Chapter 12: Epigenetic Mechanisms of Gene Regulation

epigenetics

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

Where do epigenetic changes come from?

modifications from the DNA and protein components of chromatin

chromatin marks

DNA sequence-specific interactions of proteins that recruit modifying enzymes to specific targets

What is a covalent modification of DNA?

cytosine DNA methylation

How is a methyl group transfered?

transferred from SAMe to the C-5 position of cytosine by a family of cytosine DNA methyltransferases

Where are most methyl groups found in eurkaryotes?

CpG dinucleotides

Where does cytosine methylation occur in plant DNA?

in CG or CNG, N is any base

What contains little to no 5-methyl cytosine?

C. elegans, drosophilia and yeast

What happens to the double helix after replication?

it is hemimethylated

What does a maintenance DNA methyltransferase recognize?

hemimethlyated sites

What does a maintenance DNA methyltransferase methylate?

the new strand of DNA

What does DNA methylation mark for?

gene silencing

hypermethylated genes

inactive

hypomethylated genes

active

How can you tell is DNA methylation correlates with gene activity or repression?

treat cells in culture with 5-aza-cytosine

Where are CpG islands found?

near gene promoters

CpG islands

small regions of DNA that are CG rich but normally unmethylated

What percent of promoters are housekeeping genes?

40-50%

When were CpG islands first detected?

by their sensitivites to the restriction endonuclease HpaII

HpaII

cuts only unmethylated CG regions

What histone modifications are important for transcriptional regulation?

acetylation and methylation

What are characteristics of inactive genes?

histone hypoacetylation and hypermethylation

epigenomics

the study of the genome wide pattern of epigenetic markers

What has epigenomics led to?

insights into differences in gene expression between normal and diseased cells

How many alleles do cell normally have?

two copies of autosomal genes

Are both alleles expressed?

for most genes

monoallelic expression

a single allele in a cell is expressed

What happens in most cases of monoallelic expression?

cells randomly select only one allele to encode RNA and protein

genomic imprinting

the selection of the allele is nonrandom and based on the parent of origin

What is the chromosome and selection of allele in imprinted genes?

autosomal and non random

What is the chromosome and selection of allele in x-inactivated genes?

x and random

Where are epigenetic imprints laid down in?

parental germ cell

Where does imprinting occur?

in mammals

How many different genes are known to be inprinted?

80

What is imprinting important for?

roles in development

How are imprinted genes organized?

in clusters

Prader-Willi syndrome

when the paternal alleles that would normally be expressed are missing

Angelman syndrome

when the maternal alleles that would normally be expressed are missing

What types of mechanisms can lead to missing alleles in PWS and AS?

• de novo deletion

• uniparental disomy

• ICR mutations

• mutation of UBE3A, 24%

What is the purpose of DNA methylation testing by Southern blot analysis?

to determine whether specific DNA regions are methylated by analyzing restriction enzyme digestion patterns

Which enzymes are used in DNA methylation testing by Southern blot analysis?

HindIII and HpaII

In DNA methylation testing by Southern blot, what region of the genome is analyzed?

a very small region of chromosome 15

What does the UBE3A gene encode?

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

In most tissues, from which alleles is UBE3A expressed?

from both alleles

In which specific brain regions is UBE3A maternally expressed?

neurons of the hippocampus and cerebellum

What kind of genes are paternally expressed in PWS and AS?

four protein-coding genes and several snoRNA genes

UBE3A antisense RNA

controls expression of the paternal UBE3A allele

Where does x chromosome inactivation take place in marsupials?

around the 2-4 cell stage and remains inactive

In placental mammals, what happens to the paternal X chromosome?

it erases the cells of the blastocyst

x chromosome inactivation in placental mammals

random inactivation of either paternal or maternal X chromosome

random inactivation of one X chromosome

coating of the chromosome by the untranslated XIST transcript

How is the XIST expression repressed?

by the antisense transcript Tsix on the active X

How is the XIST expression upregulated?

by the transcript Jpx on the inactive X

What are the types of recruitment of chromatin modifying complexes to the inactive X?

• histone H3 modification

• histone H4 hypoacetylation

• enrichment of variant histone macroH2A

• DNA methylation

transposable elements

has the ability to integrate into the genome at a new site within their cell of origin

Where are transposable elements abundant in?

the genomes of bacteria, plants, and animals

What is the amount of transposable elements in mammals?

nearly half the genome

What is the amount of transposable elements in some higher plants?

90%

What does transposition disrupt?

genetic function and result in phenotypic variation

What is caused in vertebrates and higher plants by transposable elements?

a low percentage of spontaneous mutations

What effects a species through potential genome modification?

a balance between detrimental effects and long-term beneficial effects

What is the primary function of eukaryotic DNA methylation?

defense of the genome from transposition of transposable elements

DNA transposons

DNA intermediate during transposition

retrotransposons

RNA intermediate during transposition

Where are DNA transposons found in?

bacteria, drosophilia, maize, and humans

How can DNA transposons switch hosts?

lateral transfer of DNA from organism to organism

What genes do some bacterial transposons contain?

antibiotic resistance genes

What do DNA transposons consist of?

a transposase gene flanked by inverted terminal repeats that bind the transposase and mediate transposition

transposase enzyme

has a catalytic domain and a DNA-binding domain

What do transposable elements provide material for?

DNA mispairing and unequal crossing-over

What are transposable elements potential causal agents of?

human disease through insertional mutagenesis

What are some possible effects of transposable elements?

• disrupt a gene-coding sequence

• disrupt splicing

• influence gene expression if insertion near promoter/enhancer elements

• contain promoters that initiate transcription of adjacent genes

• susceptible to epigenetic silencing

monoallelic gene expression

one allele of a gene is selected for expression

What is monoallelic gene expression important for?

cell differentiation

What is one major mechanism that mediates allelic exclusion?

programmed gene rearrangements

What are examples of allelic exclusion?

• yeast mating-type switching and silencing

• antigen switching in trypanosomes

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

What defines the two yeast mating types, a and α?

the expression of one of two gene cassettes

gene conversion

DNA rearrangement by homologous recombination

What determines which gene cassette is activated during yeast mating-type switching?

directionality of switching

How are silent cassettes repressed?

through epigenetic mechanisms

When does mating type switching occur?

when the HO endonuclease is expressed and the active cassette is replaced by information from a silent cassette by gene conversion

How is selective expression achieved?

the chromatin state of HMRa, HMLa, and MAT

What fatal disease does African trypanosomes cause?

sleeping sickness in humans and N'gana in cattle

What is the key to success for antigen switching in trypanosomes?

evasion of the immune system

What helps with antigen switching in trypanosomes?

periodic switching of the variant surface glycoprotein coat

How are VSGs anchored?

to the membrane by glycosyl phosphatidylinosital anchor

GPI

a complex sugar with a fatty acid myristate chain

characteristics of VSG genes

• over 1000 different genes

• only one gene is expressed at a time

• 20 possible telomeric expression sites

What are the two main branches of the immune system in vertebrates?

the innate and adaptive immune system

humoral (blood-borne) response

B cells

cell based response

T cells

How are foreign antigens recognized? 

by B and T cells by a repertoire of antigen-specific receptors

How is the diversity of antigen receptors created?

somatic rearrangement of small number of V, D, and J gene segments

immunoglobulin protein

composed of two identical light and heavy chains

What do each chain consist of?

a constant region and a variable regions

What do immunoglobin genes exist as in germline cells?

linear arrays of V, diversity (D), and joining (J) regions upstream of the C region

What do a series of site specific recombination events in B cells generate?

unique combination of V(D)J sequences that encode unique antigen receptors