Lecture 15 - Chromatin Flashcards
What is expression of genes in higher organisms dependent upon?
DNA accessibility
In what three ways can histones be modified?
Acetylation
Phosphorylation
Methylation
What does DNA methylation influence?
chromatin structure and gene expression
How is DNA packaged within cells?
DNA is associate with proteins to form chromatin
-all chromatin is condensed and highly ordered but there are differences in the types of chromatin
What are the different types of chromain?
Interphase DNA not equally packaged -
Heterochromatin: (stains darkly) little transcriptional activity
-RNA pol necessary to bind for DNA transcription but cannot access DNA as it is tightly packaged
Euchromatin: (stains lightly) active gene expression
-loosley packaged
What does the differential packaging of chromatin have effects on?
-transcriptional activity
-DNA replication e.g.
Euchromatin is replicated earlier in S phase than heterochromatin
What are the differences between heterochromatin and euchromain?
Hetero- -condensed -few genes -telomeres -centromeres -transposable/repetive elements (areas that need more stability or are silenced) Eu- -less condensed -genes that need to be expressed
How can an interphase nucleus be lysed to view the ‘beads on a string’ structure of chromatin?
Under low salt conditions
What is the composition of the nucleosome?>
- made up of an octamer of histone proteins
- 4 core proteins make dimers and come together to form an octamer (H2A, H2B, H3, H4)
- and 146bp of DNA
What are the features of the four core particles of the nucleosome?
- small (102-135 aa)
- basic (rich in lysine (can be post translationally modified) and arginine (overall + charge) residues)
- C terminal portion is well folded and involved in protein:protein interactions
- N terminal is less structure and wheremodifications occur
What are linker histones and what is their purpose?
- H1 histones
- join nucleosomes together to form a compact chromatin fibre (30nm in diameter)
- binds to the linker area between nucleosomes to allow it to fold and allows the fibre to be more tightly packaged
- ~40 fold reduction
What is necessary to make the soleinoid fibre compact further?
scaffolding proteins to form chromosomes
Is the DNA that interacts with proteins less susceptible to DNase digestions?
Yes
Enzyme needs access to DNA
-harder if associatede with proteins
What evident is there that transcribed genee are in some way packaged?
- DNA that is transcribed has the characteristic ‘beads on a string appearance’
- active genes are resistant to mild nuclease tratment
Are there subtle differences in the resistance to mild nuclease treatment between inactive and active regions of DNA?
Yes.
There is a differential sensitivity to digestion with DNaseI
How can you experimentally show that there are subtle differences in the resistance to mild nuclease treatment between inactive and active regions of DNA?
- Digest chromatin with a range of increasing concentration of DNaseI
- Isolate the surviving DNA and perform a southern blot or PCR to detect the sequences we are interested in
- Visualise on a gel
Show that:
Inactive gene will form thick, constant bands at each concentration of DNase I treatment
Active gene will show some level of resistance (bands at lower levels of DNaseI) that reduces as the concentration increasedd
What has been determined about active genes through the technique of differential DNase I sensitivity?
- actively transcribed gene are in a more open structure than non-transcribed gene
- there may be regions even within the active genes that are more open than others (DNaseI hypersensitive regions - destroyed no matter what conc. of enzyme used)
What influences chromatin structure?
1) Histone modifications (chemical groups added to the N terminal portion of histones e.g. to lysine)
- covalent modifications
- histone variants (different sort of packaging taking place in different places in the genome)
2) Chromatin remodelling complexed (potentially recurited by modifications of histones)
3) DNA methlyation status
What is the structure of histones that allows them to be modified?
- N terminal unstructured tail
- C terminal histone fold
- the histone fold is responsible for the dimerisation and is highly structured
- the N terminal tails extend out from the DNA histone core and are subject to covelant modification
What modifications can occur on the histone N terminal tails?
phosphorylation
- acetylation (neutralise + charge)
- methylation (normally repressive result in more closed structure)
- ubiquitination (destruction)
what occurs in histone acetylation?
- the free amino group on specific lysine residues are modified by one od the hydrogen atoms in the amino group being subsituted to an acetyl group
- modification is dynamic
- neutralises + charge, makes the association with DNA looser and marked so that other proteins are recruited to the area
- which could remove histones temporarily or shift them slightly away
How does histone acetylation status affect gene expression?
- Acetyl groups neutralise the positive charge of histones and therefore may weaken the electrostatic interactions between the negatively charged phosphodiester DNA backbone and the histones
- may also disrupt histone structure by a mecahnism unrelated to charge
- acetyl groups may also serve as signals to attract proteins that disrupt chromatin
What are the features of histone variants?
- histones are highly conserved in al eukaryotes
- variants of H2A and H3 are incorperated into chromatin at a low freuqncy
- variants have specific functions
- suggest that the minor differences in amino acid sequence allows distinct modifications that could influence factor binding
Give three examples of the inclusion of histone variants in the genome and what this results in
Example 1
-Histone H3.3 replaces H3 in a transcription coupled process
-thought to help maintain gene activity
Example 2
-H2AX is incorperated at low frequecy all over the genome
-when DNA is damaged it is phosphorylateede and participates in attraction of DNA repair enzymes
Example 3
-CENP-A replaces H3 at the centromeres
-binds spindle microtubules during mitosis
Are histone acetylases activators or inhibitors of gene expression?
Activators
-promote the open complex of chromatin
In what ways can DNA be methylated?
- DNA bases can be modified by the addition of methyl groups
- most common form is 5-methyl cytosine (2-7% of cytosine in mammalian DNA is modified)
- implications in which regions are active
What is the effect of DNA methylation?
- unmethylated DNA usually adopts an open chromatin configuration (sensitive to DNase I digestion)
- methylated DNA is associated with heterochromatin and inactive genes
What processes are the basis of epigenetics?
-DNA methylation and/or histone modification
What is the evidence that DNA methylation affects transcription?
Intoduction of methylated DNA into cells
- templates that are methylated are transcribed at a lower frequency than unmethylated templates
- unmethylated templates are more sensitive to DNAseI
Inhibition of DNA methyaltion
- using a base analog e.g. 5-azacytidine that is incorperated into DNA but cannot be methylated acitvates previously silent genes
- gene activation in DNA methyltransferase defective mutants
What experiment can be done to visualise histones and other nuclear proteins?
- remove soluble and loosely bound proteins
- denature tightly bound proteins and DNA
- separate by electrophoresis
- stain protein bands
- image and analyse results in relation to molecular weight markers
What experiment can be done to reveal the periodicity of nucleosomes?
Isolate mammalian cell nuclei and digest chromatin with micrococcal nuclease (MNase)
- remove soluble and loosely bound proteins
- digest nuclei (MNase)
- extract protein and recover DNA fragments
- separate DNA by electrophoresis
- image and analyse results in realtion to molecule weight markers
