Chapter 12:

Epigenetic Mechanisms of Gene Regulation

When you know you’re right, you don’t care what others think. You know sooner or later it will come out in the wash.

Barbara McClintock, quoted in Claudia Wallis, “Honoring a modern Mendel,” Time (1983), October: pp 43-44.

12.1 Introduction

• Epigenetics is the study of heritable changes in gene expression that occur without a change in the primary DNA sequence.

• Epigenetic changes arise from modifications of both the DNA and protein components of chromatin.

• Chromatin marks result from DNA sequence-specific interactions of proteins that recruit modifying enzymes to specific targets.

12.2 Epigenetic markers

Cytosine DNA methylation marks genes for silencing

• Cytosine DNA methylation is a covalent modification of DNA.

• A methyl group is transferred from S-adenosylmethionine to the carbon-5 position of cytosine by a family of cytosine DNA methytransferases.

• In eukaryotes, most methyl groups are found in “CpG” dinucleotides.

• In plant DNA, cytosine methylation occurs at either CG or CNG, where N can be any base.

• Note that C. elegans, Drosophila, and yeast contain little or no 5-methyl cytosine.

Methylation is maintained during DNA replication by a semiconservative process

• After replication, the DNA double helix is “hemimethylated.”

• A maintenance DNA methyltransferase recognizes only hemimethylated sites and methylates the new strand of DNA appropriately.

DNA methylation marks genes for silencing

• A general (but not a universal) rule.

• Hypermethylated genes are inactive.

• Hypomethylated genes are active.

• One way to demonstrate whether DNA methylation correlates with gene activity or repression is to treat cells in culture with 5-aza-cytosine.

Treatment of pancreatic cancer cells with 5-aza-deoxycytidine

• BNIP3 gene expression is silenced in some pancreatic cancer cells by methylation of the promoter region.

• After treatment, BNIP3 gene expression was restored and induced hypoxia-mediated cell death.

Cancer and epigenetics

• Aberrant DNA methylation patterns and aberrant histone modifications are found in human cancer cells.

• Hypomethylation of DNA may stimulate oncogene expression.

• Hypermethylation of DNA may silence tumor suppressor genes.

• Histone H4 “cancer signature”: loss of acetylation of lysine 16 and trimethylation at lysine 20.

Poor nutrition predisposes cells of an organism to cancer

• S-adenosylmethionine, a derivative of folic acid, is the primary methyl donor in the cell.

• A lack of folic acid leads to loss of methylation.

CpG islands are found near gene promoters

• Small regions of DNA (1-2 kb in size) that are CG-rich but normally unmethylated.

• Associated with the promoters of ~40-50% of “housekeeping genes.”

• CpG islands were first detected by their sensitivities to the restriction endonuclease HpaII which cuts only unmethylated CG regions.

• CpG islands are protected from spontaneous deamination.

• Unlike cytosine, 5-methyl cytosine is highly susceptible to spontaneous C→T deamination.

• The mammalian genome has become progressively depleted of CG dinucleotides through deamination.

Stable maintenance of histone modifications

• Histone modifications such as acetylation and methylation are important in transcriptional regulation.

• Histone hypoacetylation and hypermethylation are characteristic of inactive genes.

• Epigenomics―the study of the genome-wide pattern of epigenetic markers―has led to insights into differences in gene expression between normal and diseased cells.

Fragile X mental retardation and aberrant DNA methylation

• Patients have a trinucleotide repeat expansion (CGG) within the 5′-untranslated region of the FMR1 gene located on the X-chromosome.

Consequences of trinucleotide repeat expansion

• If the expansion exceeds 200 CGG repeats, affected individuals will exhibit disease symptoms.

• Hypermethylation of DNA and hypoacetylation of histones in the promoter region of the FMR1 gene.

• Leads to a loss of FMR1 gene expression.

• The FRM1 gene encodes the fragile X mental retardation protein (FMRP).

• FMRP is a cytoplasmic RNA-binding protein that is involved in neuronal maturation and/or development.

Diagnostic tests for fragile X

Cytogenetic analysis

• Most patients have a fragile piece hanging off one end of the X chromosome.

Direct DNA analysis to determine the length of the CGG repeat

• PCR• Southern blot

12.3 Genomic imprinting

• Cells normally have two alleles (copies) of autosomal genes.

• For most genes, both alleles are expressed.

• A small class of genes shows monoallelic expression, where a single allele in a cell is preferentially expressed.

• In most cases of monoallelic gene expression, cells randomly select only one allele to encode RNA and protein for that gene.

• An exception is genomic imprinting, where selection of the active allele is nonrandom and based on the parent of origin.

Genomic imprinting

• A gene is expressed from only one of the two parental chromosomes.

• Epigenetic imprints are laid down in the parental germ cells.

• Differential methylation of imprinting control regions (ICRs).

• Imprinting occurs in mammals, but no other vertebrates looked at so far.

• ~80 different genes currently known to be imprinted.

• Important roles in development.

• Imprinted genes are organized in clusters.

Genomic imprinting also occurs in flowering plants

• Different mechanism than in mammals.

• Removal of methylation marks from one of the parental alleles.

• Not inherited: confined to endosperm.

Genomic imprinting and neurodevelopmental disorders

• Three disorders that are the result of either direct or indirect deregulation of imprinted genes.

– Prader-Willi syndrome

– Angelman syndrome

– Rett syndrome

Defects in genomic imprinting lead to Prader-Willi syndrome (PWS) and

Angelman syndrome (AS)

• Prader-Willi syndrome occurs when the paternal allele(s) that would normally be expressed are “missing.”

• Angelman syndrome occurs when the maternal alleles that would normally be expressed are “missing.”

Several mechanisms leading to “missing alleles” in PWS and AS:

• De novo deletion.• Uniparental disomy.• ICR mutations.• In 24% of cases, AS results from classic gene

mutation of UBE3A.

Clinical diagnosis of Prader-Willi syndrome (PWS) and Angelman syndrome (AS)

• DNA methylation testing by Southern blot analysis

– Extract genomic DNA.– Digest with HindIII and HpaII (only cuts

nonmethylated restriction sites).– Analyze a very small region of chromosome

15 by Southern blot.

• Bisulfite-PCR method

– Distinguishes normal cytosine from 5-methyl cytosine.

Paternally and maternally expressed genes present at the imprinted locus

• UBE3A encodes an E6-AP ubiquitin-protein ligase involved in the ubiquitin-mediated protein degradation pathway.

• Expressed from both alleles in most tissues.

• Maternally expressed in neurons present in the hippocampus and cerebellum.

• Four protein-coding genes and several small nucleolar RNA (snoRNA) genes that are paternally expressed.

• Contribution of most to Prader-Willi syndrome remains unclear.

• UBE3A antisense RNA controls expression of the paternal UBE3A allele.

Mutations in the MeCP2 gene cause Rett syndrome

• Gene encodes methyl-CpG-binding protein 2.

• MeCP2 is part of a corepressor complex that includes a histone deacetylase.

• Causes changes in the patterns of histone acetylation and methylation in the PWS/AS ICR.

• Leads to an increase in UBE3A antisense RNA levels and reduced expression of UBE3A protein in the brain.

Establishing and maintaining the imprint

• Imprinting is reset in the germline by erasure of the DNA methylation marks in the primordial germ cells.

• Involves an active demethylation process by yet unknown enzymatic activities.

• The de novo methyltransferase DNMT3a and cofactor DNMT3L are required to re-establish imprinting.

• Exactly how the differential imprints in sperm and oocytes are established is not fully understood.

• Binding of other protein factors to ICRs could prevent methylation in either sperm or egg.

• Testis-specific factor BORIS binds to the same DNA sequences as CTCF, a protein that functions in maintaining and reading imprint marks.

Mechanisms of monoallelic gene expression

• DNA methylation and associated differences in chromatin are “read” after fertilization to ensure that the correct allele is expressed.

Three main mechanisms of monoallelic gene expression

• Altered chromatin structure in the gene promoter.

• Differential expression of an antisense RNA transcript.

• Blocking of an enhancer by an insulator.

Altered chromatin structure in the gene promoter

• Imprinting region at the SNURF-SNRPN gene of the Prader-Willi syndrome locus.

• The ICR is unmethylated on the paternal chromosome.

• Directs expression of all imprinted genes in this region, including the UBE3A antisense gene.

• The ICR is methylated on the maternal chromosome.

• In the absence of antisense UBE3A expression, the maternal UBE3A allele is expressed.

Differential expression of an antisense RNA transcript

• Insulin-like growth factor 2 receptor (Igf2r) locus

• The ICR contains the promoter of the Air gene, which produces an antisense RNA.

• Air silences the Igf2r paternal allele.

• In the absence of Air, Igf2r is expressed exclusively from the maternal allele.

Maternal ICR• DNA is methylated.• Histone H3 methylation at lysine 9 (H3-K9).• Air is silenced.

Paternal ICR• DNA is unmethylated.• Histone H3 acetylation.• Histone H3 methylation at lysine 4 (H3-K4).• Air is expressed.

Blocking of an enhancer by an insulator

• Insulin-like growth factor (Igf2)-H19 locus.

• The maternal ICR is protected from methylation by CTCF binding.

• Creates a chromatin boundary that prevents interaction of the Igf2 gene promoter with enhancers located downstream of H19.

• H19 is maternally expressed.

• The paternal lCR is methylated.

• This prevents binding of CTCF.

• The Igf2 promoter can interact with the enhancers.

• H19 is repressed because ICR methylation spreads into its nearby promoter.

• Igf2 is paternally expressed.

Genomic imprinting is essential for normal development

• Beckwith-Wiedemann Syndrome

– Biallelic expression of Igf2. – Abdominal wall defects, enlarged tongue– Various types of malignant tumors.

• Many common cancers

– Loss of imprinting of Igf2.– Inhibits apoptosis– Promotes cell proliferation.

Origins of genomic imprinting

• “Conflict hypothesis”

• Imprinting arose early in mammalian evolution.

• It is to the male’s benefit to silence genes that conserve maternal resources at the expense of the fetus.

• It is to the female’s benefit to silence genes that allocate resources to the fetus at the expense of the mother.

Support for conflict hypothesis:

• So far, imprinted genes have not been found in monotreme (egg-laying) mammals.

• Some imprinted genes do affect the allocation of resources between mother and fetus.

But…

• Many genes that are imprinted have no obvious connection to the maternal-fetal conflict.

12.4 X chromosome inactivation

• Dosage compensation or the “Lyon hypothesis”

• 1961: Mary Lyon studied coat color variegation in mice.

• Concluded that one of the two X chromosomes must be randomly inactivated in each cell of female mice.

Different organisms deal with dosage compensation in different ways

• Drosophila: genes on the male X chromosome are expressed at twice the level of the female X.

• C. elegans: expression of genes on the female X chromosome is reduced by half compared with the male X.

Random X chromosome inactivation in mammals

Marsupials• The paternal X chromosome undergoes

preferential inactivation around the 2-4 cell stage and remains inactive in all tissues.

Placental mammals• The paternal mark is erased in cells of the

blastocyst.• Random inactivation of either the paternal or

maternal X chromosome.

Molecular mechanisms for stable maintenance of X chromosome inactivation

• Random inactivation of one X chromosome is brought about by “coating” of the chromosome by the untranslated XIST transcript.

• XIST expression is repressed by the antisense transcript Tsix on the active X.

• XIST expression is upregulated by the antisense transcript Jpx on the inactive X.

• Recruitment of chromatin modifying complexes to the inactive X

– Histone H3 methylation.– Histone H4 hypoacetylation.– Enrichment of variant histone macroH2A.– DNA methylation.

Is there monoallelic expression of all X-linked genes?

• March 2005: Change to 40 years of dogma

• Experiments showed that about 15% of genes escape inactivation.

• In an additional 10% of genes, the level of expression differs from woman to woman.

• What are implications of these findings?

12.5 Epigenetic control of transposable elements

• Transposable elements are DNA sequences that have the ability to integrate into the genome at a new site within their cell of origin.

• Abundant in the genomes of bacteria, plants, and animals.

– Mammals: nearly half the genome– Some higher plants: ~90%

• Transposition (movement) may disrupt genetic function and result in phenotypic variation.

• In vertebrates and higher plants, only a low percentage of spontaneous mutations are caused by transposable elements.

• Balance between detrimental effects on an individual and long-term beneficial effects on a species through potential for genome modification.

• The primary function of eukaryotic DNA methylation may be defense of the genome from transposition of transposable elements.

Barbara McClintock’s discovery of mobile genetic elements in maize

• 1940s to 1950s: While studying chromosome breakage events in maize, McClintock noticed a high level of phenotypic variegation.

• 1983: Novel prize for the discovery of mobile genetic elements.

• Dissociation (Ds) and activator (Ac) elements.

• Variegated phenotype due to the interplay between the transposable element and a gene that encodes an enzyme involved in anthocyanin production.

Mendel’s wrinkled peas

• Transposable element in the starch-branching enzyme 1 (SBE1) gene.

Two main classes of transposable elements

• DNA transposons: DNA intermediate during transposition.

• Retrotransposons: RNA intermediate during transposition.

DNA transposons have a wide host range

• Found in many organisms, including bacteria, Drosophila, maize, and humans.

• May switch hosts by lateral (horizontal) transfer of DNA from organism to organism as opposed to inheritance of genes by vertical descent from one’s parents.

• Some bacterial transposons contain antibiotic-resistance genes.

DNA transposons move by a “cut and paste” mechanism

• DNA transposons consist of a transposase gene flanked by inverted terminal repeats that bind transposase and mediate transposition.

• The transposase enzyme has a catalytic domain and a DNA-binding domain.

• Nonautonomous transposons do not encode transposase.

• They consist of a pair of inverted repeats that function as transposase-binding sites.

• e.g. Ds element in maize.

Jumping genes and human disease

• Transposable elements provide material for DNA mispairing and unequal crossing-over and are potential causal agents of human disease through insertional mutagenesis.

Some possible effects of transposable elements

• Disrupt a gene-coding sequence.• Disrupt splicing.• Influence gene expression if the insertion is in or

near promoter/enhancer elements.• Contain promoters that initiate transcription of

adjacent genes.• Susceptible to epigenetic silencing which then

spreads to neighboring chromatin.

Apert’s syndrome

• Craniofacial abnormalities and syndactyly of the hands and feet.

• Usually results from a missense mutation in exon 7 of the fibroblast growth factor receptor II gene.

• In two cases, insertion of an Alu element in or near exon 9 as shown by PCR analysis.

Retrotransposons move by a “copy and paste” mechanism

• Remnant of ancestral retroviral infections.

• Two main groups:

– Long terminal repeat (LTR) retrotransposons

– Non-LTR retrotransposons

• Autonomous retrotransposons encode an endonuclease and a reverse transcriptase.

• First transcribed into RNA and then reverse transcribed into a cDNA and integrated into the genome.

Some LTR retrotransposons are active in the mammalian genome

• There are three distinct types of LTR retrotransposons in mouse and human.

• Nearly all are inactive in humans but active in mouse.

• The LTR-containing intracisternal A particles (IAPs) account for approximately 15% of disease-producing mutations in mouse.

Non-LTR retrotransposons include LINEs and SINEs

• Long interspersed nuclear elements (LINEs) are autonomous retrotransposons.

• Short interspersed nuclear elements (SINEs) are nonautonomous retrotransposons.

LINEs are widespread in the human genome

• Comprise 21% of the human genome.

• Active members are called “L1 elements.”– Role in X-inactivation?– Long distance modifiers of chromatin?– Material for DNA mispairing and homologous

recombination.

• The frequency of L1 transposition is estimated to be one insertion in every 2-30 individuals.

Alu elements are active SINEs

• Comprise 11% of the human genome.

• Do not encode the enzymes for transposition.

• Transposition may be mediated by L1 element reverse transcriptase.

• Alu insertions account for over 20 cases of human genetic diseases.

At least two epigenetic control methods are known to silence transposition:

• Methylation of transposable elements.

• Heterochromatin formation mediated by RNA interference (RNAi) and RNA-directed DNA methylation.

Methylation of transposable elements

• Cytosine methylation may contribute to silencing of transposable element transcriptional activity and transposition.

• Effect of transposable element methylation on plant pigmentation.

• Inactive Ac and Spm elements in maize were found to have methylated cytosines.

• Methylation of transposable elements leads to flower variegation in morning glories.

Heterochromatin formation mediated by RNAi and RNA-directed methylation

Two mechanisms for heterochromatin formation:

• Cis-acting DNA sequences called silencers are recognized by DNA-binding proteins and initiate heterochromatin formation.

• Heterochromatin formation mediated by RNAi and RNA-directed methylation.

• Transcripts generated by repetitive DNA sequences are processed into small heterochromatic RNAs siRNAs by the RNAi machinery.

• These siRNAs direct sequence-specific DNA methylation and formation of heterochromatin.

• Heterochromatin domains are inaccessible to DNA-binding factors and are transcriptionally silent.

• piRNAs in Drosophila are transcribed from heterochromatic loci in the germline.

• The piRNAs target a large number of transposons dispersed throughout the fruitfly genome.

12.6 Epigenetics and nutritional legacy

A diet lacking folic acid can activate a retrotransposon in mice

• Hair growth cycle-specific promoter in the agouti allele regulates transient switch from black pigment to yellow pigment during hair growth.

• Intracisternal A particle (IAP) insertion places agouti viable yellow (Avy) allele under control of the retrotransposon promoter.

• Constitutive expression of the agouti gene results in mice with completely yellow fur.

Paternal epigenetic effects

• Transgenerational effects in a population in Sweden.

• Limited food in paternal grandfathers was associated with a decreased risk of diabetes and cardiovascular disease in their grandchildren, but not in their children.

• Grandchildren had a higher mortality rate when their paternal grandfathers had an abundant food supply.

12.7 Allelic exclusion

Monoallelic gene expression

• One allele of a gene or gene “cassette” family is selected for expression.

• Important role in cell differentiation or diversity.

• One major mechanism mediating allelic exclusion is programmed gene rearrangements.

Three examples of allelic exclusion

• Yeast mating-type switching and silencing.

• Antigen switching in trypanosomes.

• V(D)J recombination and the adaptive immune response.

Yeast mating-type switching and silencing

• Two mating types, a and , defined by the expression of one of two gene cassettes.

• DNA rearrangement by homologous recombination (gene conversion).

• Directionality of switching.

• Silent cassettes are repressed through epigenetic mechanisms.

Homothallic life cycle of Saccharomyces cerevisiae

• A haploid yeast of mating-type a that has divided can switch to the opposite mating type.

• Opposite mating types are attached to each other by pheromones.

• The original cell and its switched partner can conjugate to form an a/ diploid cell.

• Meiosis and sporulation will regenerate haploid cells.

• Mating type switching is confined to cells that have previously divided (mother cells).

• Daughter cells must have budded and divided once before switching.

DNA rearrangement by homologous recombination (gene conversion)

• Mating type switching occurs when the HO endonuclease is expressed and the active “cassette” is replaced by information from a silent cassette by gene conversion.

• The selective expression of only one gene cassette is achieved by the chromatin state of the three mating type loci, HMRa, HML, and MAT.

Directionality of switching

• The homologous recombination enhancer (RE) regulates the directionality of switching.

• In MAT cells, the RE is turned off by -specific repressor proteins and recombination occurs preferentially with HMRa.

• In MATa cells, the RE is turned on by default and recombination occurs preferentially with HMR.

Silent cassettes are repressed by epigenetic mechanisms

• Silencers flank the silent mating loci.

• The silencers recruit specific regulatory proteins (Sir1-Sir 4 and Rap1), histone deacetylases, and heterochromatin assembly factors.

• Represses transcription, but not recombination, at the silent (donor) loci.

• Silent information regulatory (Sir) proteins are recruited both to the silencers and the telomeres.

• Assembly of the Sir complex at silencers of the silent mating loci and telomeres is proposed to occur in a stepwise fashion.

• Sir2 is an NAD+-dependent histone deacetylase.

• The enzymatic activity of Sir2 is required for association of Sir proteins with telomeric DNA regions and the HML mating-type locus.

• Sir2 plays an important role in aging.

Antigen switching in trypanosomes• African trypanosomes cause a fatal disease

called “sleeping sickness” in humans and “N’gana” in cattle.

• Key to success: evasion of the immune system

• Periodic switching of the variant surface glycoprotein (VSG) coat.

• In the lab: one in 102 to 107 switches per doubling time of 5-10 hours.

The trypanosome VSG coat

• A tight mesh of 1 X 107 identical molecules.

• The VSGs are anchored to the membrane by a glycosyl phosphatidylinosital (GPI) anchor.

• GPI is a complex sugar with a fatty acid myristate chain that may act as a “quick release” mechanism in vivo.

Trypanosomiasis: human “sleeping sickness”

Life cycle of African trypanosomes

• The African trypanosome spends part of its life cycle as a parasite in the blood of mammals and part of its life cycle in the tsetse fly host.

• Trypanosoma brucei is best investigated because it grows well in lab animals but is not infectious to the human researcher.

Symptoms of trypanosomiasis

1. Infection of blood vessels and lymph glands.

• Intermittent fever• Rash• Swelling• Complete fatigue

2. Invasion of the central nervous system.

• Inflammation of the brain outer membrane• Severe headaches• Sleep disorders• Poor coordination• Lethargy, coma, death

Treatment of trypanosomiasis

• 1865: disease nearly gone.

• 1970: new epidemic.

• 2006: At least 500,000 infected per year in sub-Saharan Africa; at least 100,000 reported deaths per year.

• No vaccine available.

• Current drugs are costly and toxic.

• Appropriate treatment protocols are hard to achieve.

The story of eflornithine

• How unwanted facial hair in women led to drug availability in Africa…

Characteristics of variant surface glycoprotein (VSG) genes

• Over 1000 different VSG genes.

• Only one gene is expressed at a time.

• 20 possible telomeric expression sites.

• Different expression sites express different sets of 12 expression site-associated genes (ESAGs).

• Cotranscribed with VSG genes as part of a long transcription unit

• Most encode surface proteins; 2 encode subunits of a transferin receptor.

VSG switching by homologous recombination

• Gene conversion is the most frequent mode of antigen switching, but switching can also occur by other mechanisms.

Mechanisms of antigen-switching in trypanosomes

• Gene conversion.• Point mutations during gene conversion.• Reciprocal recombination.• Formation of a chimeric gene and movement

into an active expression site by a series of recombination events.

• Switching of the active expression site.

Epigenetic regulation of active expression site

• Monoallelic VSG gene expression is maintained by an epigenetic control mechanism that silences all but one of the 20 possible telomere-linked expression sites (ES).

• Transcription is localized in a nuclear compartment called the expression site body where only one ES is allowed to enter at a time.

• VSG gene transcription is mediated by RNA polymerase I.

The modified base “J”

• A fraction of thymine is replaced by thymine with a bulky glucose moiety attached.

• J is present in the inactive telomeric VSG gene expression sites, but not in the active expression site.

• Exact role of J remains to be determined.

V(D)J recombination and the adaptive immune response

Two main branches of the immune system in vertebrates

• The innate immune response.

• The adaptive immune response.

The adaptive immune response

• Humoral (blood-borne) response: B cells

• Cell-based response: T cells

• Foreign antigens are recognized by B and T cells via a vast repertoire of antigen-specific receptors.

• The diversity of antigen receptors is created by somatic rearrangement of a small number of V, D, and J gene segments.

Immunoglobulin genes and antibody diversity

• The immunoglobulin (antibody) protein is composed of two identical heavy chains and two identical light chains.

• Each chain consists of a constant (C) region and a variable (V) regions.

Mapping of cloned germline and rearranged immunoglobulin gene segments

• Late 1970s: Evidence that the variable and constant portions of the light chain gene arose by novel DNA rearrangements.

• R-looping experiments by Susumu Tonegawa’s group.

• In germline cells, immunoglobulin genes exist as linear arrays of V, diversity (D) (only in heavy chains), and joining (J) regions upstream of the C region.

• A series of site-specific recombination events in B cells generate unique combinations of V(D)J sequences that encode unique antigen receptors.

Mechanism for V(D)J recombination

• V(D)J recombination is mediated by the RAG1/2 recombinase.

• A target recombination signal sequence (RSS) flanks each gene segment.

• After cleavage of both strands is complete, the segments join by a nonhomologous end-joining pathway.

Epigenetic regulation of monoallelic recombination and expression

• Each cell contains a paternal and maternal allele of the heavy chain locus and of each type of light chain locus.

• There is epigenetic control of the initial selection of the allele to be arranged, as well as maintenance of allelic exclusion.

Assembly of a functional heavy chain gene

• V(D)J recombination is a temporally ordered process.

• D-J recombination occurs on both alleles.

• V-D-J recombination takes place on only one allele, mediated by progressive histone hyperacetylation.

• A feedback signal inhibits further rearrangement of the second heavy chain allele.

• The recombinase complex is directed to the light chain locus.

Assembly of a functional light chain gene

• The two alleles are differentially methylated.

• One allele becomes early-replicating in the S phase and the other late-replicating.

• The early-replicating allele is usually the one that undergoes rearrangment.

• The late-replicating allele is moved to heterochromatic subdomains of the nucleus.

Did the V(D)J system evolve from a transposon?

• Many parallels between V(D)J recombination and transposition.

• Postulated that the V(D)J system might have evolved from a transposon.