Eukaryotic Gene Control Flashcards
The difference between prokaryotes and eukaryots and their default settings.
- Prokaryotic genes are generally ‘ON’ by default, and have to be repressed to switch them off.
- Eukaryotic genes are ‘off’ by default and need activating factors to active transcription.
Short -term regulation in Eukaryotes (reversible)
- Regulatory events to quickly turn gene sets on or off in response to environmental changes.
- Proteins interact transiently with DNA control elements.
- Transient changes in chromatin structure.
Long-term regulation (semi-irreversible)
- Associated with cell determination, differentiation, and more generally, embryonic development.
- Permanent changes in chromatin conformation.
- DNA methylation
Activation of gene expression depends on:
- Chromatin conformation that is accessible.
- Control elements present on DNA
- Transcription factors
Control elements present on DNA
- Large variety: basal promoter, enhancers, silencers
2. Different control regions in combination with different TFs provide specificity.
Transcription factors (TFs)
- Are proteins that interact with DNA control elements.
- Interact with other TFs as well as cofactors
- Interacts with RNA polymerases
What is the role of the strong positive charge of histones.
It neutralizes negative charge on DNA, allows folding.
First order structure of chromatin structure in the presence of H1.
More tightly packaged
First order structure of chromatin structure in the absence of H1.
Less tightly packaged
How are nucleosomes structure and position can be altered by chromatin remodelling.
- Nucleosome is highly dynamic, DNA is constantly unwrapping and rewrapping itselt around the nucleosome..
- This is done by the protein complexes called chromatin remodelling complexes (CRCs)
- Local chromatin structure differes between regions of dna.
Chromatin remodelling complexes (CRCs)
- CRCs are complexes of proteins that bring about changes in chromatin compaction.
- CRCs can act as either activating or repressing complexes.
- CRCs disrupt DNA-histone interactions
Effects of chromatin remodelling on nucleosomes when nucleosomes completely disassembles.
Is lost from the DNA (nucleosome eviction)
What are the effects of chromatin remodelling on nucleosomes.
Causes chromatin to be more densely or more loosely packages.
Histone modifications (HMs)
- Histone proteins can be chemically modified after it has been translated.
- Modification occur mostly on N-terminal tails that potrude from the nucleosome .
- Modificataion occur mostly on the tails of histones H3 and H4
- Many different histones sites can be modified
Why does histone modification mostly occur at the N-terminal.
Tails are accessible to ezymes that lay down the marks, and removed these marks. Tails can also interact with regulatory proteins that recognise and bind to the tags.
The histone code
Modifications to N-terminal tails of histones can be written and read by the cell to influence gene expression.
How is Acetylation (Ac) of histons added to the histones
AC group is added to lysine (K) at histone tails. Can involve many Ks in each of the four core histones types.
How is methylation (me) of histons added to the histones
Targets lysine at N-terminal tail adding 1,2 or 3 methyl groups to lysine
What enzymes catalyse and remove acetylation from Histones.
Catalyse: histone acetyl-transferase (HAT)
Remove: histone deacetylase (HDAC)
Function of acetylation (Ac) of histones
Acetylation decrease nett positive charge, making the chromatin more open.
This indicates transcriptionally active DNA.
What enzymes catalyse methylation (me) of histones
Histone methylase (HMT)
Relationship between methylation and acetylation
methylation and acetylation are mutually exclusive (both cannot be present at same K residue)
Ubiquitination of histones
- ubiquitin is a small protein of 76aa
- Links only to histones H2A or H2B
- Only at a single K in each histone, some distance away from Me and Ac groups (decrease positive charge of the histone)
- May recruit HMT enzymes to promote H-me
Phosphorylation (P) of histones targets which amino acids
Serine
Threonine
Sumoylation of histones
- Small ubiquitin-related MOdifier (SUMO) protein may also be linked to lysine to modify the histone
- Sumoylation leads to recruitment of HDAC enzymes and hence deacetylation of histones
Core histones
- H2A
- H2B
- H3
- H4
(H1 not core)
What other amino acid does methylation affect other than lysine.
Arginine residues in the core histones. (This does not reduce positive charge of basic AA. )
Where does phosphorylation occur in the histones.
- Occurs on all core histones.
2. N-terminus, close to Ac and me-k residues.
What is the function of phosphorylation
P is negatively charged, it will neutralise the positive charge of histones.
10nm is what kind of packaging of chromatin
Transcriptionally active DNA is packaged into a first- order beads-on-a string structure involving nucleosomes.
What structure is most chromatin packed in ?
30nm
Example of histone modification that causes transition from 30nm to 10nm fibers.
- Positive region at N-tail of H4 interacts with negative region on H2A and H2B in adjacent nucleosomes, promotes closer packinging.
- Acetylation of H4 reduces positive charge of this region, promotes a more open chromatin structure.
What is the role of H1 in transformation in chromatin structure.
The central region of H1 interacts with linker DNA between nucleosomes and it seals two turns of DNA making it more compact chromatin.
What does it mean if a region of DNA is enriched with H1?
The region of DNA is not being transcribed.
How does H1 compact chromatin.
- H1 recruits Dnmt enzymes (methylates DNA) and inhibits recruitment of HTM enzymes (methylates histones) therefore DNA is methylated in these regions and chromatin is tightly packed.
What histone variant inhibits recruitment of H1 and opens chromatin structure.
H3.3
30nm fiber is further condensed by ?
Looping
Loops are attached to nuclear protein scaffold via?
Matrix-attachment regions (MARs) in DNA at base of each loop
What does the binding of Matrix-attachment regions (MARS) and transcription factor.
Maintain transcriptionally active loop structure
Does RNA move toward the loop of chromatin or does the chromatin move toward the RNA polymerase.
RNA polymerase does not migrate to the DNA in these accessible loops for transcription. Rather, the loop extend to transcription factories where there are many RNA pol 2 enzymes. Genes move, RNA polymerase remains clustered in distinct nuclear sites.
Locus-control regions(LCR)
Are DNA elements that regulate chromatin structure
LCRs function
- Controls chromatin structure of a large region of DNA.
- Responsible for tissue/ cell type specific activation of genes under its control.
- alteres chromatin structure to open conformation, genes are then poised for transcription.
LCR controls successive expression of Beta-goblin genes.
Beta - globin locus consists of several genes, each is expressed at a particular stage and in tissue-specific manner during embryonic development.
What does hemoglobin switching involve?
Interaction between chromatin, remodeling complexes, LCR, globin genes and different transcription factors.
Insulators flanking LCR
- Blocks the spread of chromatin-opening effects of LCR.
2. Prevents LCR from stimulating expression of adjacent genes
What do CTCF proteins bind to
Insulators
How does CTCF work.
- each insulator flanking Beta-globin locus binds to CTFC.
- The two CTCF proteins interact to create the beta-globin loop, for active transcription in red blood cells.
- Neighbouring loci remain in an inactive chromatin state.
Heterochromatin
Very tightly packed from of chromatin.
Euchromatin
30nm or 10nm DNA regions that will be transcribed in specific cell types.
What will happen with the loss of insulator.
This will cause inappropriate spread of heterochromatin, which will then prevent expression of genes.
Why do chromatin condense further during mitosis.
For replicated chromosome to pair up correctly.
Stages in compaction of chromatin from DNA double helix to metaphase chromosome.
- 2nm DNA double helix wrapped around histone octamer.
- 11nm nucleosomes are formed into three-dimensional zigzag structure via histones H1 and other DNA -binding proteins.
- 30nm fiber are anchored via radial loops to the nuclear matrix.
- 300nm radial loop domain are further compacted
- 700nm radial loop becomes a metaphase chromosome
Methylation of H3K4.
Positively affects gene expression
Acetylation H3K9
Positively affects gene expression
Methylation of H3K9
Negatively affects gene expression
What is epigenetics
Potentially heritable modification to DNA without any changes to the DNA sequence.
How does mitotically heritably of epigenetics states help to maintain cells identity.
Heritability ensures that daughter cells have the same set of epigenetic/ chromatin marks = long term-stable. Therefore same set of genes are expressed, same function.
What happens to cell identity if epigenetic were mitotically removed?
A different set of genes will be expressed with a different function.
Tissue homogeneity
Tissue being of the quality or state of being all the same or all of the same kind.
Tissue heterogeneity
Tissues that are in character or content.
When and where does epigenetic reprogramming occur.
- It occurs in germ cells and in very early development.
2. Also occurs at other periods during differentiation
Do epigenetic changes happen before or after a gene becomes active.
Before
Steps of epigenetic changes.
- Alters chromatin structure from 30nm to more open 10nm.
2. Invlove modifications to both genomic DNA and histones
What does epigenetic changes result in ?
It results in a epigenome which varies between cells and under different conditions.
What does the chomatin of active genes (actively being transcribed) look like.
- Less dense DNA conformation
- Associated with histone as 10nm beads-on-a-string structure.
- Nucleosomes are still present behind and in front of RNA polymerase while a gene is being transcribed.
- Nucleosomes can be displaced/ removed in promoter regions.
What does the chomatin of inactive genes (not being transcribed) look like.
30nm solenoid structure
What percent of cytosines in mammalian genomes are methylated .
2-7%
Dnase 1 hypersensitive sites
Nucleosome free promoter regions
Dnase 1 sensitive sites
10nm chromatin (beads on a string)
Dnase 1 insensitive sites
30nm or higher chromatin
Why do promoter regions have 10-20x more hypo-methylated CG dinucleotides compared to the rest of the genome.
For constant active transcription
Function of Dnmt1
Recognizes strands where only 1 C in the sequence is methylated and methylates the opossite C.
Why does the pattern of methylation stay the same from embryonic development
Dnmt does not recognize unmethlated Cs therefore after replication umethylated Cs will remain umethylated.
Which part of the nucleosome interact with other parts of the nucleosome and are are subjected to specific modifications.
Amino terminal tails
What do chromatin remodeling complexes ( CRC) do?
- Each CRC contains an ATPase component which actually hydrolyzes ATP to generate the energy to displace the nucleosome or modify its structure.
- Can promote the exchange of histone molecules .
Why is exchange of histones significant.
Variant forms of histones exist. Therefore different histones can have different effects on a strand of DNA.
What does the enzyme histone acetyltransferse (HATs)
Catalyzes the acetylation of histones
Which residues on the amino acid terminal chain does ubiquitin bind to.
- Amino acid 119 in H2A
2. Amino acid 120 in H2B
Function of H3 variant CENP-A
Organizing chromatin structure at the centromere.
Function of H2A variant, H2AX
Associated with regions where DNA damage needs to be repaired
Function of H2A variant, H2AZ and H3 variant H3.3
Localize to regions where genes are being acively transcribed and may play a role in producing a more open chromatin structure.
Convertion of 10nm to 30 nm is done how?
- A positively charged N-terminus of histone 4 interacts with a negativel charged n-terminus (attracting the to eachother .
- H1 histone
How does DNA methylation promote closed chromatin structure.
Methylation status of DNA influences which proteins are recruited to DNA, and so alters chromatin conformation.
What is a MeCP protein
- Proteins that have a DNA- binding domain and transcriptional repression domain.
- MeCP proteins can recruit other factors that condense the chromatin.
MeCP2 protein function
- MeCP2 binds only to methylated on DNA.
2. MeCP2 recruits history deacetylase (HDAC) that removes acetyl groups from histones.
Function of HDAC
Removes acetyl groups from histones
How do we study histone modification.
Chromatin immunoprecipitation (ChIP)
ChIP-PCR is used for ?
Assay a single locus
ChIP-seq used for?
Assay whole genome
Reader protein function
The signature on histone can be read by reader proteins.
Function of a writer protein
Writer proteins write the signature of histones
Function of eraser proteins
Eraser proteins erase the signature
Co-activator proteins CBP and p300, TAFII subunit of TFIID are examples of
Prots that have HAT activity. So they add Ac groups to histones.
What are Co-repressor protein of the thyroid hormone receptor examples of
Prots with HDAC activity. Resulting in the removal of Ac groups for histones.
Process of opening chromatin structure.
Activator protein binds to chromatin, this will acetylate the histone by HATs.
Process of closing chromatin structure.
Repressor protein binds to open chromatin, this will lead to the de-acetylation of histones by HDACs
What is the effect of acetylation weakening the strength of association between histones in adjacent nucleosomes.
- Looser association facilitates access of activator protein to linker DNA regions.
- Looser association promotes nucleosome displacement and this facilitates access of activator protein to nucleosome-free DNA.
What are the effects of acetylation directly influencing binding of acetyl-reading proteins to histones.
- Acetylated histone is recognised by an activator protein, resulting in more open chromatin.
- Acetylation disrupts association of histones with repressor protein, resulting in more open chromatin.
Regulation of HDAC.
- In myoblasts MEF2(activator protein) are bound to HDAC (removes acetylation) closing chromatin.
- When myotube needs to be transcribed HDAC is phosphorylated, causing HDAC to dissociated and MEF2 can acetylated to open up chromatin.
Function of HP1
Binding to H3K9 and spreading of tight heterochromatin
What is the effect of phosphorylation on H1 and chromatin structure.
Phosphorylation prevents the binding of HP1 to H1 resulting in open chromatin.
Function of poly comb complex(PC)
It has histone methyltransferase (HMT) activity promotes methylation of histones H3 resulting in a tightly packed chromatin.
Function of trithorax proteins
Promotes demethylation of H3 resulting in a more open chromatin
Different types of ways a reader protein can associate with the histone.
- Activity of reader can be influenced by neighbouring marks on one histone.
- Different readers recognise different marks on the same histone. (Cis)
- Different readers recognise different marks on different tails (trans)
How does IncRNA cause closed chromatin structure.
It recruits protein complexes that cause closed chromatin structure.
- It recruits PRC2 polycomb which causes histone methylation.
- It recruits Dnmt1 which maintains DNA methylation.
- It recruits Dnmt3a/3b which methylated DNA de novo
What complexes does siRNA become part of ?
- RISC: post-transcriptional repression: RNA degrading
2. RITS: transcriptional repression: Chromatin structure
When does DHS appear ?
DHSs appear in the promoter area of each individual gene, just before expression of that gene at appropriate time during development.
What does DHSs stand for ?
DNase 1 hypersensitive sites
What causes hypersensitive sites?
- Displacement of a nucleosomes
2. Alteration in nucleosomes structure
DNase 1 hypersensitive sites become available and facilitates?
Hypersensitive sites facilitate entry of transcription factors (TFs)
How are Heat shock-genes induced
- In promoters, DHSs are created by GAGA factor that binds to its GAGA response element causing a displacement of a nucleosome that allows for a exposed heat shock element.
- This happens before heat exposure so that genes are ready when body is heat exposed and response is quick.
- Only after cell is exposed to heat does the heat-shock protein bind to the heat shock element and cause transcriptional activation.
How are steroid-induced genes induced .
- Steroid-receptor complex binds to the DNA and creates a DHS at the steroid-inducible in the presence of steroid.
- This allows the already active Transcription factor to bind to the steroid-inducible gene
How does the steroid-receptor complex work.
It recruits a chromatin remodelling complex such as SWI/SNF which uses ATP hydrolysis to displaces nucleosomes and remodel the chromatin .
Pioneer transcriptional factor
Can bind to tightly closed chromatin and open it so that others can subsequently bind.
How do pioneer transcriptional factors work (Example FoxA1)
FoxA1 can bind to the estranged DNA binding element (ERE) in tight chromatin form, this then opens chromatin. Now oestrogen receptor protein can bind to ERE.
