Genomic Programming Flashcards
Define euchromatin DNA
Open, active
Define heterochromatin DNA
Closed, inactive
What is the role of epigenetic regulation
enables us to read genetic information in different manners, enabling us to generate different cells of the body
Layers of the embryonic stem cell (ESC)
mesoderm (middle layer)
endoderm (internal layer)
ectoderm (external layer)
What is the Waddington epigenetic landscape
- cell begins at bottom of a single potential well
- as development proceeds, the single well splits into many more, representing possible differentiation states of the cell
What are nucleosomes
- fundamental subunit of chromatin
- composed of 8 histones (octomer)
- H3 and H4 form a trimer
- H2A and H2B form a histone octomer
- responsible for DNA compacting and organisation
What type of bonding binds DNA to nucleosome
hydrogen bonding -> the negatively charged phosphate backbone of the DNA binds strongly to the positively charged histones and therefore is tightly compacted due to the neutralisation of the negative charge
Role of H1 histone
binds linker DNA
How can we obtain individual nucleosomes
by treating chromatin with endonuclease micrococcal nuclease (MNase) -> cuts DNA duplex at junction between nucleosomes (linker DNA)
Describe events that take place when chromatin is digested with MNase
- DNA is cleaved into integral multiples of a unit length
- fractionation of DNA fragments by gel electrophoresis reveals ‘ladder’
Define histone octamer
complex of two copies of four histones (H3, H4, H2A, H2B)
Define linker DNA
non-nucleosomal DNA present between nucleosomes
Structure of nucleosome
- octomer
- H32-H42 tetramer (horseshoe shape)
- H2A H2B pairs fit in as two dimers each binding to opposite face of H3-H4 tetramer
- protein forms spool with superhelical path which corresponds to binding site for DNA
- twofold symmetry
- binds mostly to phosphodiester backbones
Define the globular core
histone fold domain of core histones that contribute to the central protein mass of the nucleosome
Role of the N-terminal and C-terminal* tails
- contain sites for covalent modification
- highly flexible
- not present in crystal structures
*for H2A and H2B
Covalent modifications of nucleosomes
- transient
- typically occur at the histone tails (lysines)
- methylation, acetylation, or phosphorylation (relatively small)
- can be modified at numerous sites
- ADP-ribosylation, ubiquitylation, sumoylation (larger)
Effect of lysine acetylation
- occurs on free epsilon amino group of lysine
- neutralises positive charge residing on NH3 form of the epsilon amino group; reduces electrostatic grip on DNA and exposes transcription factor binding sites
- makes protein bigger
Effect of methylation
- retains positive charge whether mono-, di- or trimethylated
- adds hydrophobic interaction
- recruits proteins involved in gene repression or by inhibiting the binding of transcription factor(s) to DNA
Location and effect of phosphorylation
- occurs on the hydroxyl group of serine and threonine
- introduces 2 negative charges
How can arginine residues be modified
- can be mono- or dimethylated
- removing positive charge on Arg weakens interaction with DNA, resulting in opening of chromatin (unstable state)
Name a noncovalent modification of the histone tail
proline izomerisation
- regulates lysine methylation and gene expression
Define the histone code
the hypothesis that combinations of specific modifications on specific histone residues act cooperatively to define chromatin function
Function of p53
- regulates expression of numerous chromatin-modifying enzymes
- regulates expression of several key rate-limiting enzymes of the cell metabolism (glycolysis,TCA, folate, methionine)
How does tissue regenerate following injury/damage
- totally or partially
- stem and progenitors cells around the damage site proliferate and trigger the genome programming process to regenerate damaged tissue
Factors that damage cells/tissues
- injuries
- malnutrition
- ischemia
- infection
- sun-exposure
- pH
- cold/heat
- pollutants
Excessive cell proliferation
cancer
low cell proliferation
ageing
Role of damaged cells in tissue regeneration
- induce the repair process and send signals to immune cells to protect from infection and eliminate damaged cells
- send signals (alarmins) to surrounding stem or progenitor cells to induce proliferation and differentiation
- all processes must occur simultaneously
Importance of epigenetic regulations
- enable stem cells and progenitor cells to re-enter into proliferation and differentiate to regenerate tissue
- enables them to read genetic information in different manners (plasticity/genome reprogramming)
Role of differentiated cells in tissue regeneration
can de-differentiate and become stem cells to proliferate and differentiate in the different cell types to regenerate tissue
A chromatosome is comprised of…
a nucleosome + H1 histone
How many times do the eight histone proteins wrap the DNA
1.65 times
Define solenoid
H1 histones aggregate causing 6 nucleosomes to coil together
Chromosome territories
- chromatin fibres form chromosome territories in the nucleus of a nondividing cell
- correlated with gene densities
- gene rich tend to be located toward the interior of the nucleus
How is the DNA/nucleosome interaction regulated
post-translational modifications (PTM) e.g. acetylation, methylation, phosphorylation; induced by cell signalling (receptors) and energy metabolism (glycolysis)
Function of enzyme Histone Acetyl-Transferase (HAT)
transfers Acetyl-CoA produced by glucolysis on lysine to regulate the strength of the binding of the histone to DNA and open chromatin to allow for gene expression
What regulates the expression of histone-modifying genes
cellular receptors and energy metabolism (glycolysis)
Effect of deacetylation of lysine
- regulates interaction with negatively charged phosphate on DNA
- HAT transfers Acetyl group of Acetyl-CoA to epsilon-amino group of lysines (+) present in N-terminal (tails) of histones. This is a reversible process by HDAC
Effect of lysine ubiquitilation
increase size of lysine (steric regulation) regulating interactions
Arginine (R) and Lysine (K)
- positively charged amino acid
- can be mono, di, or tri-methylated (Me) which increases size and hydrophobicity, affecting binding of histone to DNA
Serine (S) and Threonine (T)
- amino-acids
- can be phosphorylated (Ph) to repulse histone and DNA
Location of histone modifications
N-terminal (NH2) and C-terminal domains (COOH)
Effect of Arginine deimination/citrunillation
neutralises the positive charge of R, weakening the interaction with DNA and thus opening the chromatin
Methylation of positively charged AAs
regulates hydrophobic interaction with histones, incrementally weakening the interaction between histones and DNA
Expression of histone-modifying enzymes
- tissue-dependent manner
- open/close access to gene
Role of promoters
- recruit RNA Polymerase to DNA sequence
- contain specific DNA sequences such as response elements (TATA box) and transcription factors
What is the role of the TATA box
provides secure initial binding site for RNA Polymerase
What are enhancers
DNA sequences localised in non-coding regions and coding regions to increase the likelihood that transcription of a particular gene will occur
- cis-acting (attached to DNA)
- can be located up to 1 Mbp away from gene, upstream or downstream from promoter
What are silencers
made of DNA and bind TF to inhibit or abolish gene transcription, thus silencing the gene
Role of DNMT in DNA modification
Governs DNA methylation. 3a and 3b set the patterns, Dnmt1 maintains them
Role of demethylase (APOBEC, TETs) in DNA modification
- CG rich region of DNA (CpG islands)
- maintains DNA methylation homeostasis
The enzymatic activity of chromatin-modifying enzymes is dependent on…
- concentration of the metabolite to transfer to targeted histone/DNA
- concentration of target (histone/DNA)
- concentration of chromatin modifying enzyme itself
- allosteric regulator
Levels of chromatin-modifying enzyme regulation
- at gene and protein expression levels
- at enzymatic activity level
How can chromatin-modifying enzymes be regulated at the gene and protein expression level
transcription, splicing, protein translation, protein stability, subcellular localisation
How does energy metabolism regulate genome programming
modifications catalysed by histone-modifying enzymes and DNA-modifying enzymes are directly dependent on the concentration of their substrates produced by the energy metabolism
Metabolite products of the TCA cycle
- Acetyl-CoA
- ATP
- SAM
- NADH
- FADH
- ketolutarate
- UDP-GlcNAc
Products synthesised from the folate cycle and methionine cycle
amino acids, nucleic acids, S-Adensoyl Methionine (SAM) and glutathione
Role of glutathione
neutralise oxidative free radicals
Why is SAM so important?
unique donor of methyl in cells to methylate proteins/histones/DNA/RNA/lipids
Role of Acetyl-CoA
generates cell building blocks and energy (cellular wood)
Lipid biosynthesis results in …
- makes cell signalling molecules
- makes hormones
- makes phospholipids to generate cell membranes
- enables storage of energy as fat
Addition of Acetyl-CoA on histones
program genome (open/close gene)
(histone acetyl-transferase, HAT -> can be reversed by HDAC to decrease transcription)
Usually coupled with DNA methylation
Addition of Acetyl-CoA on transcription factors/splicing factors
regulate timing and gene expression level of the different open genes
Energy metabolism is responsible for regulating…
- programming of genome and gene expression via production of metabolites used by chromatin-modifying enzymes
- activities of non-histone proteins (TFs) as metabolites are used by kinases/phosphatases, ubiquitin transferase and also chromatin-modifying enzymes to induce PTMS of non-histone proteins that bind to open genes and induce expression
Describe the feedback loop between energy and genome expression
gene expression is directly regulated by energy metabolism and conversely energy metabolism is directly regulated by gene expression
Input factors altering metabolite homeostasis
- nutrition
- oxygen
- heat/cold
- infections
- obesity
Output factors altering metabolite homeostasis
- physical exercise
- sun exposure, pollution, tobacco and toxins
- infection
- injuries
How can sun exposure, pollution, tobacco and toxins alter metabolite homeostasis
- generation of free radicals
- lipid peroxidation of cellular membranes
- protein aggregation
- DNA breaks
- RNA breaks
