Department of Biosciences, Sri Sathya Sai University, Prasanthi Nilayam 515 134 A P, India , *Department of Bioinformatics, Sathyabhama University, Old Mahabilam Road, Chennai, 600 019 T N, India, ** Dept. of Genetics and Molecular Medicines, Vasavi Medical and Research Centre, 6-1-91 Kharaitabad, Hyderabad 500 004
Correspondence: [email protected]
Article submitted on June 27, 2008.
Article published on July 18, 2008
An article submitted by S.Krupanidhi, (18 July 2008): Epigenetic Microenvironment Awakes Genes. Published by Biology-Online.org.
The field of epigenetics is an emerging area. It aims to strengthen the genome based therapeutics. The reviewing of literature indicates that despite the invariant nature of genomic DNA, the epigenetic microenvironment dictates tissues’ specificity. Mendel’s concept of alleles and the newer concept of epialleles and epimutations which are in vogue in the genomic era are vividly elaborated. The role of epigenetics in the reprogramming and maintaining stable methylated states of CpG islands during growth and differentiation are reviewed. Epigenetic regulation of imprinted genes and tumor suppressor genes towards gene imprinting and silencing and their transgenerational phenotypes and therapeutic avenues respectively have generated impetus to unfold methylation maps through yet another mammoth and forthcoming Human Epigenome Project.
Key Words: Epigenetics, Microenvironment, Cellular differentiation, Epialleles, Imprinted genes, DNA methylation, Histone deacetylation, Cancer and Human Epigenome Project.
Gregor Mendel, a monk in the Augustinian monastery at Brunn, Austria in 1866, intuitively attempted an independent observation using garden pea plants. He must have made a serendipitous observation of a few set of characters in a number of plants comprising of a few generations and deduced profound Laws of Inheritance. They invariably constituted the basis for the genesis of the field of genetics. The concepts of genes, genome and epigenome have appeared at a later time embedding the principles of Mendel’s Laws of Inheritance. Incidentally, the characters chosen by Gregor Mendel to demonstrate the patterns of inheritance must have been under the influence of epigenome. However, providentially there must not have been epimutations for the chosen characters in the short-term experiments on pea plants conducted by Mendel. Hence, the results obtained during his observations were unique that they followed in an arithmetic fashion viz. 1:2:1, 3:1, and 9:3:3:1 etc. (8) without expressing variegation among the chosen pairs of characters namely tall and dwarf plants and round and wrinkled seeds.
The tissues of multicellular organisms are genetically homogeneous but phenotypically heterogeneous. This phenomenon is due to the unusual variable expressivity of characters in the absence of genetic heterogeneity. The variation in the expressivity of a character must have been due to the conditioning imparted during development and retained through mitosis. The stable phenotypic alterations resulted due to conditioning without involving a change in DNA code of an allele is said to be “epigenetics” as both alleles and the conditioning factors are together heritable wherein no mutation is involved. However, this phenomenon is unusually distinct from the maturation of B-cells whose genome undergoes hyper-somatic mutations to specialize for the secretion of a customized assembled immunoglobulin as per the prompting of T-helper cell. At times, the extent to which environmental factors condition epigenetic responses is somewhat labile culminating in phenotypic mosaicism (variegation) among tissues and cells. Thus, the epigenetic factors determine the variable expressivity of alleles and biased expressivity (imprinting) of either paternal or maternal alleles in the progeny.
In 19th Century, Casper Friedrich Wolff introduced the term “epigenesis” to comprehend the mysterious workings of Nature that awakes the structures to form de novo from the apparent homogeneous (structureless) mass of a zygote (9). The jargon of epigenesis is further brought into limelight through one of the branches of biology viz., Developmental Biology, which deals with the causative features of embryonic development wherein it is elaborated as: the study of changes in gene function due to combinatorial factors that are heritable among the cells of an embryo without bringing alterations in the concerned gene sequence, which means that the factors other than DNA supplement in the process of Heredity. Further, the heritable epigenetic changes are called ‘epimutations’. B. McClintock observed one of the first epimutations in maize through transposon activity (10). Demethylation of a tandem repeat in the promoter accompanied the ectopic expression of the late flowering gene, fwa in ddm1 plants and this resulted in a dominant epimutation (10). Methylated genes in Neurospora crassa behaved as epimutant alleles wherein methylation – associated silencing of transcript elongation were shown in Neurospsora (10). The term epiallele also comes into vogue whose epigenetic factors are stable at least through transgenerations. For example, the transgene (Tg-13 HBV – E36-pas) when inherited paternally is unmethylated but maternal transmission of the same transgene results in methylation and transcriptional silencing (11). Thus, as envisaged by Jenuwein and Allis (12), the phenomenon of epigenesis implies a fundamental regulatory system beyond nucleotide sequence information of DNA emphasizing that Mendel’s alleles are more than just a DNA moiety.
The two principal mechanisms that regulate gene expression are DNA methylation and histone deacetylation (Fig.1 a). Among the amino acid residues on the histone proteins, lysine and argentine are relatively in good proportion and they are vulnerable for methylation and other enzymatic modifications (Table.1). DNA methylation in mammals plays a crucial role in their cell-cycles, developmental stages, X-chromosome inactivation, telomere length adjustment, gene silencing, aging, carcinogenesis and a few human genetic disorders. Histone acetylation is associated with transcriptional activation because the affinity of acetylated histone protein for DNA is reduced and chromatin package is thus relaxed (Fig.1 b). Furthermore, there is a positive correlation between DNA methylation and gene inactivity.
Does DNA methylation cause the loss of transcriptional activity of a specific region of chromatin? The same question was addressed experimentally in that a DNA – demethylating agent viz., 5-azacytidine brought about the reactivation of silenced endogenous genes viz., ribosomal RNA genes (13). In a few ingenious protocols designed by Buschhausen et al.,(14) it was shown that the injected methylated and non- methylated versions of the Herpes simplex virus thymidine kinase (HSVTK) gene into the rodent cells were reported to be active during the first 8-hours. Later, the inhibitory effects of DNA methylation were noticed on the methylated version of HSVTK. With these observations, it was hypothesized that initially microinjected versions of TK were not fully incorporated into the chromatin in the first 8-hours and that at a later stage the methylated version became silent upon incorporation into the chromatin. The time-dependent repression of methylated versions of DNA fragments was reported upon injection of m ethylated DNA fragments into Xenopus oocytes (15).
Histone methylation results in various transcriptional consequences depending on which histone is affected. For example, histone H3 methylation at lysine 9 is associated with heterochromatin, a more compact version of chromatin and ultimately silencing of genes. Additionally, inhibition of histone deacetylation results in the re-expression of methylated copy of the hygromycine resistance gene in the fungus, Neurospora crassa (16) suggesting that deacetylation may either directly or indirectly cause loss of DNA methylation. Even though myeloid specific mouse M-lysozyme gene is methylated and silenced in non-myeloid cells, upon treating them (fibroblasts, T-lymphocytes, etc) with trichostatin-A (a specific inhibitor of histone deacetylse), the expression of the same is revived. The level of revival of M-lysozyme gene was comparable with that achieved by the demethylating agent viz., 5-azacytidine. In yet another incident, it is shown that the silencing of Metallothionein-1 gene is induced due to the methylation of CpG islands in mouse lymphosarcoma P1798 cells (17). Thus, there are multi-subunit chromatin-protein complexes prone to anchor a variety of epigenetic factors which collectively and preferentially work together for the awakening of genes.
The paternal genome undergoes a remarkable transformation in the oocyte cytoplasm. It constitutes the remodeling of sperm chromatin through the removal of protamines and replacement by acetylated histones followed by genome-wide demethylation (18). Typically, the process of demethylation is intricately coupled to chromatin remodeling in the zygote. The chromatin during spermatogenesis undergoes methylation and compacted with protamines (19) – which are crucial to facilitate normal fertilization. Whereas, genome of oocyte possesses oocyte-specific linker histones. The chromatin of female pro-nucleus is more repressive than male pro-nucleus, which possibly protects the oocyte genome against extensive epigenetic modifications imposed on the paternal genome in the zygote. Interestingly, the repetitive sequences (LINES, SINES, etc) are not uniformly methylated in gametes.
The somatic cell proliferation during organogenesis and histological differentiation follows mitosis which invariably results into two equal daughter somatic cells. They share equally both epigenetic factors and chromatin. Hence, the two daughter cells look similar in all respects. In contrast, the zygote undergoes cleavage and yields two blastomeres: one of the two is left and another is right blastomere whose developmental programme invariably is found to be different (Fig.2). The possible implications for the same are – 1) unequal sharing of epigenetic factors between the first two blastomeres and 2) unequally conditioned chromatin material sharing equally between them. Soon after fertilization, the paternal genome is actively demethylated in contrast to maternal genome facilitating for paternal imprinting. The basic reprogramming events of paternal demethylation in zygote appear to be conserved in eutherian mammals. In mouse and bovine embryos, the de novo methylation occurs in the inner mass of cells of the blastocyst and in 8 to 16 cell stage respectively. Post-zygotic demethylation and remethylation bring about the modifications in epigenetic microenvironment. Thus, reprogramming of epigenetic factors is not only observed in germ cells and early embryos but also in embryonic stem cells (18).
In radial development, the third cleavage results into two tiers of unequal blastomeres viz., upper tier comprising of 4 micromeres and a lower tier with 4 macromeres indicating the influence imparted by epigenetic factors which are being shared unequally between them. Hence, their developmental fates vary profoundly. The micromeres tend to develop into epidermis. During involution of micromeres through the blastopore, there is a supplementation of microenvironmental factors (BMPs, Noggin, etc) from Nieuwkoop centre in frog gastrula and direct them to develop into neural derivatives causing tissue specificity (2). Another classical example is one of the mesodermal derivatives namely somites and their differentiation. After 96 hours of incubation, the somites of chick embryo undergo differentiation as dermatome, sclerotome and myotome by sharing cellular microenvironmental factors (FGFs) among them resulting into fully differentiated states viz., dermis, skeleton and muscles respectively. These observations reveal that in advanced embryos, genome methylation patterns are stable and heritable, whereas in early embryos (zygote, cleavage and gastrula) the methylation patterns are reprogrammable to manifest a broad developmental potential. The imposed methylation patterns among blastomeres are irreversibly continued by maintenance DNA methyltransferase (DNMT1) and this would make the germ layers to progress in predetermined programmes of gene expression (18).
The inbuilt genome-wide developmental reprogrammes in the life-history of an organism provide several avenues to design epigenetic modifications of chromatin structure to simulate for somatic nuclear cloning experiments to succeed. Because epigenetic conformations of any somatic nuclei markedly vary from that of the nuclei of mature gametes and it is unique that the cytoplasm of oocyte can reverse the epigenetic modifications to reestablish a state of totipotency.
CpG dinucleotides are found in clusters and thus constitute CpG islands. In vertebrates, 60 to 90% of all CpGs are methylated. The remaining non-methylated CpGs include functional promoters in the region more towards 5’ end (20). They are found to contain highly acetylated histones, H3 and H4. There are regions of which each is greater than 200 bp with high GC content (>0.6). Methylation of cytosines at the carbon 5’ position of CpG dinucleotides is a characteristic feature of many eukaryotic genomes (13). The salient property of CpG Island is that it is unmethylated in the germ line. It is suggested that CpG island methylation has a dominant effect upon comparison with histone deacetylation in silencing genes (13). The lactoferrin promoter that resides immediately upstream from the estrogen response element contains 5 CpG sites within the region from 590 to 330 bp (21). Further, it is reported that CpG island in the estrogen receptor gene is hypermethylated in human breast cancer cells and also in sporadic colorectal tumerogenesis. Mujumder, et al., (22) have shown that Metallothionein 1 gene is silenced by methylation of CpG islands present within 216 bp to +1 bp with respect to transcription start in mouse lymphosarcoma P 1798 cells. Furthermore, the intriguing feature is that there is an association between the promoter regions of many tumor suppressor genes and de novo methylation of an entire CpG island (23) which is the primary cause for the genesis of tumor.
There is a family of highly conserved proteins namely methyl CpG binding proteins sharing a common binding domain (MBD family) selectively docks to methylated CpG dinucleotides. Huck and Adrain (13) indicated that the transcriptional silencing is also mediated by methyl CpG binding protein (MeCP2) which is found to interact with Sin3/ histone deacetylase co-repressor complex. Thus, methylation of CpG island results in the alteration of chromatin structure followed by the direct impediment in the binding of positive factors to the regulatory elements (15) and ultimately rendering the sites inaccessible to the basal transcriptional machinery i.e., prevention of interaction of transcription factors with the promoters.
Genome imprinting is unique among mammals. It is a phenomenon that a few genes are expressed according to their parental origin. Imprinted genes facilitate us to comprehend the intricacies of a particular lineage and prediction of behaviour as well as human diseases. These genes which are privileged to become transgenerational imprints are initiated through gametogenesis, inherited by mature gametes and infused in the following generation. As elaborated by Elizabeth Pennisi (24), the age old mule breeders noticed that a mare crossed with a donkey yields a mule, whereas a hinny resulted out of the cross between stallion and donkey. These breeding experiments gave insight to ponder over the phenomenon of paternal or maternal imprint of characters among offspring i.e., ‘parent-specific effects’ in offspring. In other words, the characters from any one of the parents have preponderantly marked or imprinted in the progeny.
There are nearly 40 genes whose expression depends on their parents of origin. A few to mention here are: 1) Igf2r and 2) H19 - they are active only when inherited from the maternal side and 3) Igf2 expresses only when inherited from father – all of which are invariably influenced by epigenetic tags. Furthermore, a number of disease causing genes also follow the pattern of imprinting. They include necdin and UBE 3A genes present on chromosome 15. They cause Prader –Willi and Angelman syndromes respectively (24). The tumor suppressor gene, p73, which is involved in the brain cancer neuroblastema, also comes under this category. It has also been shown that imprinted genes are clustered in specific chromosomes. For e.g., H19, Igf2 and six other imprinted genes are found in close proximity on human chromosome 11 (11p15.5). Another set of imprinted genes viz., DKK1 and GTL2 spatially lie together on human chromosome 14 (14q3.2) arranged in the same temporal order as found in mouse, which facilitates for reciprocal expression. The enzyme viz., maintenance DNA methylase (Dnmt1) possibly plays a crucial role in the sustenance of genomic imprinting.
Thus, the clustered maternally or paternally expressed imprinted genes with biased methylation towards any one of the parental chromosomes would result in the trans-generational expression of such genes reflecting the appearance of corresponding trans-generational behaviour or phenotype. Moreover, the genomic imprinting confers a developmental asymmetry on the parental genome through epigenetic preferences on the cluster of genes in blast cells and embryo (25). The heritable epigenetic asymmetry regulates one of the parental alleles clustered through specific cis-acting imprinting centers. These modifications are manifested in the germ line and inherited as imprinted alleles which would be more graciously termed as epialleles. Therefore, the mechanism of genomic imprinting serves as a model system for the evaluation of epigenetic microenvironment on the schedule of awakening of gene function.
Geneticists have been fascinated by mammalian alleles that are variably expressed, even in the absence of gene sequence variability (Table 2). Rakyan Vardhman et al (11) have noticed the expression of two epialleles viz., murine Avy (Agouti variable yellow) and axin fused (Axin Fu) alleles. Variable expressivity (variegation) of these two epialleles was attributed to their epigenetic state. Ex, Isogenic mice that carry Avy allele display a range of cot colours from completely yellow to wild type agouti. A yellow coat correlates with a hypomethylated state and therefore an active epiallele. In a similar vein, Auxin fused gene in mouse exhibits phenotypic classification as follows: normal tail non kinky, slightly kinky, kinky and very kinky. Such variations could be explained through epigenetic phenomenon.
There is growing evidence revolving around ‘trio of events’ viz., human diseases, genetic alternations and acquired epigenetic abnormalities. Much has been discussed in earlier sections highlighting the significance of promoter CpG island methylation and gene silencing. As shown by Harikrishnan et al., (26) that the methylated DNA binding protein (MeCP2) is associated with Brahma (Brm), a catalytic component of SW1/SNF chromatin-remodeling complex. Thus, it is clear that the cytosine methylation is mediated by MeCP2. Further, there is a potential link between cytosine methylation and chromatin silencing - one of the crux events for the initiation of tumor and it is hypothesized to constitute a distinct phenotype viz., ‘CpG island methylation phenotype’ (CIMP). Histone modification, such as, loss of acetylation at lysine 16 and trimethylation at lysine 20 of histone H4 are the epigenetic events prone to human cancer. Besides, there is another interesting polycomb repressor complex (PRC 2) in the initiation of genome silencing. This contains histone methyltransferases which possibly translocate methyl groups to lysine residues 9 and 27 of histone H3 (27). It is reported by Peter and Stephen (28) that the polycomb gene (BMI1), a component of PRC1 is unusually over expressed in several human cancers. In addition, transcription of a number of tumor suppressor genes such as p16, BRCA1, p53, hMLH-1 has now been shown to be inhibited due to the hypermethylation of their corresponding promoter sites (29).
Another side of the coin in gene silencing is that the methylation of CpG dinucleotides prevents transcription factors such as c-Myc to recognize their foot-prints on DNA (30). The above accumulated experimental evidences strongly indicate that the entire methylated epigenome is customarily dysregulated causing cancer to develop. These epigenetic dictums in cancer have led to the development of an entirely new therapeutic approach in which the focus is to reverse gene (tumor suppressor gene) silencing (28). Thus, the drugs inhibiting DNA methyl transferase enzyme such as azanucleoside (31), 5-fluoro-2’-deoxycytidine (32) and Zebularine (33) are under active consideration of FDA, USA for treatment of cancer.
Though the DNA sequence is invariant across the tissues, yet the epigenetic microenvironment dictates tissue-specific variation. The mammoth Human Genome Project unraveled the nucleotide sequences for nearly 40,000 genes. Their differential and possibly preferential expression is invariably under the guidance and involvement of epigenetic factors. The degree of evaluation of these epigenetic factors, their quantification and relations with the corresponding regions of genome need to be understood for the successful prognostic measures in the prevention of human diseases associated with aging, inborn errors and cellular differentiation (Table.3). Therefore, attempts are under progress since two years for an international consensus in the epigenetic community to establish an organized Human Epigenome Project, which may perhaps focus on identification, cataloguing and interpreting genome wide methylation patterns - which are intricately involved in diverse biological processes and etiology of many diseases. The cataloguing of such methylation variable positions will invariably improve our understanding of epigenome biology and our ability to diagnose diseases (34).
A beginning is made to set up Human Epigenome Consortium by the collaboration of Sanger Centre, Epigenomics AG and the Centre National de Genotypage. Adopting automated bisulphite DNA sequencing technique, they are advancing to unfold 150 loci in the MHC region on chromosome 6. One of the pioneers in the field viz. Thomas Jenuwein, Vienna, (Austria), is heading European Epigenome Network. Another group viz., High throughput Epigenetic Regulatory Organisation in Chromatin (HEROIC) led by Henk Stunnenberg is aiming at developing tools for the analysis of complex chromatin-DNA interaction. One of the private/public partnerships being funded by the German government is running under the leadership of Joerg Hoheisel. Recently, the American Association for Cancer Research Sponsored a Workshop (35) to formulate a proposal for a Human Epigenome Project with a working group aiming at the evaluation of epigenetic factors concerned with the tissues of pathological states.
Callinan and Feinberg (36) cited an example that was reported by Fraga et al., (37) illustrating the prevalence of epigenetic disparities between monozygotic twins with a consequent upsurge during ageing. This is a remarkable example displaying phenotypic discordance for complex diseases such as psychiatric disorders and thus highlighting the contribution of individual’s epigenotype to the phenotypic manifestation of the inherited genotype. The public data base for epigenomics is available at http://WWW.epigenome.org.
OOne of the authors (SK) gratefully acknowledges UGC and DST, New Delhi for providing financial support through SAP DRS and FIST programmes respectively. Authors acknowledge Mrs. Deepa Srikumar, NIH, Bethesda, USA, for having responded our request and provided us the literature.
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Relative proportions of Lysine (K) and Arginine (R) in histones.
The relative dominance of epialleles uAvy / mAvy and wild type allele (A/A) are shown (11). Note: Homozygous hypermethylated epiallele, mAvy (epigenetic effect) behaves as a wild type, whereas un/hypo methylated epiallele, uAvy / A in heterozygous state restrains agouti (despite being wild type) and behaves as dominant (suppressing wild type) with a phenotype viz., yellow colour. The homozygous un/hypo methylated state imparts yellow coat colour with higher intensity.