epigenetics flash cards

includes- model systems and techniques, muscle development, chromatin?, gastrulation, micro RNA

what is Saccharomyces cerevisiae

bakers yeast

why is saccharomyces cerevisiae a good model organism

haploid genome- 14 × 10⁶ base pairs

16 chromosomes

short gestation time- 1.5-2 hours at 30^oC

yeast and mammalian neuro have similar cellular processes (although separated by millions of years of evolution)

example of saccharomyces cerevisiae

Study of chromatin control

• variegated- varying colours, phenotypes of ade-2-yeast strain

what is Drosophila melanogaster

fruit fly

describe features of a Drosophila melanogaster

haploid genome- 16.5 × 10⁷BP

8 diploid chromosomes

about 13500 genes

short life cycle of 2 weeks between generations

genetic, cytological, and biochemical analysis possible

systematic genetic screens

done of whole genome for mutations that affect the patterning of the early embryo

how are random mutations induced?

ethyl Methane Sulfonate

ethyl groups of EMS react with guanine in DNA, forming base O-6 methylguanine

EMS induced mutations are C -T point mutations

results in a C/G to T/A substitutions

variegating phenotype

expression of a gene is altered by its position on the chromosome

can be caused by- position-effect variegation, heterochromatin formation, and gene silencing

what is the result of genetic screens

identification of

• enhancer of variegation E[var]

• suppressors of variegation S[var]

(both connected with modulation of chromatin)

what is a Caenorhabditis elegans

small nematode

what are the features of Caenorhabditis elegans

haploid genome- 10 × 10⁷BP

6 chromosomes

about 19735 protein coding genes

short life cycle

1mm in size

grown in a petri dish

genetic, cytological, and biochemical analysis is possible

How many cells are in Caenorhabditis elegans

adult- 959 somatic cells

newly hatched larva- 558 cells

complete linage of every cell known

Discovery of RNA interface in animals - in Caenorhabditis elegans

1- E.coli, expressing double stranded RNA, eaten by worm

2- dsRNA injected into the gut

RNAi exploits naturally degrading mechanisms highly conserved in eukaryotes

introduction of double stranded RNA triggers sequence- specific destruction of complementary mRNAs

what are Danio rerio

Zebrafish

describe Danio rerio features

haploid genome- 1.5 ×10⁹BP

25 chromosmes

~26206 protein coding genes

life cycle of about 3 months

200 eggs/week

organs form within 24 hours

transparent embryos

adult 4-5 cm

what is a Gallus gallus

chicken

Features of the Gallus gallus

genome size- 1 × 10⁹BP

78 diploid chromosomes

~20 000 protein coding genes

huge eggs

what is whole mount in-situ hybridization

a technique used to view the location of nucleic acids in situ

What is the process of whole mount in-situ hybridization

RNA made complementary to specific mRNA → whole in cell membrane → digoxigenin label on uridine → RNA binds to mRNA of egg

alkaline phosphatase-conjugated antibody to digoxigenin- binds to RNA mRNA complex

colourless compound that becomes purple dye when phosphate is removed added to egg

what is Xenopus laevis

African claw-toed frog

describe features of Xenopus laevis

Haploid genome- 3.1 × 10⁹BP

36 tetraploid chromosomes

relative long life cycle- 1 year to reach sexual maturity

about 12cm in size

kept in a bucket of tap water

what are Xenopus laevis used for?

manipulation, experiments of early embryo

cytological and biochemical analysis

why are Xenopus laevis not used for genetic analysis

due to multiple sex chromosomes

what is a Mus musculus

House mouse

Features of a Mus musculus

haploid genome- 2.5 × 10 ⁹BP

40 diploid chromosomes

~ 22 000 protein coding genes

relatively short life cycle- takes about 8 weeks to reach sexual maturity

gestation period of about 3 weeks

very good for manipulation of genome and ES cells

mammal 80-90% protein hormonology shared with humans

suitable for genetic analysis

what is a chimeric

an organism containing a mixture of genetically different tissues

making a chimeric mouse with- ES cells

ES cells are pluripotent- can give rise to all embryonic tissues, including germ cells

ES are maintained and genetically manipulated in a petri dish

what are Organoids?

small, self organised, three-dimensional tissue cultures

culture systems contain self-renewing stem cell populations

the SC population can differentiate into multiple organ-specific cell types that exhibit spatial organisation similar to the corresponding organ

Optogenetics

use light to modulate events in a targeted manner in living cells or organisms

use genetically-encoded proteins that changed conformation in the presence of light

alters cell behaviour

what is a genomic library

a collection of subdivided portions of a larger genetic element, commonly created by the partial digestion of genomic DNA with restriction endonuclease and cloning the fragments into vectors

what is a cDNA library

a cloned DNA library

a collection of cloned DNA fragments synthesised from mRNA templates, representing only the expressed genes in a specific cell type or tissue at a particular time.

what are the principles of gene targeting

Homologous recombination

• the natural process of homologous recombination of the cell is used to exchange or merge DNA strands that share an identical or highly similar sequence

what are the types of target vector

insertion and replacement

insertion vectors

short fragments of DNA to be added into the hosts DNA sequence

Replacement vector

Larger DNA fragments that replace sections of the host DNA

what is site specific recombination

the cleavage of a target sequence and then ligate it to cleave DNA of a second site

what are specific recombination strategies

Cre-loxP causes recombination of locus cross over in p1

Lox-P sites are inserted on both sides of the target sequence via homologous recombination

The cre protein binds to the lox-P sites then circularizes and excise the intervening DNA fragment

How are chimeric mice generated

Interspecies cell combination

Mouse stem cells are combined with SC of a rat (with no pancreases)

this produces a rat with a mouse pancreas and beta cells

KO strategies

KO is a knockout

a technique used to study specific genes by inactivating them to see the effect on the organism

Describe the origin of muscle cells

epithelial cells from the somite’s undergo epithelial mesenchymal transformation (EMT)

remaining epithelial cells are the dermomyotome- Pax3 gene expressed from muscle precursors

Primary myotome is formed from future muscle cells that migrate around the dermomyotome and form a layer underneath

the cells then differentiate to express muscle cell specific markers

How do muscle cells develop

Primordial factor

myotome, determination→

dividing myoblasts, multiplication →

cell alignment, multiplication stops →

initial myotube formation, fusion and differentiation →

maturation of myotube, most remaining myoblasts fuse →

muscle fibre and stem cell, maturation → contractions begin

Muscle stem cells

Derived from somites

Self-renewal and differentiation capacity of satellite cells (form of stem cell), that when activated differentiate into myoblasts and fuse with each other to form myofibers

express transcription factors Pax7 and myf5

cells activated following injury

activity is similar to that seen in embryonic muscle development

what are somite’s

paired blocks of mesodermal tissue that give rise to vertebrae, skeletal muscle, and dermis in vertebrae embryos

limb muscles in chickens

Derived from somites, pax3 expressing myoblasts migrate from somites to limb buds

forelimbs come from somites 16-21 in chickens

hindlimbs from somites 26-33

formation of head muscles

NOT from somites (apart from tongue)

jaw, facial, extraocular

formed from unsegmented paraxial head mesoderm

shown using inter-species grafts and tracking cell fate

muscle development summary

trunk muscles derived from somites

within the somite they differentiate in the myotome

limb muscles from somites but migrate into limbs before differentiating

head muscles from unsegmented paraxial mesoderm

what signals control the development of embryonic muscle cells

MRF- myogen regulatory factors

myf5, MRF4, myogenin

expression of alpha globin

low up until 6 weeks prenatal where it makes around 50% of total globin production

expression of beta globin

gradual increase in expression up to 30 weeks prenatal, making up under 50% of total globin synthesis

expression of gamma globin

there is an increase to ~45% of total production at 12 weeks prenatal

it then gradually decreases to 0% at 36 weeks prenatal

expression of delta globin

expression starts at around 36 weeks prenatal, making up about 5% of total globin synthesis

expression of epsilon globin

starts at 30% at 0 weeks, then decreases to 0% by 12 weeks prenatal

expression of zeta globin

initially its starts at 30% production, then decreases to 0% by 12 weeks prenatal

Principles of genome organisation- transcription from a eukaryotic gene

activator protein binds to enhancer

mediator connects activator to transcription machinery

chromatin-remodelling complexes open up DNA

histone-modifying enzymes make DNA easier to read

general transcription factors assemble at promotor

RNA polymerase II begins transcription

what are enhancers

regulatory DNA sequence

Enhance or inhibit transcription associated genes

often located remotely from promotors

what are features of enhancers

enable correct spatio-temoral gene activation

determine cell-type specific expression

can function regardless of orientation, position, distance

contain TF binding sites

have both promotor and enhancer activity

key regulator- BCL11A

central hub that represses fetal y-globin and activates B-globin

key regulator- KLF1

activates BCL11A and other adult-stage genes

linked to chromatin remodelling

key regulator- MYB

supports erythroid development and helps maintain adult gene expression

key regulator GATA1

coordinates erythroid differentiation

interacts with enhancers

key regulator LRF

works with BCL11A to silence fetal genes

key regulator TF2/4

nuclear receptors fine tune switching

epigenetics and chromatin modifier

DNMT1- adds methyl group to fetal genes

lysine-specific histone demethylase- removes repressive marks to achieve adult genes

nucleosome remodelling deacetylase- compacts chromatin to suppress y-globin

microRNAs- post transcriptional regulators that influence BCL11A and developmental timing

enhancer-mediated regulation of genes- inactive enhancer

gene off

enhancer is present in the DNA but not accessible

no TFs binding

no looping occurs between enhancer and promotor

gene stays silent as a result

enhancer-mediated regulation of genes- active enhancer

TFs bind to enhancer

enhancer physically loops to contact promotor

this recruits co-activators and RNA polymerase

gene can bind as a result

chromatin states and histone marks- inaccessible chromatin

DNA tightly wrapped around nucleosomes

no TFs can bind

gene is off

chromatin states and histone marks- primed/inactive enhancer

marked by H3K4me1

chromatin is partially open

ready for activation but not active yet

chromatin states and histone marks- repressed/poised enhancer

has H3K4me3, a repressive mark

often found in developmentally regulated genes

poised- can activate it later

what is chromatin

a complex of DNA, histones, and non-histone proteins found in the nucleus of a eukaryotic cell- the material of which chromosomes are made

chromatin organisation

organised into open regions with active gene transcription and closed regions where genes are repressed

chromatin remodelling

active structure remodelled by chromatin remodelling complexes

alters structure of nucleosome

chromatin packing

DNA double helix

beads on string of nucleosomes

30 nm chromatin fibre of packed nucleosomes

section of chromosome in an extended form

condensed section if chromosomes

entire mitotic chromosome

what is a TAD

topologically associated Domains

segregation of chromatin into active and inactive domains

what a dynamic nucleus

what is polycomb

PcG family of proteins establish a silenced chromatin state

what is trithorax

trxG family of proteins propagate gene activity

what are body plan genes

regulatory genes that control the development of body parts in an animal

how do body plan genes work

spatially restricted DNA binding TFs control patterned expression of many genes

these direct regulators soon disappear while the patterned expression of the target is maintained

the TrxG and PcG group are responsible for maintaining expression in the ON and OFF states respectively

maintenance of body plan gene expression

Specific TF that establish specific expression patterns are only transiently present during development

what is the function of TrxG and PcG proteins

mechanisms of ATP- dependant chromatin remodelling

• nucleosome sliding

• histone exchange

• nucleosome eviction

• altered nucleosome structure

covalent modifications of histones

Histone tails

each core histone has an N-terminal tail that extrudes from the surface of the nucleosome

may help pack nucleosomes

subject to several forms of covalent modification

methods of histone modification

acetylation

methylation

phosphorylation

all are reversable

histone methylation

occurs at lysine residuals in histones H3 and H4

certain methylated lysine residuals are associated with activating transcription, other are involved in the repressive process

histone concepts

histone modification provide functional marks on the nucleosome

specific marks are recognised

there is cross-talk between marks

• combination of marks is important

histone modification plays a role in inheritance of patterns of gene expression

how packing of DNA can be inherited

during replication, heterochromatin provides a template for its own reassembly

DNA and chromatin replicates in self-templating manner

• leads to propagation of differential chromatin states for multiple generations

alpha- globin expression

low until 6 weeks prenatal

makes up 50% of globin synthesis

beta-globin expression

increase is gradual until 30 weeks prenatal

~45/50% of globin production

gamma-globin expression

increase to 45% at 12 weeks prenatal

gradual decrease to 0% at 36 weeks postnatal

delta-globin expression

starts at 36 weeks prenatal

makes up 5% globin synthesis

epsilon-globin expression

30% at 0 weeks

0% by 12 weeks prenatal

zeta-globin expression

30 % at 0 weeks

0% by 12 weeks prenatal

principles of genome organisation

activator protein binds to enhancer

mediator connects activator to the transcription machinery

chromatin-remodelling complexes open up the DNA

histone-modifying enzymes make DNA easier to read

general transcription factors assemble at the promotor

RNA polymerase II begins transcription

traits of enhancers

regulatory DNA sequence

enhance or inhibit transcription associated genes

located remotely from promotors

what is epigenetics

the structural adaptation of chromosomal regions so as to register, signal or perpetuate altered activity states

what does segmented mean

the serial repetition of parts along the body axis

what is gastrulation

process of a singled layered blastula reorganised into a multi-layered gastrula

ectoderm

outer surface, CNS, neural crest

mesoderm

dorsal (notochord), paraxial (bone tissue), intermediate (tubule of kidney), lateral (RBC), head (facial muscle)

endoderm

digestive tubes (stomach cells), pharynx (thyoid cells), respiratory tube

what are somites

balls of epithelial tissue that bud off from the unsegmented paraxial mesoderm

start forming at the anterior end from unsegmented mesoderm and added to at the posterior

fibroblast growth factors

control cell motility and elongation of the embryo

excess in posterior prevents segmentation- somites not formed