How the Idea of the Code Came About

•The history of the study of histone modifications began over 40 years ago with Murray’s identification of lysine methylation in the N-terminal domains of calf histones.

• Lysine acetylation and serine phosphorylation were discovered soon after in histone tails from such broadly displayed sources as human lymphocytes, rat liver, peas, and calf thymus1.

• In 1975, ubiquitylation and ADP-ribosylation were added to the spectrum, with novel histone modifications such as SUMO (the small ubiquitin-related modifier) being discovered as recently as 20031,2,3. • These covalent posttranslational modifications (PTMs) allow for regulating contacts with the underlying DNA2,4 (Fig 1A). • In 2000, awareness that enzymes created both remarkable diversity and biological specificity through these distinct histone modification patterns led Strahl and Allis to propose that distinct histone tail PTMs may act sequentially or in combination to form a “histone code” that could be read by other proteins or protein modules4,5 (Fig 1B,C).

• The most extensively studied histone tail modification is acetylation by histone acetyltransferases (HATs) and deacetylation by histone deacetylase (HDACs) of the ε-amino groups of conserved lysine residues associated with transcriptional activity2,5.

• It is thought that the neutralization of the histone tails’ basic charge through acetylation reduces their affinity for DNA and alters histone-histone interactions between adjacent nucleosomes as well as histone interactions with other regulatory proteins2,5 (Fig 2).

• Convincing molecular evidence directly linking acetylation and transcription was finally obtained when acetyltansferase (HAT) activity was discovered in the conserved transcriptional regulator Gcn5. Since then, numerous other coactivator proteins existing as families of acetyltransferases (comprised of more than 20 enzymes!) have been found to possess HAT activity2,4.

• It is likely that short preferred consensus motifs exist for individual HATs and HDACsthat assist in establishing the histone code5.

Discovering the Modifications

• The study of the N-terminal tail domains dominated research in the field for over 4 decades.

• One classical method for analyzing PTMs used specialized gel systems and/or the incorporation of radioactive precursor molecules followed by protein hydrolysis and analysis of the resulting amino acid.

• Edman degredation was also used as a primary method for discovering histone modifications however it favored the analysis of the first 20-30 amino acids of the tail.

• Both these methods required multiple arduous purification steps making them non-applicable for the analysis of histones from small cell numbers or for mapping PTMs at defined genomic loci which prompted researchers to search for novel more sensitive reagents3.

• In the late 1980’s specific antibodies were developed that could be used in western blots to identify PTMs and these immunofluorescence studies finally allowed a localization of specifically modified histones within particular regions of the genome3.

• Unfortunately, similarities between different modification sites caused cross reactivity in antibodies which weakened specificity. Also, in the presence of combined PTMs, many antisera showed a strong interference in epitope binding, necessitating the development of specific antibodies for each individual combination. This proved to be virtually impossible due to the large number of modifications within a short stretch of amino acids.

• Finally, modern mass spectrometry provided the key technique for PTM analysis because of its high resolution and the development of “soft” ionization techniques.

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Beyond Histone Tails

• Mass spectrometry is an instrumental technique that determines mass to charge ratios (m/z) of ionized molecules and/or fragments. These m/z are used to calculate weights of proteins, protein fragments and peptides6.

• A translated genomic database can be used to determine the predicted molecular weight of a given protein and its peptides and deviations in the predicted weight from observed masses, termed ‘mass shifts’ are often indicative of PTMs.

• Identification of these proteins and peptides can be determined from fragmentation patterns obtained by mass spectrometric analysis (MS).

• There are two general approaches to mass spectrometry of protein complexes: “bottom-up” and “top-down”7.Current methodology utilizes a combination of these two approaches to get the most accurate determination of PTMs8.

• The bottom-up approach allows for comprehensive and quantitative measurement of distinct histone modifications while the top-down approach aides one in determining the interdependencies between clustered modifications. Mass spectrometry not only allowed a much faster detection of PTMs, but also revealed a much higher abundance of PTMs than previously expected3 (Fig 1).

• In 2002, the research monopoly of the histone code tail ended with the application of mass spectrometry, opening the door to the study of the modifications of the core histone domain1.

• These core PTMs are postulated to have different mechanisms of action than their tail domain counterparts1. Their importance has been illustrated with point mutations for single modifiable residues which demonstrated sometimes dramatic effects on transcriptional silencing, formation and/or maintenance of chromatin structure and DNA damage repair9.

• In fact the role these modifications play may be more critical than tail domain PTMs given that deletion of tail domains in H3, H2A or H2B proteins was found to have no effect on the ability of these proteins to organize into nucleosomes10.

• Clustering of these modifications in particular locations led Marsfelder and Parthun to organize them into three classes; (1) the solute accessible face: believed to play a role in the regulation of chromatin (i.e. assembly of silent chromatin structure), the ability of non-histone proteins to bind to the nucleosome and influencing nucleosome-nucleosome interactions1, One modification on the solute accessible face, methylation of histone H3 lysine 79, has also been shown to play a role in the functionality of DNA damage checkpoints11.(2) the histone lateral face: that regulates histone-DNA interactions by altering the mobility of the nucleosomes, which results in a change in higher order chromatin structure or accessibility of specific sequences of DNA such as those needed to be available for DNA damage repair1. Many of these modifications act in concert with the cell cycle to enhance the general dynamic nature of eukaryotic chromosomes12,13.(3) the histone-histone interface: essential in the regulation of nucleosome stability. Weakening of these interactions can allow access of DNA damage repair machinery and orderly assembly of nucleosomes onto DNA following replication, transcription and DNA repair1.

Deciphering the Histone CodeDandan Li, Karen Lohnes, Kathleen Sanders, Nuttinee Teerakulkittipong

Alteration of Histone Modification Patterns in Cancer Cells

Today there is more and more evidence that cancer cells present an altered pattern of histone modifications within constitutive heterochromatin15. Aberrant transcription of the genes that result in improper targeting of HATs or HDACs to certain loci, functional inactivation of HATs, overexpression of HDACs or epigenetic changes due to DNA hyper- and hypomethylation, can induce abnormal expression of genes that regulate cellular differentiation, the cell cycle and apoptosis, thereby enhancing the potential to mediate neoplastic transformation, tumor onset and progression14.

New Approach for the Treatment of Cancer by Chromatin Remodeling

Expression of the aberrant gene in cancer cells always involves epigenetic gene silencing mechanisms that occur mainly through two pathways:(1) Dense hypermethylation of the CpG islands located in the promoter region of tumor suppression genes. This has been targeted in cancer therapeutics using DNA demethylating agents that restore the functionality of silent genes, but a significant percentage of these drugs have toxic effects15,16. (2) Different transcriptional repressors aberrantly target the HDACs of the gene promoter and cause histone hypoacetylation. The archetypical gene silenced in this manner in human cancer is the cyclin-dependent kinase inhibitor (p21WAF1)17. This causes loss of the P21WAF1 gene expression in a broad spectrum of tumor types, and its experimental overexpression in deficient cancer cells can cause growth arrest18.

HDAC inhibitors: Novel drugs for the treatment of cancer

The biochemical structure and mechanism of HDAC inhibitors have been classified. The hydroxamic acids are probably the broadest set of HDAC inhibitors. Crystallographic studies of histone deacetylase-like protein complexes with zinc and hydroxamic acid (HDLP–Zn2+–HA) show the structure of the histone deacetylase catalytic core, as revealed by a homologue from the hyperthermophillic bacterium (Aquifex aeolicus) that shares 35.2% identity with human HDAC1 over 375 residues as shown in Fig 319. The structures of HDAC inhibitors deacetylase-trichostatin A (TSA) reveals a hydrophobic linker that allows the hydroxamic acid moiety to chelate the Zn2+ at the bottom of HDAC catalytic pocket, while the bulky part of the molecule acts as a cap for the tube as shown in Fig 4, and establish the mechanism of HDAC inhibition19.

The actions of histone deacetylase (HDAC) inhibitors are to induce histone hyperacetylation, reactivate suppressed genes, and cause pleiotropic cellular effects that inhibit tumor-cell growth and survival. Almost all HDAC inhibitor mechanisms cause induction of cell cycle arrest which leads to mediated cell differentiation or apoptosis in vitro20,21. Many also have potent antitumor activities in vivo by activation of the host immune response22 and inhibition of angiogenesis14,23. The molecular and biological functions of these agents are being used in preclinical cancer models and clinical trials that represent a new wave of anticancer drugs and are exciting prospects for a more rational approach to chemotherapy.

Fig 5. A comparison of treatment of normal and certain tumor cell lines with HDAC inhibitors23.

Fig 4. TSA binds inside the pocket making contacts to residues at the rim, walls and bottom of the pockets19.

Fig 3. The A. aeolicus HDLP has 35.2% sequence homology to human HDAC119.

Coffin Lowry Syndrome: A Disease Associated with Histone Phosphorylation

Coffin-Lowry syndrome (CLS) is a genetic disorder characterized by craniofacial dysmorphisms, progressive skeletal abnormalities, severe mental retardation and short stature24,25. It is caused by a mutated gene, RSK-2, which is located on the X chromosome (Xp22.2-p22.1), and is the sole gene known to be associated with CLS26. RSK-2 is a growth factor-regulated serine/threonine kinase that can change the activity of many transcription factors by phosphorylation27. Rsk-2 is involved in a Ras-dependent mitogen-activated protein kinase (MAPK) cascade that results in the transcriptional activation of immediate-early responsive genes. During the immediate-early response of mammalian cells to mitogens, rapid and transient phosphorylation of histone H3 occurs. Various stimuli induce a MAPK cascade and RSK-2 is required for the epidermal growth factor (EGF)-stimulated phosphorylation28 of H3. Fibroblasts derived from a CLS patient cannot exhibit EGF-stimulated phosphorylation of H3, and transcriptional activation in response to stimuli is altered in CLS cells28. H3 appears to be a target of Rsk-2, suggesting a direct role for H3 phosphorylation in regulating gene transcription possibly through chromatin remodeling and resulting in decondensation29.

Fig 6. Specific phosphorylation site of histone H3 by Rsk-2 in vitro29.

Fig 7. Deficiency of histone H3 phosphorylation by EGF-stimulated pathway and decrease of Rsk-2-associated H3 kinase activity in CLS cells29.

Fig 8. Rsk-2 deficiency effects on mitogen-stimulated but not mitotic phosphorylation of histone H3. Indirect immuno-fluorescence microscopy of transformed normal (the first pair of panels) and CLS (the second pair of panels) fibroblasts incubated with EGF and costained with DAPI and pS10 antiserum. The last pair of panels shows another fields of CLS fibroblasts in which a cell in mitosis is visible. The location of phosphorylated H3 in cells is indicated by arrowheads29.

Conclusion

The discovery of the histone code and subsequent attempts to decipher it has opened many avenues of research into the essential role that the histone modifications play in gene expression. Unraveling the mechanisms and consequences of this epigenetic control has both extended our understanding of chromatin regulation and offered far reaching implications for human biology and disease.

References

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Fig 1. Models for “on-off” transcription states reflected by differential histone tail modifications4,5.

Fig 2. The hypothesis of the “histone code”. Diverse modifications occur at selected histone amino acid residues. Some patterns are linked to biological events (such as acetylation and transcription). Distinct H3 (red) and H4 (black) tail modifications are believed to act in sequential and combinatorial fashion in their regulation of unique biological functions.