-inducible crosslinkers, and functional groups for bioorthogonal conjugations or posttranslational modifications. Orthogonal ribosomes provide a parallel translational machinery in Escherichia coli that has lost its evolutionary constraints. Evolved variants of these ribosomes translate amber or quadruplet codons with massively enhanced efficiency. Here, I review these recent developments emphasizing their tremendous potential to facilitate biochemical and cell biological studies.     

Applications of Genetic Code Expansion in Studying Protein Post-translational Modification

Various post-translational modifications can naturally occur on proteins, regulating the activity, subcellular localization, interaction, or stability of the proteins. However, it can be challenging to decipher the biological implication or physiological roles of site-specific modifications due to their dynamic and sub-stoichiometric nature. Genetic code expansion method, relying on an orthogonal aminoacyl-tRNA synthetase/tRNA pair, enables site-specific incorporation of non-canonical amino acids. Here we focus on the application of genetic code expansion to study site-specific protein post-translational modification in vitro and in vivo. After a brief introduction, we discuss possibilities of incorporating non-canonical amino acids containing post-translational modifications or their mimics into target proteins. This approach is applicable for Ser/Thr/Tyr phosphorylation, Tyr sulfation/nitration/hydroxylation, Lys acetylation/acylation, Lys/His mono-methylation, as well as Arg citrullination. The next section describes the use of a precursor non-canonical amino acid followed by chemical and/or enzymatic reactions to afford the desired modification, such as Cys/Lys acylation, ubiquitin and ubiquitin-like modifications, as well as Lys/Gln methylation. We also discuss means for functional regulation of enzymes involving in post-translational modifications through genetically incorporated non-canonical amino acids. Lastly, the limitations and perspectives of genetic code expansion in studying protein post-translational modification are described.     

Histone modifications in transcriptional regulation

Covalent modifications of the amino termini of the core histones in nucleosomes have important roles in gene regulation. Research in the past two years reveals these modifications to consist of phosphorylation, methylation and ubiquitination, in addition to the better-characterized acetylation. This multiplicity of modifications, and their occurrence in patterns and dependent sequences, argues persuasively for the existence of a histone code.