RR7: Chromatin, Epigenetics and the histone code

Question 1

how is DNA organised and packaged in cells?

Answer 1

not in its naked form
bound up by histone proteins
folded into a characteristic structure: the nucleosome
nucleosomes arrange into a very specific manner to fold up DNA in a very specific space
the chromatin is a major barrier in gene expression: blocks the DNA from being accessed

Question 2

what are the two forms that chromatin exists under and what do those forms mean?

Answer 2

heterochromatin: highly dense, compacted chromatin
genes within the heterochromatin are usually silenced, DNA binding transcription factors cannot access the DNA
euchromatin: feathery, diffuse form
genes that are being expressed, accessible to transcription factors

Question 3

how was the work done on yeast indicative that there are silent and expressed regions?

Answer 3

yeast exists in either haploid or diploid form
becomes diploid from two haploids when growth conditions are sub optimal
haploid yeast can only mate with another yeast that is of a different mating type
each haploid yeast expresses a specific gene that will confer a mating type identity: type alpha or typa a
alpha cell has to mate with A cell
the way they identify themselves as one mating type or the other is based on a complex series of events that will reciprocally move one section of a chromosome into an actively expressed region
the sections that are moved contain the mating type identity
you become one or the other by transfer, recombination event
moved to the middle, genes are silenced on the side
in that region, if other genes are introduced, they will not be expressed
something physical about the silencing mechanism

Question 4

what happens when you introduce a methylating bacterial enzyme into yeast silent gene regions?

Answer 4

this enzyme methylates specific regions around the GACT motif
none of the residues will be methylated in that silent region, suggesting that the enzyme cannot access those targets in the DNA, and thore residues in the silenced section will not be methylated
there is something physical that makes the DNA inaccessible to proteins

Question 5

what is of genes in the telomeres? and how was it tested

Answer 5

genes in telomeres are also silenced (another physical constraint)

Question 6

what are the key elements that confer the silencing? and how was that tested

Answer 6

investigators introduced mutations randomly
try to find those mutants that dont obey normal biological rules
mutations in histones allow the expression of these sequences in the silencer regions/telomeres
suggests that the histones play a key role in cofferring the silencing mechanism

Question 7

how are the silent regions set up in the telomeres and the silencer regions? (proteins and mechanism)

Answer 7

• suite of proteins that are critical in setting up these silent regions in the telomeres and in the silencer regions of the yeast
• RAP1 and silent information regulators (SIRs) SIR 1, 3, 4
• RAP1 is the first subunit, and then the formation of a complex
• RAP1 recognises the sequences, DNA binding protein
• othe proteins come into the complex probably through protein protein interaction or with other things
• once those sequences are recognised, the SIR proteins come in and start to interact together
• SIR 2, 3, 4 come in and form a complex around the region where RAP1 initially bound
• recruited through protein protein interaction but also because they recognise hypoacetylated histones (acetylated to a small extent, tails that don’t have any post translational modification on them) in the region around that RAP1 binding
• SIR2 is super important, has an enzymatic activity
• once it comes into the complex and recognises those hypoacetylated histones it starts to change them
• it ensures that all of the tails around that region are HYPOacetylated, in doing so, ensures that the histone tails can interact with the DNA and it tightens the winding, chromatin gets more condensed/compacted
• as there is more hypoacetylated histones, the more the complex starts to grow, and expand around those regions
• end up with a higher order chromatin structure that is very tightly compacted based on an initial interaction with RAP1 interacting with the sequences and the recruitment of the complex

Question 8

what is observed when you do immunofluorescence of SIR3 (antibody against SIR3) and telomeric sequences?

Answer 8

they coincide almost perfectly in a nucleus
suggests that SIR3 protein interacts with those sequencing, through an initial RAp1 identification/recognition of those sequences

Question 9

what is a characteristic of end terminal histone tails and what are the different post translational modifications that it can be subject to?

Answer 9

largely unstructured
phosphorylation
acetylation
ubiquitylation
methylation
these lead to specific ouputs

Question 10

what does acetylation do?

Answer 10

acetylation to the histone tails on lysine residues will act to open/loosen up the chromatin, and increase transcriptional efficiency
the histone’s grip on the DNA backbone is weaker
neutralises the electrostatic interaction between the positive histone tails and the negative DNA phosphate backbone

Question 11

what does phosphorylation lead to?

Answer 11

specific serine or threonine residues can also give rise to specific changes

Question 12

what does methylation lead to?

Answer 12

methylation of lysines
methylation of lysine 4 (mono, di or trimethylations) is written as H3K4 (on histone 3)
methylation is almost always associated with some aspect of activation, depending on the type of methylation and how many methyl groups are added

Question 13

what is H3K9 associated with?

Answer 13

heterochromatin
you cannot generalise with methylation

Question 14

what is H3K27 associated with?

Answer 14

mediated by the polycomb group
formation of heterochromatin

Question 15

what is H3K36 associated with?

Answer 15

associated with activation

Question 16

what types of changes are these? (methylations, acetylations)

Answer 16

epigenetic traits
not changing the genome sequence, only its expression
the daughter cell has to have the same expression patterns as the parent cell

Question 17

what are some examples of those epigenetic traits that need to be passed on?

Answer 17

the inactive X chromosome in females
developmental restrictions and imprints
paternal imprints are different from maternal ones due to different reproductive strategies

Question 18

how are cytosine methylations recognised?

Answer 18

recognised by complexes such as mSin3 (m=mammalian)
Sin3 will come in with associated histone modifying enzymes and change the histones from around those regions
these marks have to be recognised and then rewritten in the following generation
key proteins that recognise (reader) and then rewrite (writer) or summon something else that might rewrite
either a reader, a writer or both

Question 19

what is a situation where the reader is also the writer?

Answer 19

when ensuring that genes that have been heterochromatinised remain that way even after cell division
during cell division you make DNA but also new histones
old methylated histones are divided between the new and the old strand
specific proteins that recognise those marks, associate with them if there are methylations next door, and they will methylate those unmethylated histones
leads to the same methylation pattern after cell division
the reader was also the writer

Question 20

how can those regions of the genome affected by histone modifications be identified?

Answer 20

investigators have generated antibodies that recognise each specific mark that occurs on these positions in histones
done by carryout out ChiP seq experiments using those antibodies
can examine the genome for these regions

Question 21

explain how transcriptional repressors can act through histone deacetylation complexes

Answer 21

• DNA binding transcription factors that recognise key sites that repress DNA
• recognise the cis acting elements
• recruits a large protein complex that contains proteins, one of which has a histone deacetylase activity
• important for removing acetyl groups on neighbouring histone tails
• become highly positive, interact with DNA backbone, close the chromatin around those regions, more compact
• SIR2 is also a histone deacetylase
• co repressors
• interact with DNA binding transcriptional repressors

Question 22

how do co-activators work? (with histone acetyl transferases)

Answer 22

• these DNA binding transcription factors sometimes require additional factors in order to achieve their max/optimal output
• these additional factors impinge on the chromatin/histones around specific DNA sites, recognised by those factors
• a DNA binding transcription factor called Gcn4
• interacts with a very well defined UAS (upstream activator sequence) and has a transcription activation domains
• Gcn4 and GAL4 were found to interact with an important other complex called the saga complex
• important for optimal transcriptional activity for Gcn4
• one of the proteins that work with Gcn4 is a histone acetylase, part of an activator complex that interacts with Gcn4 and interacts with the chromatin around where Gcn4 will bind
• histone acetyl transferase will modify all the histone tails around that region
• unlike SIR2 (which removes the acetyl groups), Gcn5 adds acetyl groups, which releases the histone tails from the electrostatic interaction with the DNA backbone and then it opens up the chromatin, by virtue of this enzymatic activity
• interaction between DNA binding transcription factor, activation domain, and this accessory protein that acts as a co-activator
• activates the transcription of downstream genes: interact with DNA + modify the chromatin region around that binding site, opening it up and making it accessible to transcription factors (GTFs and RNA pol II)
• co activators

Question 23

how was condensation/decondensation of chromatin studied with the lacI gene?

Answer 23

• activation domains can recruit in a number of proteins that affect the modification of the histone tails which changes the higher order confirmation of chromatin around specific regions of the genome
• wanted to investigate this in a more in vivo manner
• introduced lać repressor elements
• put these lacI elements into a cell, and examine what it looks like within the cell
• how is the lac repressor interacting with the DNA
• in the regions where it is heavily heterochromatinised (known to be as such) it forms very tight compact puncta
• indicative of the higher order conformation of the chromatin
• in the second experiment, they used the same lacI configuration but this time they introduced a lac repressor fused with one of the strongest known transcriptional activation domains (VP16), transfect all of that into cells
• now the chromatin opened up, there are different regions
• recruits chromatin co activators and remodellers
• these remodellers, in an ATP dependent manner, push chromosomes out of the way so that there is a new configuration around the regions
• critical for giving rise to these major changes

Question 24

what are pioneer transcription factors and how do they work?

Answer 24

• there are specific trans. Factors that take advantage of this ability to modify the histones in nearby regions
• important for setting motion of hierarchy of gene expression during embryogenesis
• these factors can recognise their DNA sequences despite the fact that they’re all wound up in very compact regions
• important in the early stages of embryogenesis, to activate the transcription of specific genes that will distinguish various cell types
• pioneer trans. Factors
• able to recognise because those sequences are on the exterior of the nucleosomes
• interact so strongly that the free energy associated with that interaction can lead to an unwinding of the chromatin
• also recruit/are associated with co-activators that will thereafter modify the histones in neighbouring region through acetylation, opening up the chromatin, allow other DNA binding trans.factors to access
• lead to a cascade of trans.factors

Question 25

what does the mediator complex do?

Answer 25

• the mediator can recognise those regions because of all those transcriptional activation domains and end up giving rise to a loop and summoning RNA pol II
• how some of the early trans.events take place