Lecture 18 (RR6): Chromatin, Epigenetics, and the Histone code Flashcards
Heterochromatin
Heterochromatin
* Heterochromatin is a condensed form of chromatin that localises at the nuclear envelope often near the nuclear pores
* Heterochromatin is considered transcriptionally inactive due to it being highly wound —> inaccessible to transcription factors.
* Transcriptionally inactive regions of the genome are maintained in a “heterochromatinized” state. Their transcription could be detrimental to the cell/organism.
* It is densely stained in the picture.
NOT TRANSCRIBED HEAVILY
Euchromatin
- Euchromatin is delicate and thread-like.
- It is abundant in actively transcribing cells. Associated with parts of the genome that are being transcribed.
- It may represent DNA that is unwound to provide a transcriptional template.
- Only stains very lightly —> light stain in the picture.
REPRESENTS GENOME WHERE IT IS BEING TRANSCRIBED.
Arrangement of mating-type loci on chromosome III in the yeast S. cerevisiae
- Three genetic loci on chromosome III genetically control the mating type of Saccharomyces cerevisiae (yeast). There are 2 silent loci and one MAT ocus.
- In order to specify mating type, the sequences from HMLα and HMRa have to recombine into the center locus (mating type locus map). Once it is in there, it will be expressed. Only one can go into that locus at the time. When the α or a sequences are present at the MAT locus, they can be transcribed into mRNAs whose encoded proteins specify the mating-type phenotype of the cell.
- The HMLα and HMRa loci (on the right and left/not in the middle) must be silenced otherwise the cells will be diploid a/α and cannot mate.
- Transcriptional repression depends on silencer sequences. This silencer works outside the context of mating and can even block expression of tRNA genes (RNA Pol III). Anything introduced into these silencing regions will not be transcribed and therefore expressed.
- These silencing regions (they bind proteins that are critical for repression of the silent loci) are resistant to DNA degradation. These regions protect the DNA from degradation. This suggests that enzymes cannot get in there to digest its substrate DNA. Block access to transcription factors.
- Genetic experiments indicate that histones affect repression (involved in silence genes), while regions around telomeres behave similarly.
What factors are requird for the repression of the silent mating type loci?
-
RAP1
-binds to DNA in the region of the silencer
-also binds to repetitive sequence in telomeres
-acts as a scaffold which is recognized by other key proteins -
SIR1 (Silent Information Regulator)
-cooperates with RAP1 and is important for binding the silencer region in the silent mating type loci- required at the silent mating type loci
-
SIR2, 3, and SIR4
-bind to hypoacetylated histone tails (H3, H4)
-forms large complexes with telomeric DNA
-Required at the telomeres and the silent mating type loci
-Recognize RAP1: RAP 1 binds first then SIR3 and SIR4 bing to RAP1.- SIR3 and SIR 4 interact with RAP1 and that complex is recognized by another silent information regulator called CIRC2.
- Sir2 is the last protein to come in. It has enzymatic activity. It’s capable of removing Acetyl groups from the neighbouring histone tails around that region. When it removes these tails the histones become hypoacetylated (bidirectional - heterochromatin can spread both ways up to a boundary) and then recruit in more SIR3 and SIR4 which creates a spreading effect. The hypoacetylation leads to change in the histone and its ability to interact with the DNA. It becomes highly condense, folds in and becomes tightly bound to DNA. This change in configuration closes down the region and blocks access to a number of proteins (transcription factors).
What are possible modifications that can occur on the histone tails?
- The hypoacetylation is just one type of change that can occur on these histone tails.
- Histones are made up of a domain that forms the core in the nucleosides and they have a termini (an end-terminus that is highly disordered and away from the core of the nucleosome). Sometimes the c-terminus tails are also involved in post translational modifications.
- Specific modifications on tails of H3 and H4 induce changes in chromatin structure typical of EUCHROMATIN vs HETEROCHROMATIN…….but be careful not to generalise!!!
- Types of possible modifications that can occur on the tail: phosphorylation, oscillations, methylations, ubiquity lotions. Not all of these occur at specific residues and give rise to marks on the histone tails.
- REMEMER: acetylation is almost always associated with an increase of transcriptional efficiency. It opens us the chromatin.
- phosphorylation, ubiquitylation and methylation can go both ways
Example:
Methylation on H3 K4 (lysine)->ACTIVE
Methylation on H3 K9 (lysine)->INACTIVE
These are examples of epigenetic modifications
What are epigenetic traits?
- EPIGENETIC TRAITS - transmitted independently of the DNA sequence itself
Examples of things that are usually epigenetic traits:
* Inactive X chromosome (Xist, histone methylation and heterochromatin spreading)
* Developmental restrictions (legs vs antennae…Polycomb)
* Imprints (DNA methylation): imprints are mammalian specific
* DNA marks (methylation), often CpG islands, are read by specific proteins then used to modify histones in proximity through mSin3 recruitment. Altering the histone tail leads to reduced transcription.
* Histone marks (ie..H3 K9 methylation) can nucleate histone methyltransferases to repress gene activity across an entire genetic region. These marks can be (often are) heritable following cell divisions.
What are epigenetic marks?
- We can refer to these enzymes as epigenetic writers.
- Epigenetic writers will put down epigenetic marks on the histones so that epigenetic readers can be brought into the system and change gene expression appropriately.
- To ensure that every cell daughter acquires the appropriate fate following division, epigenetic marks must be faithfully inherited and acted upon accordingly by proteins that recognize these modifications or epigenetic readers.
- They will recruit the appropriate effectors to carry out the correct response after recognizing that particular post translational modification.
- epigenetic marks have to be propagated
Can epigenetic readers be epigenetic writers too?
Heterochromatin maintenance requires H3K9me3 during DNA replication
* A specific histone methyltransferase (HMT) recognizes H3K9me3 to methylate neighbouring naïve histones.
* This HMT therefore acts as both an epigenetic reader and an epigenetic writer
1) When DNA replication takes place, the two strands that arise following replication will inherit the 2 histones that were on the original strand of DNA that were marked accordingly whether it was silenced or active. They get distributed randomly between the two strands.
2) During the S-phase or DNA replication, you are also making lots of new histones (that are naive) and will be incorporated onto those strands as well.
3) Epigenetic readers come in and recognize the particular site (in this case trimethylations -Me3) and that there are histones beside them that are not marked with the same post translational modifications.
4) So, it will recruit enzymes that will make the changes on the naive histones to make sure that all the histones on the mother and daughter strands are the same.
ChIP Immunoprecipitation
- Antibodies against modified histone tails can be used for Chromatin Immunoprecipitation (ChIP)
- To understand where all of these epigenetic marks are within the genome, we can use antibodies that recognize all of these modifications on the histones.
1) Carry out these chromatin immunoprecipitations using your antibody against that particular mark.
2) You fragment all of the chromatin and precipitate it so that you have at the bottom of your test tube an antibody that recognizes the histone tail modification and the DNA associated with it.
3) Then you can sequence the DNA and you will find out all of the sequences that are associated with that particular mark. Either use PCR or NGS (new generation sequencing).
ChIP and NGS
By using reversible crosslinking agents, proteins bound to chromatin can be isolated using antibodies and the sequence of the bound DNA can be determined.
1. Using known primers if you want to know whether a specific gene is affected
2. By using NGS the entire genome can be analyzed to determine what regions of the genome are being affected.
First graph: Using NGS the entire genome can be surveyed to provide information about the genes (loci) affected by the marks.
An example, where 3 different antibodies recognize each of the histone marks, whether it is H2K4 methylation, dimethylation or trimethylation. The map of the reeds shows that on chromosome 17:
- H3K4 trimethylation is very isolated around the end region, which is generally transcription start sites. That is where this specific mark likes to be put down.
- H3K4 dimethylation is around the start site, proximal and then far upstream.
- H3K4 monomethylation are generally associated with enhancer regions. They also have far upstream regions like dimethylation.
** Second graph:** ChIPs performed with antibodies against transcription factors help to delineate enhancer positions and other key regulatory elements. The same experiment with a collection of DNA binding transcription factors. They also find themselves enriched in the same regions far upstream as the H3K4 demethylation and monomethylation and they also like binding the proximal promoter elements near the transcriptional start sites.
Transcriptional Co-activators
- Positive charge of the N-terminal histone tail interacts electrostatically with the DNA phosphate groups.
- Acetylation neutralizes the electrostatic interaction and permits complex formation.
- Some transcriptional activators can overcome the repressed chromatin state by inducing acetylation of histone tails through associated proteins
- DNA-binding domain of the activator interactss with UAS of the gene it regulates. The activation domain then interactss with a histone acetylase which opens up the chromatin (it neutralizes the charge between the histone tails and the DNA backbone).
- ie… Gcn4p and Gcn5p CBP, p300
Co-repressors
- DNA binding sequence transcriptional repress or interacts with its know DNA binding sequence.
- Positive charge of N-terminal histone tail interacts electrostatically with the DNA phosphate groups.
- Acetylation neutralizes the electrostatic interaction and permits complex formation.
- Rpd3p is a histone deacetylase and demonstrates deacetylase activity. This means it will take off the acetyl groups from the histone tails around that region. These are referred to as Hdacks.
- Specific targeting requires Ume6p (its DNA-binding domain binds to URS!) which then binds Sin3p.
- Deacetylation of histone N-terminal tails on nucleosomes in the region of the Ume6-binding site inhibits binding of general transcription factors at the TATA box, thereby repressing gene expression.
Chromatin remodelers
- Chromatin remodelers can trigger the decondensation of chromatin.
- If you introduce the operator sequences from the lack Operon into cells. They are recognized by a DNA binding protein called lac inhibitor (lacI). Lac I interacts in the condensed regions.
- However, if you add an activation domain and you make a hybrid DNA binding transcription factor that recognizes those same lac operator sequences. When you introduce that hybrid transcription factor you see a major change in the chromatin configuration, it opens right up and becomes like euchromatin.
- This is entirely dependant on the activators function. SWI is a chromatin remodeler that pushes the nucleosome one way or the other to relocalize things, to allow access to specific regions in the genome.
Pioneer transcription factors
- Pioneer transcription factors are DNA Binding transcriptional activators that function by interacting with DNA via sequences exposed on the outside of the DNA-wound histone octamer.
- They recruit enzymes that alter the configuration of the neighbouring histone tails. This opens the chromatin to permit association with the general transcription factors.
- Allow for transcription, after opening the chromatin, usually giving rise to a cascade of other transcriptional events.
- They use histone acetyl transferases to change the chromatin, they can summon in chromatin remodelers and change the configuration of the chromatin to allow for transcriptional cascade to take place.
- Pioneer transcription factors wear two HATs
- The Mediator complex is recruited to the site of transcriptional initiation
Summary: Pioneer transcription factors go in and open up the chromatin. It allows for the mediator complex to bring in RNA polymerase II and the general transcription factors to regions around that segment DNA where the chromatin was largely opened up, doing this enhances the initiation of the transcription and the genes that are nearby. This gives us a handful of different mechanisms through which its thought that DNA binding transcriptional activators function.
Pioneer transcription factors are hats. They interact with DNA on the outside.
They interact with the DNA (they go in and interact with the DNA first).
HATs
Histone acetyltransferases (HATs) are epigenetic enzymes that install acetyl groups onto lysine residues of cellular proteins such as histones, transcription factors, nuclear receptors, and enzymes.
