The somatic cells of the human body contain 20,000 to 25,000 genes. Yet in any of these cells, only a relatively small percentage of all genes are active.

In the more than 200 different cell types present in the

body, different cell-specific gene sets are transcribed, while the rest of the genome is transcriptionally inactive.

In addition, programs of gene expression become more

and more restricted during development and differentiation as embryonic cells gradually become specialized adult cells with distinct phenotypes.

The prevailing view has been that regulation of gene expression is coordinated by promoter, promoter–proximal, enhancer, and other cis-regulatory elements as well as DNA-binding proteins and transcription factors.

The newly emerging field of epigenetics is providing us with a basis for understanding how heritable changes other than those in DNA sequence can influence phenotypic variation.

These advances greatly extend our understanding of the molecular basis of gene regulation and apply to wide-ranging areas including genetic disorders, cancer, and behavior.

An epigenetic trait : is a stable, mitotically and meiotically heritable phenotype that results from changes in gene expression without alterations in the DNA sequence. Epigenetics: is the study of the ways in which these changes alter cell- and tissue-specific patterns of gene expression. The epigenome: refers to the epigenetic state of a cell. During its life span, an organism has one genome, but this genome can be modified in diverse cell types at different times to produce many epigenomes.

Current research efforts are focused on several aspects of epigenetics:

how an epigenome arises in developing and differentiated cells.

what mechanisms maintain these states, and how they are transmitted via mitosis and meiosis, making them heritable traits.

In addition, because epigenetically controlled alterations to the genome are associated with cancer and some common diseases such as diabetes and asthma, efforts are also being directed at the development of drugs that can modify or reverse disease-associated epigenetic changes in cells.

Several systems and pathways that result in the establishment, maintenance, and inheritance of the epigenetic state are recognized. These pathways are organized into three categories.

The first category includes environmental signals called epigenators that are received by the cell and that stimulate a response via an intracellular pathway.

Epigenetic pathway

1-Epigenators

Responses to epigenator signals are called epigenetic initiators. Components of this second category produce epigenetic changes. These initiators include protein–protein signal transduction pathways, DNA binding proteins, and noncoding RNAs. The actions of initiators define the location at which epigenetic changes in chromatin will take place. DNA sequence recognition is a necessary part of this response.

2- epigenetic initiators

Once the epigenetic modifications have occurred, they are maintained by molecular elements called epigenetic maintainers.

Components of this third category conserve and sustain the epigenetic changes in the present and future generations.

Epigenetic maintainers are not sequence-specific, they operate anywhere in the genome, and they depend on initiators to specify the loci at which chromatin modifications will take place.

Maintainers include DNA methylation and histone modifications.

3- epigenetic maintainers

Unlike the genome, which is identical in all cell types of an organism, the epigenome is cell-type specific and heritable.

Like the genome, the epigenome can be transmitted to daughter cells by mitosis and to future generations by meiosis.

There are three major epigenetic mechanisms:

(1) Reversible modification of DNA by the addition or removal of methyl groups

(2) modification of histones by the addition or removal of chemical groups

(3) regulation of gene expression by small, noncoding RNA molecules.

Epigenetic Alterations To The Genome

In mammals, methylation of DNA takes place after replication and involves the addition of a methyl group (–CH3) to cytosine, a reaction catalyzed by methyltransferase enzymes.

DNA methylation also occurs during the differentiation of adult cells.

In both instances, methylation takes place almost exclusively on cytosine bases adjacent to a guanine, a combination called a CpG dinucleotide.

Many of these dinucleotides are clustered in regions, called CpG islands, located in and near promoter sequences adjacent to genes.

1-Methylation

Islands adjacent to essential genes (housekeeping genes) and cell-specific genes are unmethylated, making these genes available for transcription.

Other genes with adjacent methylated CpG islands are transcriptionally silenced.

The methyl groups in CpG islands occupy the major groove of DNA, and block the binding of transcription factors necessary to form transcription complexes.

The bulk of methylated CpG dinucleotides are found in repetitive DNA sequences located in heterochromatic regions of the genome, including the centromere.

Methylation of these sequences contributes to silencing the transcription and replication of transposable elements such as LINE and SINE sequences which constitute a major fraction of the human genome.

Heterochromatic methylation also maintains chromosome stability by preventing translocation and other chromosomal abnormalities.

As part of dosage compensation, X chromosomes in mammalian females are inactivated by converting them to heterochromatin. These inactivated chromosomes have altered patterns of DNA methylation. As mentioned above, CpG methylation in euchromatic regions causes a parentspecific pattern of gene transcription.

histone modification is an important epigenetic mechanism of gene regulation.

Recall that chromatin is composed of DNA wound around an octamer core of histone proteins to form nucleosomes.

Amino acids in the N-terminal region of these histones can be covalently modified in several ways, including acetylation, methylation, and phosphorylation.

These modifications occur at conserved amino acid sequences in the N-terminal histone tails.

Chemical modification of histones alters the structure of chromatin, making genes accessible or inaccessible for transcription.

2- Histone Modification

Amino acids in the N-terminal region of these histones can be covalently modified in several ways, including acetylation, methylation, and phosphorylation.

Features of Histone Modifications

Methyl Acetyl Phospho

Normally, when histones are modified by acetylation, a reaction catalyzed by the enzyme histone acetyltransferase (HAT), chromatin structure becomes “open,” making genes on these modified nucleosomes available for transcription This modification is reversible, and acetyl groups can be removed by another enzyme, histone deacetylase (HDAC), changing the chromatin to a “closed” configuration, and silencing genes by making them unavailable for transcription

•Covalently attached groups (usually to histone tails)

Methyl Acetyl Phospho

After transcription, small interfering RNA (siRNA) molecules associate with protein complexes to form RNA-Induced Silencing Complexes (RISCs).

RISCs bind to mRNA molecules that carry sequences complementary to siRNA in the RISC.

If the siRNA is not perfectly complementary to the mRNA, the binding interferes with translation, resulting in downregulation of gene expression.

If, however, the siRNA in the RISC is perfectly complementary to sequences in the mRNA, the mRNA is cleaved and destroyed, effectively silencing the gene.

3- RNA Interference

Recently, it has been discovered that siRNAs can silence genes by directly interfering with transcription initiation. This does not involve any changes in existing epigenetic promoter modifications, nor does it require new modifications. Instead, siRNAs complementary to promoter regions bind to a promoter. Binding blocks the assembly of the preinitiation complex by preventing binding of transcription factor TFIIB and RNA polymerase.

In sum, epigenetic modifications alter chromatin structure by several mechanisms including DNA methylation, histone acetylation, and RNA interference, without changing the sequence of DNA.

These epigenetic changes create an epigenome that in turn, can regulate normal development or generate responses to environmental signals.

Mammals inherit a maternal and a paternal copy of each autosomal gene, and either or both copies of these genes can be expressed in the offspring.

Imprinted genes show expression of only the maternal

allele or the paternal allele. This parent-specific pattern of allele expression is laid

down during gamete formation. Differential methylation of CpG rich regions produce

allele-specific imprinting and subsequent gene silencing.

Epigenetics and Imprinting

In mice, fewer than 100 genes were thought to be imprinted, but recent work has identified more than 1300 imprinted genes.

In humans, more than 150 candidate genes are thought to be imprinted, but the findings in mice suggest that many more imprinted genes remain to be identified in humans.

Once a gene has been methylated and imprinted, it remains transcriptionally silent during embryogenesis and development.

At the level of individual genes, having only one functional allele makes these genes highly susceptible to the deleterious effects of mutations.

Because imprinted genes are clustered, mutation in one gene can have an impact on the function of adjacent imprinted genes, amplifying its impact on the phenotype.

Mutations in imprinted genes can arise by changes in the DNA sequence or by epigenetic changes, called epimutations, both of which are heritable changes in the activity of a gene.

Mutations and imprinted genes

For example, females receive a maternal and a paternal set of chromosomes. In somatic cells and in germ cells, the maternal chromosome set has female imprints, and the paternal set contains male imprints.

When gamete formation begins in

female germ cells, both chromosome sets have their imprints erased and are each reprogrammed by changing the pattern of methylation to carry a female imprint pattern that is transmitted to the next generation through the egg.

Imprinting patterns are reprogrammed each generation

Most human disorders associated with imprinting have their origins during fetal growth and development. Imprinting defects cause Prader–Willi syndrome, Angelman syndrome, Beckwith–Wiedemann syndrome, and several other diseases (ST Table 3.1). However, given the number of candidate genes and the possibility that additional imprinted genes remain to be discovered, the overall number of imprinting-related genetic disorders may be much higher.

In humans, most known imprinted genes encode growth factors or other growth-regulating genes.

Generally, maternally expressed alleles of imprinted genes suppress growth, and paternal alleles enhance growth.

One autosomal dominant disorder of imprinting, Beckwith–Wiedemann syndrome (BWS), offers insight into how disruptions of epigenetically imprinted genes lead to an abnormal phenotype.

BWS is a prenatal overgrowth disorder with abdominal wall defects, enlarged organs, large birth weight, and predisposition to cancer.

BWS is not caused by mutation, nor is it associated with any chromosomal aberration.

Instead it is a disorder of imprinting and is caused by abnormal methylation patterns.

BWS في جدارالبطن، تضخم قبل الوالدة مع عيوب زيادة الوزن ما هو اضطراب

.بالسرطان عاليلالصابة كبير، واالستعداد الوزن عند الوالدة في األعضاء،

Beckwith–Wiedemann syndrome (BWS),

Genes linked to BWS are located in a cluster of imprinted genes on the short arm of chromosome 11.

This cluster contains more than a dozen imprinted genes, some of which are paternally expressed, while others are maternally expressed and all genes in this cluster regulate growth during prenatal development.

The imprinted region is subdivided into two separately regulated domains, one of which contains the closely linked genes IGF2 (insulin growth factor 2) and H19.

Normally, the paternal allele of IGF2 is expressed, and the allele on the maternal homolog is imprinted and silenced. In the case of H19, the situation is usually the reverse.

The protein encoded by IGF2 is a growth factor, and the product of the H19 is a long, noncoding RNA that is a growth repressor.

Expression of these genes is normally controlled by an imprinting control region (ICR) located within its chromosomal domain.

Many affected individuals have a loss of imprinting of the maternal IGF2 allele. This causes both the maternal and paternal alleles to be transcriptionally active, resulting in the overgrowth of tissues characteristic of this disease.

Epidemiological studies investigate the role of environmental factors in normal phenotypic variation and as risk factors for disease.

For some complex diseases, there are strong links with

environmental factors such as the association between smoking and lung cancer.

Following the discovery of cancer-associated genes, including tumor-suppressor genes and proto-oncogenes, research into the genetic basis of cancer focused mainly on mutant alleles of genes involved in several cellular functions, including the cell cycle, differentiation, and apoptosis.

Epigenetics and Cancer

Converging lines of evidence are clarifying the role that epigenetic changes play in the initiation and maintenance of malignancy.

The relationship between epigenetics and cancer was

first noted in the 1980s by Feinberg and Vogelstein who observed that colon cancer cells had much lower levels of methylation than normal cells derived from the same tissue.

Subsequent research by many investigators showed that global hypomethylation is a property of all cancers examined to date.

In the ensuing years, it has become clear that the epigenetic states of normal cells are greatly altered in cancer cells and that other epigenetic changes, including selective hypermethylation and gene silencing, are also present in cancer cells. Cancer is now being viewed as a disease that involves both epigenetic and genetic changes that lead to alterations in gene expression (ST Figure 3–6).

DNA hypomethylation reverses the inactivation of genes, leading to unrestricted transcription of many gene sets including oncogenes.

Hypomethylation of repetitive DNA sequences in heterochromatic regions is associated with an increase in chromosome rearrangements and changes in chromosome number, both of which are characteristic of cancer cells.

In addition, hypomethylation of repetitive sequences leads to transcriptional activation of transposable DNA sequences such as LINEs and SINEs, further increasing genomic instability.

While widespread hypomethylation is a hallmark of cancer cells, hypermethylation at CpG islands and inactivation of certain genes, including tumor-suppressor genes (ST Table 3.2), are also found in many cancers, often in a tumor-specific pattern.

For example BRCA1 is hypermethylated and inactivated in breast and ovarian cancer, and MLH1 is hypermethylated in some forms of colon cancer.

Inactivation of tumor-suppressor genes by hypermethylation is thought to play an important complementary role to mutational changes that accompany the transformation of normal cells into malignant cells.

For example, in a bladder cancer cell line, one allele of

the cell cycle control gene CDKN2A is mutated, and the other, normal allele is inactivated by hypermethylation of its CpG island.

The inactivation of both alleles allows these cells to

escape control of the cell cycle and divide continuously. In many clinical cases, the combination of mutation

and hypermethylation occurs in familial forms of cancer.

However, genes other than tumor-suppressor genes are also hypermethylated in some cancer cells; these include genes that control or participate in DNA repair, differentiation, apoptosis, and drug resistance.

In addition to altered patterns of methylation, many cancer cells also have disrupted histone modification profiles. In some cases, mutations in the genes encoding members of the histone-modifying proteins histone acetyltransferase (HAT) and histone deacetylase (HDAC) are linked to the development of cancer.

initiating epigenetic changes leading to cancer may occur in stem cells residing in normal tissue. Three lines of evidence support this idea. First, epigenetic mechanisms can replace mutations as a way of silencing individual tumor-suppressor genes or activating oncogenes. Second, global hypomethylation may cause genomic instability and the large-scale chromosomal changes that are a characteristic feature of cancer. Third, because epigenetic modifications can silence multiple genes, they are more effective than serial mutations of single genes in transforming normal cells into malignant cells.

A model of cancer based on epigenetic changes in colon stem cells as initiating events in carcinogenesis followed by mutational events is shown in ST Figure 3–7

Environmental agents including nutrition, chemicals, and physical factors can alter gene expression by affecting the epigenetic state of the genome.

In mice, coat color is controlled by the dominant allele Agouti (A). In homozygous AA mice, the allele is active only during a specific time during hair development, producing a yellow band on an otherwise black hair shaft, resulting in the agouti phenotype.

A nonlethal mutant allele (Avy) causes yellow pigment formation along the entire hair shaft, producing a yellow fur color. This allele is the result of the insertion of a transposable element near the transcription start site of the Agouti gene.

Epigenetics and the Environment

A promoter element within the transposon is responsible for this change in gene expression.

The degree of methylation in the transposon’s promoter is

related to the amount of yellow pigment deposited in the hair shaft and varies from individual to individual.

The result is a wide variation in coat color in genetically identical mice (ST Figure 3–8), ranging from yellow (unmethylated) to pseudoagouti (highly methylated).

In addition to a gradation in coat color, there is also a gradation in body weight. Yellow mice are more obese than the brown, pseudoagouti mice.

Epigenetics and the Environment

To evaluate the role of environmental factors in modifying the epigenome, the diet of pregnant mice was supplemented with methylation precursors, including folic acid, vitamin B12, and choline.

In the offspring, variation in coat color was reduced and shifted toward the pseudoagouti phenotype.

The shift in coat color was accompanied by increased methylation of the transposon’s promoter.

Epigenetics and the Environment

Example 1: These two genetically identical mice were

born of genetically identical mothers who were fed

differently in pregnancy and they will have very

different lives

Their identical mothers were fed different amounts of

methylating nutrients or soy genistein during pregnancy

Yellow Mouse Agouti Mouse

High risk cancer, diabetes, obesity & reduced lifespan

Lower risk of cancer, diabetes, obesity and prolonged life

LTR Hypomethylated

Transposon sequence LTR Hypermethylated

Transposon sequence Maternal Supplements

With Genistein

zinc methionine

betaine choline, folate

B12

Epigenetics Occurs

Increasing Methylation Change in coat color

Change to lower lifetime weight Change to improved lifetime health

Increasing soy supplement genistein alters gene expression and thus phenotype

PNAS November 14, 2006 Vol. 103 no. 46 17-71-17072