Transcript UBE3A - Wiley

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 sequencespecific 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 Sadenosylmethionine 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-azacytosine.
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 starchbranching 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 antibioticresistance 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 crossingover 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 telomerelinked 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 antigenspecific 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.