Histone modifications and traditional Chinese medicinals
© Hsieh et al.; licensee BioMed Central Ltd. 2013
Received: 5 November 2012
Accepted: 22 May 2013
Published: 27 May 2013
Chromatin, residing in the nuclei of eukaryotic cells, comprises DNA and histones to make up chromosomes. Chromatin condenses to compact the chromosomes and loosens to facilitate gene transcription and DNA replication/repair. Chemical modifications to the histones mediate changes in chromatin structure. Histone-modifying enzymes are potential drug targets. How herbs affect phenotypes through histone modifications is interesting.
Two public traditional Chinese medicine (TCM) databases were accessed to retrieve the chemical constituents and TCM natures of 3,294 TCM medicinals. NCBI taxonomy database was accessed to build the phylogenetic tree of the TCM medicinals. Statistical test was used to test if TCM natures of the medicinals cluster in the phylogenetic tree. A public chemical-protein interaction database was accessed to identify TCM medicinals whose constituent chemicals interact with human histone-modifying enzymes. For each histone modification, a correlation coefficient was calculated between the medicinals’ TCM natures and modification modulabilities. Information of the ingredient medicinals of 200 classical TCM formulas was accessed from a public database.
It was found that 1,170 or 36% of the 3,294 TCM medicinals interact with human histone-modifying enzymes. Among the histone-modifying medicinals, 56% of them promote chromatin condensation. The cold-hot natures of TCM medicinals were found to be phylogenetically correlated. Furthermore, cold (hot) TCM medicinals were found to be associated with heterochromatinization (euchromatinization) through mainly H3K9 methylation and H3K4 demethylation. The associations were weak yet statistically significant. On the other hand, analysis of TCM formulas, the major form of TCM prescriptions in clinical practice, found that 99% of 200 government approved TCM formulas are histone-modifying. Furthermore, in formula formation, heterochromatic medicinals were found to team up with other heterochromatic medicinals to enhance the heterochromatinization of the formula. The synergy was mainly through concurrent DNMT and HDAC inhibition, co-inhibition of histone acetylation and H3S10 phosphorylation, or co-inhibition of H3K4 demethylation and H3K36 demethylation.
TCM prescriptions’ modulation of the human epigenome helps elucidation of phyto-pharmacology and discovery of epigenetic drugs. Furthermore, as TCM medicinals’ properties are closely tied to patient TCM syndromes, results of this materia-medica-wide, bioinformatic analysis of TCM medicinals may have implications for molecular differentiation of TCM syndromes.
KeywordsDNA methylation Histone code Chromatin structure Traditional Chinese medicine Yin-yang Phylogenetics Synergy DNMT HDAC HAT H3S10 phosphorylation H3K4 methylation H3K9 methylation
Histones are evolutionarily conserved proteins that abound in the cells of eukaryotes including plants and animals . They form protein families and two copies of each of the structurally similar histones H2A, H2B, H3 and H4 assemble into histone octamers. DNA sequence wraps around the octamers to form nucleosomes, which constitute the subunits of chromatin. Nucleosome-nucleosome and histone-DNA interactions take place to tighten or loosen the chromatin structure, prohibiting or permitting access of the transcriptional machinery, such as RNA polymerase II and regulatory factors, to the DNA sequence. Gene activities and thus genomic functions can change independent of the DNA sequence. Chromatin structure is altered by covalent modifications to the amino acid residues in the unstructured tails of histones. For example, acetylation of the lysines in H3 and H4 N-termini neutralizes the otherwise positively charged histones, weakening the coupling between histones and negatively charged DNA sugar-phosphate backbone. The relaxing chromatin is associated with active gene transcription , so is cytosine hypomethylation, a covalent modification to the DNA that is found in association with histone acetylation . An equally important property of histone modifications and DNA methylation is that modification patterns, once established, propagate through cell divisions. Different combinations of covalent modifications over the chromatin give rise to different cellular phenotypes. A histone code, supplementary to the DNA sequence, for cellular functions was therefore recently proposed .
Traditional Chinese medicine (TCM) has developed a system of theories and practices since at least 2,000 years ago and remains popular in some Far East Asian areas. In contrast to the reductionist approach of modern western medicine, TCM diagnoses a patient via inspection (e.g. tongue color), listening/smelling, questioning and palpation (e.g. pulse-reading) . Emotional, mental and environmental factors are usually also taken into account. Outcomes of the diagnostics are summarized as TCM syndromes (called Zheng in TCM) which are usually classified under the eight outlines: yin or yang, internal or external, cold or hot, deficiency or excess . Yin and yang in TCM refer, respectively, to the materialistic and functional qualities of the body (parts). External and internal indicate the origin or direction of syndrome development. Cold and hot are manifestations of the syndrome through metabolism and body heat. Deficiency means lack of activities, such as immunodeficiency, of the body organ(s). Two examples of TCM syndromes are Lung-Stomach-yin deficiency with excessive heat and concurrent yin-yang deficiency, both being commonly diagnosed by TCM in type II diabetic patients . A major feat of TCM is that Chinese herbal formulas that counteract the TCM syndromes have been developed so that once the patient’s TCM syndrome is identified, the Chinese herbal formula specific to the syndrome is readily prescribed [7–9]. Due to its diagnostic system, TCM is considered a holistic, personalized yet less specific therapy compared to modern western medicine.
As histone modifications and cytosine methylation play a role in the activity of genes, aberration in the pattern of modifications to histones and DNA, called epigenome, can lead to disease. Indeed, increasing evidence for dysregulated epigenomes in developmental, autoimmunological, metabolic and neurodegenerative disorders has been reported [10–13]. In particular, region-specific hypermethylation over a hypomethylated genome is characteristic of cancer cells . Drugs that target the altered epigenome for cancer treatment have been under investigation. For example two compounds (vorinostat and romidepsin) that inhibit histone deacetylases have been FDA-approved for cutaneous T-cell lymphoma. As histone modifications and DNA methylation are evolutionarily conserved regulatory mechanisms for cell functions, chemicals, e.g. secondary metabolites, in plants and animals that modulate the human epigenome may be found. It is therefore not surprising that a recent study, of bioinformatic nature yet at the pharmacopeia scale, found 30% of ~3,000 traditional Chinese medicinals, the majority of which are herbs, were human epigenome-interacting . Human epigenome-interacting herbs were extensively utilized in TCM formulas so that 99% of the studied formulas were epigenome-interacting. Furthermore, the epigenome-interacting herbs were found to serve in the formulas as the major herbs, called Monarch in TCM herbalism, that target patient’s main syndrome .
While modern western medicine remains the first choice for most patients, TCM, and other traditional medicines, has long been regarded as preventive or alternative for individuals at sub-healthy conditions or as complementary for patients at terminal conditions. As personalized medicine starts gaining impetus in modern western medicine , TCM has long been practicing tailored treatment, which is exemplified by the many possible different TCM syndromes that can be diagnosed to patients of a same disease. A medicine that is integrative of modern western medicine’s pathway-targeting and TCM’s epigenome-modulating therapy will be beneficial to patient’s well-being. A better understanding of TCM syndromes is essential. The diagnosed TCM syndrome of an individual can remain stable beyond the time span of a cell cycle. Evidence from recent studies demonstrates that histone modifications faithfully predict cell fates in various cellular contexts . A link between TCM syndrome and epigenomic change can therefore be hypothesized. In an attempt to address the hypothesis, we propose to look at TCM medicinals first because of the following two observations: 1) TCM doctrine dictates that TCM syndromes (e.g. hot) should be treated by the counteracting (cold) medicinals ; and 2) the cold-hot annotations of most TCM medicinals are available as they have been characterized and documented ever since the first TCM materia medica 2,000 years ago . We undertook the analysis in the following way. We first tested if the cold-hot and yin-yang TCM properties are, in a modern biological sense, viable through their relatedness in the phylogenetic tree of the medicinals. We next identified, using public chemical-protein interaction resources, the TCM medicinals whose chemical ingredients modulate DNA methylation and histone modifications of human cells. The TCM medicinals’ modulabilities of histone marks, which open, condense, or poise chromatin, were then correlated with their TCM properties. A significant correlation would lend support for the proposition of a link between epigenome patterns and TCM syndromes. We went on to study any synergistic effect of combing TCM medicinals in forming TCM formulas, under the perspective of chromatinization. Our findings may have implications for herbal pharmacology and epigenetic therapy.
Public databases and resources
Information about Chinese medicinals, including their TCM properties, constituent chemicals and scientific names in binomial nomenclature, was obtained from two open-access TCM databases. The Shanghai TCM database (http://www.sirc-tcm.sh.cn/en/service_1_1_1.html) at Shanghai Tcm Data Centre is funded by Shanghai municipal government and contains information of 8,896 traditional Chinese medicinals. We further supplemented the TCM information from a Singaporean TCM database (http://tcm.cz3.nus.edu.sg/group/tcm-id/tcmid.asp) containing 1,313 TCM medicinals . The NCBI taxonomy database (http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html/) incorporates the phylogenetic knowledge from a variety of sources and contains 201,995 species from plants, animals and fungi . A phylogenetic tree of Chinese medicinals was then built, consisting of 1,208 Chinese medicinals, where 95% of them are plants. The chemical-protein interaction database STITCH 2 (http://stitch.embl.de/)  contains 897,803 pairs of chemical-proteins associations between 14,732 human proteins and 53,092 chemicals. Information of 200 government-approved TCM formulas is available at http://www.ccmp.gov.tw/en/information/formula.asp. We converted the CAS registry numbers, for chemical identification, used in the TCM databases to the PubChem IDs used in the STITCH 2 database by the use of the NCBI PubChem server (http://pubchem.ncbi.nlm.nih.gov/).
Phylogenetic correlation of TCM properties
where y i is the cold-hot or yin-yang score of medicinal i, Y the mean score of all the N medicinals, w ij a weight that is inversely proportional to the distance d ij between medicinals i and j on the phylogenetic tree: w ij = 1/d ij . A P-value of the observed I can be calculated from the null distribution of I’s assuming no phylogenetic correlation of y’s. Moran’s I and P-value were calculated by the Moran.I function and d ij by the cophenetic function in the R package ape .
Determination of histone-modifying TCM medicinals
The human enzymes that are responsible for the histone modifications under study are listed in the Additional file 1: Table S1. The residue-specific modification annotations were through links from Ensembl Human to Entrez Gene databases. A medicinal that modifies histone modification X if it contains chemicals that interact with the human enzymes responsible for the X modification, where the chemical-enzyme interaction was from the STITCH 2 chemical-protein interaction database . Note that an interaction in STITCH 2 can be either activation, inhibition or not specified. In the case of not specified, we assumed it to be an inhibition, which is usually the case for chemical protein interactions. The X potency of the medicinal is the total number of chemical-protein interaction pairs that modify the X modification.
Unsupervised hierarchical clustering of the TCM medicinals
A medicinal is represented by an array of 18 attributes, that is, the potencies to modify the 18 histone marks. Comparison between the medicinals is then made by the Euclidean distance between the attribute arrays. Hierarchical clustering works by firstly placing the two most similar medicinals together, followed by merging the two into a ‘medicinal’. The pairing and merging are repeated on the remaining N–1 medicinals till all the medicinals are merged. Clustering of the TCM formulas was conducted the same way with the attribute array of a formula from the sum of the attribute arrays of the medicinals composing the formula. N is 1,170 or 199, the number of histone-modifying medicinals or formulas. We performed the hierarchical clustering by the heatmap function in R with default parameter settings.
Positive phylogenetic correlation of the cold-hot/yin-yang TCM properties
Likewise, after quantifying the yin-yang of the TCM medicinals, we colour-coded the tree in Additional file 1: Figure S5 and Additional file 1: Figure S6. Eyeball inspection and statistical test of the tree revealed a significant local clustering of the yin-yang on the tree (P = 1.3 × 10-6). In sum, the phylogenetic analysis of the two basic TCM properties supports TCM’s classification of medicinal properties, which would in turn support TCM’s classification of pathological states, through the TCM doctrine of syndrome-prescription mapping. After substantiating TCM medicinals’ effects, specifically the cold-hot and yin-yang, we are in a position to elucidate the underlying molecular mechanisms.
Thirty-six percent of TCM medicinals are histone-modifying with over half of them chromatin-condensing
In TCM theory, perfect health is in yin-yang balance. However, the innate constitute of an individual can be slightly yin (e.g. cold) or yang (hot). Once the balance is broken (by inherited predisposition or environmental stress), a TCM syndrome develops. As the affliction progresses, the TCM syndrome can change its grade or to another TCM syndrome until it subsides under proper TCM treatment. The phenomenon of slow yet long-term effects of Chinese herbs and reversible dynamics of TCM syndrome development incites a link between Chinese herbalism and epigenetics .
Histone modifications and the associated chromatin conformation
mitogen- and stress-activated protein kinase (MSK), I kappa B alpha kinase (IKK)
MAP kinase phosphatase (MKP)
ATP-dependent chromatin remodeling
ATP-dependent chromatin remodeling complex
hetero- or euchromatic
Histone modifications exert their functional importance through chromatin conformation. If a medicinal brings about more chromatin unpacking than condensing modifications, the medicinal is called chromatin unpacking. If a medicinal effectuates an equal number of unpacking and condensing marks, it is chromatin neutral (or poising). Figure 2 shows that among the 1,170 histone-modifying medicinals, 56%, 28% and 16%, respectively, condense, unpack and poise chromatin.
Cold- and yin-TCM medicinals are associated with chromatin condensing while hot- and yang-TCM medicinals with chromatin unpacking
For each of the 18 modifications in Table 1, we correlate the modifying potencies of the 1,170 TCM medicinals with their cold-hot scores which were defined previously for the phylogenetic analysis. At the significance threshold of 0.05, we found cold-hot score to be negatively correlated with H3K9 methylation (Pearson correlation coefficient r = −0.07, P = 0.038) and H3K4 demethylation (r = −0.06, P = 0.049), both of which are modifications that condense chromatin. Since the higher the score, the warmer the medicinal (as defined in Methods), the result indicates that cold medicinals condense chromatin.
Similarly, for each of the histone modifications under study, we correlate the modifying potencies of the 1,170 TCM medicinals with their yin-yang scores defined previously for the phylogenetic tree of Additional file 1: Figures S5 and Additional file 1: Figure S6. At the significance threshold of 0.05, we found yin-yang score to be negatively correlated with H3K4 demethylation (r = −0.08, P = 0.012), which is a chromatin condensing modification. In other words, yin TCM medicinals condense chromatin.
When we redefined the yin-yang scores by using the TCM flavours only (i.e. without considering the cold-hot natures) and repeated the correlation analysis, we found, at the significance threshold of 0.05, yin-yang scores to be negatively correlated with H3K36 methylation (r = −0.07, P = 0.020) and H3K4 demethylation (r = −0.07, P = 0.033) and positively correlated with H3S10 phosphorylation (r = 0.07, P = 0.029). The result is again in concordance with the picture of a chromatin condensing effect of yin TCM medicinals.
Similar medicinals combine in a TCM formula to reinforce the hetero- or euchromatinization
A TCM syndrome, once diagnosed, is readily prescribed a TCM formula consisting of multiple medicinals. The syndrome-formula mapping has been developed and documented by TCM healers through empiricism for thousands of years [7–9]. To study how formulas evolved, we analysed 200 government-approved and insurance-covered TCM formulas whose composing medicinals are known. The analysis was straightforward in that we worked on individual formula, instead of medicinal, as an entity. Additional file 1: Figure S7 shows the distribution of histone-modifying medicinals among the medicinals which make up the 200 formulas. Additional file 1: Figure S8 shows the clustering of the histone modifications employed by the medicinals composing the 200 formulas. The figures indicate that although the proportion of histone-modifying medicinals (Additional file 1: Figure S7) is higher in the set of medicinals making up the formulas, the modes of histone modifications by them (Additional file 1: Figure S8) are no different from those by the universe of 1,170 medicinals (Figure 3). The 200 TCM formulas can thus be considered representative in the sense that the medicinals in them are not epigenetically peculiar.
TCM natures (i.e. cold, cool, warm and hot) and flavors (i.e. pungent, sweet, sour, bitter and salty) have been essential properties of Chinese herbs since the early days of TCM in that the properties are closely connected to the classes of TCM syndromes they are supposed to treat. For example, yin-deficient physical constitutes and TCM syndromes are treated with yin-nourishing herbs. Similarly, cold-inclined individuals will be prescribed with warm herbs where cold can be considered warm-deficient. It is therefore of paramount importance that the TCM properties live up to the expectations of modern biology. Evolutionarily closely related species, such as Asian ginseng (Panax ginseng) and American ginseng (Panax quinquefolius), have similar genomes and may be expected to deliver similar pharmacological effects to the human body compared with herbs of dissimilar genomes. Since the nucleic acid or amino acid sequences are of a modern discovery, results of the phylogenetic analysis argue that TCM cold-hot and yin-yang are qualified properties for scientific investigation. Indeed, an in vitro study reported bitter, sour (yin) herbs to be associated with higher anti-oxidant activity than pungent, sweet (yang) herbs .
In the current computational study, we showed that more than one third of TCM medicinals and almost all TCM formulas are potentially histone-modifying. We went on to elucidate the association between the chromatinization of the histone modifications and the yin-yang of the TCM medicinals. TCM cold-hot and yin-yang are among the fundamental classifications of TCM syndromes on patients. Genome-wide profiling of histone modifications can be achieved nowadays by such high throughput techniques as chromatin immunoprecipitation followed by high-density chip hybridization (ChIP-chip) or high-throughput sequencing (ChIP-seq) [32, 33]. An association between chromatin conformations and TCM syndromes provides a quantitative measure for TCM syndrome diagnosis which has traditionally been more qualitative and thus subjective [34, 35]. Normalization and standardization of the TCM syndromes within a disease have been a top priority within the TCM community since the 1980s . A metabolomic marker identification of a TCM syndrome in rats was recently reported . Although both epigenome and metabolome can represent cellular conditions, transcriptome is more often considered cause of the phenotype. Indeed, a network-based study identified hormones and immune factors as predominant players in cold and hot TCM syndromes . It was demonstrated that, in various cell lines, gene expression could be predicted by chromatin features [17, 39]. Thus, our suggestion of a connection between chromatin conformations and TCM syndromes has impacts on TCM syndrome research. Further studies in this avenue are urged.
Herbs and formulas differ from conventional drugs in their multi-compound composition. The effects of many of the compounds on cells are, however, unclear, let alone studies of their interactions. By focusing on histone modifications and chromatin conformations the herbs impart, we showed that herbs in a formula cooperate to condense or unpack the chromatin and that a chromatin condensing/unpacking formula is composed of herbs that are chromatin condensing/unpacking. The finding is in agreement with a recent study in which warm (cold) herbs were found to connect with other warm (cold) herbs in the herb network constructed from TCM formulas . The findings may shed light on herb combination rules for the development of complementary and new herbal prescriptions for such complex and emerging disorders as cancer and SARS.
Our study relied on open-access databases, namely the NCBI taxonomy database, Shanghai and Singapore TCM databases and German chemical-protein interaction database. Simple yet naive assumption about the medicinal chemicals’ reaching the nuclei was also made. Albeit the limitations, the materia-medica-wide bioinformatic approach taken here helps yield insights that can prove difficult to gain otherwise.
With the data of 3,294 TCM medicinals from public resources and with the help of a chemical-protein interaction database, we found that 36% of the medicinals interact with human histone-modifying enzymes. Among the histone-modifying medicinals, 56% of them condense chromatin. Further exploration of the connection between histone modifications and TCM medicinals demonstrated that the cold-hot nature of TCM medicinals, one of the central properties of TCM, is phylogenetically correlated. Cold or yin (hot or yang) medicinals were then found to be associated with heterochromatinization (euchromatinization) through mainly H3K9 methylation and H3K4 demethylation. Studies of TCM formulas found that 99% of 200 government approved TCM formulas are histone-modifying. Furthermore, in formula formation, medicinals are combined in a way that heterochromatic medicinals team up with other heterochromatic medicinals to enhance the heterochromatinization of the formula. TCM prescriptions’ modulation of the human epigenome helps elucidation of TCM pharmacology and discovery of epigenetic drugs.
Traditional Chinese medicine
This work was supported by the National Science Council (Taiwan) grant #: 101-2112-M-008-003-MY3.
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