The character of a cell is defined by its constituent proteins, which are the result of specific patterns of gene expression. Crucial determinants of gene expression patterns are DNA-binding transcription factors that choose genes for transcriptional activation or repression by recognizing the sequence of DNA bases in their promoter regions. Interaction of these factors with their cognate sequences triggers a chain of events, often involving changes in the structure of chromatin, that leads to the assembly of an active transcription complex (e.g., Cosma et al. 1999). But the types of transcription factors present in a cell are not alone sufficient to define its spectrum of gene activity, as the transcriptional potential of a genome can become restricted in a stable manner during development. The constraints imposed by developmental history probably account for the very low efficiency of cloning animals from the nuclei of differentiated cells (Rideout et al. 2001; Wakayama and Yanagimachi 2001). A “transcription factors only” model would predict that the gene expression pattern of a differentiated nucleus would be completely reversible upon exposure to a new spectrum of factors. Although many aspects of expression can be reprogrammed in this way (Gurdon 1999), some marks of differentiation are evidently so stable that immersion in an alien cytoplasm cannot erase the memory. The genomic sequence of a differentiated cell is thought to be identical in most cases to that of the zygote from which it is descended (mammalian B and T cells being an obvious exception). This means that the marks of developmental history are unlikely to be caused by widespread somatic mutation. Processes less irrevocable than mutation fall under the umbrella term “epigenetic” mechanisms. A current definition of epigenetics is: “The study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence” (Russo et al. 1996). There are two epigenetic systems that affect animal development and fulfill the criterion of heritability: DNA methylation and the Polycomb-trithorax group (Pc-G/trx) protein complexes. (Histone modification has some attributes of an epigenetic process, but the issue of heritability has yet to be resolved.) This review concerns DNA methylation, focusing on the generation, inheritance, and biological significance of genomic methylation patterns in the development of mammals. Data will be discussed favoring the notion that DNA methylation may only affect genes that are already silenced by other mechanisms in the embryo. Embryonic transcription, on the other hand, may cause the exclusion of the DNA methylation machinery. The heritability of methylation states and the secondary nature of the decision to invite or exclude methylation support the idea that DNA methylation is adapted for a specific cellular memory function in development. Indeed, the possibility will be discussed that DNA methylation and Pc-G/trx may represent alternative systems of epigenetic memory that have been interchanged over evolutionary time. Animal DNA methylation has been the subject of several recent reviews (Bird and Wolffe 1999; Bestor 2000; Hsieh 2000; Costello and Plass 2001; Jones and Takai 2001). For recent reviews of plant and fungal DNA methylation, see Finnegan et al. (2000), Martienssen and Colot (2001), and Matzke et al. (2001).

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DNA methylation patterns and epigenetic memory

Cells of a multicellular organism are genetically homogeneous but structurally and functionally heterogeneous owing to the differential expression of genes. Many of these differences in gene expression arise during development and are subsequently retained through mitosis. Stable alterations of this kind are said to be 'epigenetic', because they are heritable in the short term but do not involve mutations of the DNA itself. Research over the past few years has focused on two molecular mechanisms that mediate epigenetic phenomena: DNA methylation and histone modifications. Here, we review advances in the understanding of the mechanism and role of DNA methylation in biological processes. Epigenetic effects by means of DNA methylation have an important role in development but can also arise stochastically as animals age. Identification of proteins that mediate these effects has provided insight into this complex process and diseases that occur when it is perturbed. External influences on epigenetic processes are seen in the effects of diet on long-term diseases such as cancer. Thus, epigenetic mechanisms seem to allow an organism to respond to the environment through changes in gene expression. The extent to which environmental effects can provoke epigenetic responses represents an exciting area of future research.

/pdf/epigenetic-regulation-of-gene-expression-how-the-genome-55fnpf16ec.pdf

Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals

Patterns of DNA methylation and chromatin structure are profoundly altered in neoplasia and include genome-wide losses of, and regional gains in, DNA methylation. The recent explosion in our knowledge of how chromatin organization modulates gene transcription has further highlighted the importance of epigenetic mechanisms in the initiation and progression of human cancer. These epigenetic changes -- in particular, aberrant promoter hypermethylation that is associated with inappropriate gene silencing -- affect virtually every step in tumour progression. In this review, we discuss these epigenetic events and the molecular alterations that might cause them and/or underlie altered gene expression in cancer.

The fundamental role of epigenetic events in cancer

A Bivalent Chromatin Structure Marks Key Developmental Genes in Embryonic Stem Cells

DNA methylation is frequently described as a 'silencing' epigenetic mark, and indeed this function of 5-methylcytosine was originally proposed in the 1970s. Now, thanks to improved genome-scale mapping of methylation, we can evaluate DNA methylation in different genomic contexts: transcriptional start sites with or without CpG islands, in gene bodies, at regulatory elements and at repeat sequences. The emerging picture is that the function of DNA methylation seems to vary with context, and the relationship between DNA methylation and transcription is more nuanced than we realized at first. Improving our understanding of the functions of DNA methylation is necessary for interpreting changes in this mark that are observed in diseases such as cancer.

Functions of DNA methylation: islands, start sites, gene bodies and beyond

Genes constitute only a small proportion of the total mammalian genome, and the precise control of their expression in the presence of an overwhelming background of noncoding DNA presents a substantial problem for their regulation. Noncoding DNA, containing introns, repetitive elements, and potentially active transposable elements, requires effective mechanisms for its long-term silencing. Mammals appear to have taken advantage of the possibilities afforded by cytosine methylation to provide a heritable mechanism for altering DNA-protein interactions to assist in such silencing. Genes can be transcribed from methylation-free promoters even though adjacent transcribed and nontranscribed regions are extensively methylated. Gene promoters can be used and regulated while keeping noncoding DNA, including transposable elements, suppressed. Methylation is also used for long-term epigenetic silencing of X-linked and imprinted genes and can either increase or decrease the level of transcription, depending on whether the methylation inactivates a positive or negative regulatory element.

https://science.sciencemag.org/content/sci/293/5532/1068.full.pdf

The Role of DNA Methylation in Mammalian Epigenetics

CpG islands are useful markers for genes in organisms containing 5-methylcytosine in their genomes. In addition, CpG islands located in the promoter regions of genes can play important roles in gene silencing during processes such as X-chromosome inactivation, imprinting, and silencing of intragenomic parasites. The generally accepted definition of what constitutes a CpG island was proposed in 1987 by Gardiner-Garden and Frommer [Gardiner-Garden, M. & Frommer, M. (1987) J. Mol. Biol. 196, 261–282] as being a 200-bp stretch of DNA with a C+G content of 50% and an observed CpG/expected CpG in excess of 0.6. Any definition of a CpG island is somewhat arbitrary, and this one, which was derived before the sequencing of mammalian genomes, will include many sequences that are not necessarily associated with controlling regions of genes but rather are associated with intragenomic parasites. We have therefore used the complete genomic sequences of human chromosomes 21 and 22 to examine the properties of CpG islands in different sequence classes by using a search algorithm that we have developed. Regions of DNA of greater than 500 bp with a G+C equal to or greater than 55% and observed CpG/expected CpG of 0.65 were more likely to be associated with the 5′ regions of genes and this definition excluded most Alu-repetitive elements. We also used genome sequences to show strong CpG suppression in the human genome and slight suppression in Drosophila melanogaster and Saccharomyces cerevisiae. This finding is compatible with the recent detection of 5-methylcytosine in Drosophila, and might suggest that S. cerevisiae has, or once had, CpG methylation.

https://www.pnas.org/content/pnas/99/6/3740.full.pdf

Comprehensive analysis of CpG islands in human chromosomes 21 and 22

Almost 1-2% of the human genome is located within 500 bp of either side of a transcription initiation site, whereas a far larger proportion (≈25%) is potentially transcribable by elongating RNA polymerases. This observation raises the question of how the genome is packaged into chromatin to allow start sites to be recognized by the regulatory machinery at the same time as transcription initiation, but not elongation, is blocked in the 25% of intragenic DNA. We developed a chromatin scanning technique called ChAP, coupling the chromatin immunoprecipitation assay with arbitrarily primed PCR, which allows for the rapid and unbiased comparison of histone modification patterns within the eukaryotic nucleus. Methylated lysine 4 (K4) and acetylated K9/14 of histone H3 were both highly localized to the 5′ regions of transcriptionally active human genes but were greatly decreased downstream of the start sites. Our results suggest that the large transcribed regions of human genes are maintained in a deacetylated conformation in regions read by elongating polymerase. Common models depicting widespread histone acetylation and K4 methylation throughout the transcribed unit do not therefore apply to the majority of human genes.

https://www.pnas.org/content/pnas/101/19/7357.full.pdf

Distinct localization of histone H3 acetylation and H3-K4 methylation to the transcription start sites in the human genome.

Clusters of CpG dinucleotides in GC rich regions of the genome called "CpG islands" frequently occur in the 5' ends of genes. Methylation of CpG islands plays a role in transcriptional silencing in higher organisms in certain situations. We have established a CpG-island-extraction algorithm, which we previously developed [Takai and Jones, 2002], on a web site which has a simple user interface to identify CpG islands from submitted sequences of up to 50kb. The web site determines the locations of CpG islands using parameters (lower limit of %GC, ObsCpG/ExpCpG, length) set by the user, to display the value of parameters on each CpG island, and provides a graphical map of CpG dinucleotide distribution and borders of CpG islands. A command-line version of the CpG islands searcher has also been developed for larger sequences. The CpG Island Searcher was applied to the latest sequence and mapping information of human chromosomes 20, 21 and 22, and a total of 2345 CpG islands were extracted and 534 (23%) of them contained first coding exons and 650 (28%) contained other exons. The CpG Island Searcher is available on the World Wide Web at http://www.cpgislands.com or http://www.uscnorris.com/cpgislands/cpg.cgi.

Daiya Takai

Papers

The Role of DNA Methylation in Mammalian Epigenetics

Comprehensive analysis of CpG islands in human chromosomes 21 and 22

Distinct localization of histone H3 acetylation and H3-K4 methylation to the transcription start sites in the human genome.

The CpG island searcher: a new WWW resource.

Role of nucleosomal occupancy in the epigenetic silencing of the MLH1 CpG island.