Landsberger: Lecture XIII Flashcards
Role of Epigenetics in the Regulation of Gene Expression and in our Health
What is epigenetics?
how cells interpret what is written on DNA
it is the information that is deposited on top of a nucleotide in order to regulate gene expression and other processes
Epigenetics is…
information that affects our phenotype (physiological or pathological conditions), which is given both by genetic (our genotype) and epigenome
What mechanisms are involved in epigenetics?
DNA methylation
histone composition
Why are the 2 mechanisms involved in epigenetics important?
they amplify the information contained in our genome and inform the cell about when, where and how a gene has been transcribed
Even though we have the same number of genes as Drosophila, what makes us more complicated?
we use more gene regulation
What allows us to have so many different types of cells?
when different cells express the same protein at different levels
What is Adrian Bird’s theory?
cells evolved from prokaryotes to eukaryotes and started to generate multicellular organisms
this along with an increase in genome size and genome complexity allowed for genes to code for protein and ncRNA
if a cell or organism has to increase its complexity, it is very good in controlling its gene expression
the cells learn how to use the genes in a proper way, and it can only achieve this by telling a gene to be expressed or repressed
What are the mechanisms that have been put into place to better control gene expression?
presence of nucleus (debatable “check-point”): if a gene must be translated, its mRNA will pass through the nuclear pore and go to the cytoplasm to be translated; if it does not need to be translated, it will be degraded in the nucleus (it is not certain if this is how it works)
chromatin: nucleosomes inhibit gene expression because when the DNA is wrapped around the nucleosomes, it cannot be read by TF → this means that in eukaryotic nuclei, DNA is repressed by default because it is assembled into chromatin
Describe transcription in prokaryotic cells:
transcription is a basal transcription: the DNA is almost naked and therefore transcription depends only on RNA Polymerase and on the sequence of the promoter (only sometimes by activators and repressors)
Describe transcription in Eukaryotic cells:
the promoter (TATA box and the core promoter) is assembled into nucleosomes, so TBP is unable to bind the TATA box so there is no transcription going on and DNA is repressed so the cell only activates what it needs
What is unique about our cell?
we have layers of epigenetics: post-translational modification of histones, DNA methylation, RNA-editing…
Why does epigenetics exist?
it allows us to fine tune our gene expression and it introduces the repressing mechanism to not express what is not needed
the different layers of repression have to be removed only when a gene must be expressed
How do our genes become so complex even though we only have 1 genome?
we have many epigenomes that permit our cells to differentiate subsets of the same cell type
How is epigenetics different from genomic code?
it is flexible and dynamic: it is affected by the environment
During the experiment with mice in regards to how epigenetics is affected by the environment, what 2 mothers were used?
High LG: good mothers
Low LG
What were the results of the experiment with the mice and how epigenetics is affected by the environment?
good mother → puppies are less sensitive to stress and have less chance to develop cardiovascular pathologies (they become good mothers)
bad mother → puppies were more sensitive to stress and have a greater chance of developing cardiovascular pathologies (they become bad mothers)
Why does the mother’s behavior affect cardiovascular pathology? How was this analyzed?
gene expression of specific genes that respond to stress (hippocampus) was analyzed and it was found that:
high LG → promoter of these genes was not methylated
low LG → promoter sequence was methylated
*this occurs in the first 6 days of life in these animals, which translates to 3 human years
Recap the mouse experiment that involves environmental factors:
environment in first days of life affects methylation level of genes → phenotypic outcome of kid is affected →capacity to be resilient affects health
Did they translate this to humans?
yes and it makes sense because children who suffer abuse usually suffer from depression and the same methylation pattern was found in humans
Why was the monozygotic twins experiment carried out?
to understand how the environment affects epigenetics
What were the results of the monozygotic twin experiment?
twins are born with similar epigenetic patterns, but the pattern can change over the years, which can lead to sensitivity of diseases → if 2 monozygotic twins are seperated, they have a different epigenetic pattern compared to monozygotic twins that have not been seperated
What was the found in the NASA twin epigenetic experiment?
gene expression is different
epigenetic change based on the 2 situations (one in space the other in America)
when the one that went to space came back to Earth, most of the epigenetics and gene expression quickly came back to the normal level (5 months)
*this suggests the environment affects and our epigenetics are dynamic
What is a fun fact about a discovery from the NASA twins?
telomeres get longer in space but shorten upon returning to Earth
Describe how epigenetics is different in bees:
Queen bee has the same epigenetics as the workers, but she eats a lot of royal jelly which contains donors for methylation
Describe how epigenetics affects turtles:
the sex of the turtle depends on the temperature of the sand
How does epigenetics work?
it is a code that has to be translated and read
What do we need to understand epigenetic code?
writers: enzymes that modify the histones or DNA
readers: protein that binds to a histone or nucleotide only because it has specific modification and after binding they exert their function
modifiers (erasers): needed since the code is dynamic; changes our epigenetic modifications in order to adopt new situations
Histones are ___
highly dynamic entities and essential for every fundamental process
What are the functions of chromatin?
help decompress DNA during cell division so that it can be divided into 2 new cells
packages DNA so that it can fit in the nucleus
regulates transcription by strengthening/weakening and therefore restricts/allows DNA information to be transcribed into RNA
provides DNA protection from degradation/breakage/somatic recombination
*this refers to the 1st level of chromatin condensation which is the 10nm fibers
Review the chromatin structure:
What is the nucleosome?
contains DNA + histones
What is chromatin made up of?
repeating units of 147 bp of DNA that make 1.7 turns around the core of the histone proteins
What is the histone core?
octamer composed of 2 copies of each of the 4 core histones
What are the 4 core histones?
H2A
H2B
H3
H4
If TFs want to interact with the DNA wrapped around the histone, what must be done?
they must interact with the outside face of the DNA but it is still hard since the DNA is bent (not straight)
Describe the histone core domain (fold domain):
made of 3 alpha helices and an N-terminal tail that does not have a structure (X-ray of tail is not feasible; therefore, it cannot be synthesized)
What do experiments aimed at what the regions of the histone are important for generating the chromatin structure use?
clone the history of the protein that do not have the tail (just the core or histone fold domain)
*results show this region is enough to form an octamer structure around which DNA can wrap around
How can the 1st level (10nm fibers) of a chromatin structure be formed?
only by using the histone core domain/core histones
What does the N-shape domain contain?
long alpha helix in the middle and 2 sides
it is a protein-protein interaction domain that stabilizes the interaction between histones
the histone tails are directed toward the outside of the domain
How can you prove that DNA/protein is on the inside/outside of a structure?
!!!
you treat it with hydrolytic enzymes, such as DNAse1 or protease1
these digest it into smaller, more accessible parts
why?
histone tails are very sensitive to low level of trypsin and the core is not…this was sufficient to prove the core was protected by DNA (on the inside) and the tails are directed towards the outside
Why are histone tails important?
subsequent level of organization of chromatin
*if you want to form the 30nm fiber, you need histone tails
**histone tails are the most important site for post-translational transcription
How can you prove that nucleosomes are inhibitory for binding TF?
you can use Electrophoretic Mobility Shift Assay (EMSA) to prove proteins are bound to DNA or a piece of DNA
How does EMSA work?
it is used to understand if a specific transcription factor (SP1) is binding to a receptor
in vitro, the promoter (or DNA) is purified and amplified using PCR
it is the. incubated in 2 separate tubes (1 with naked DNA and the other with TF of interest) + DNA
after incubation, if TF is binding to sequence, the solutions from the 2 tubes and 2 controls are loaded on a gel-electrophoresis and protein motility is observed
the control DNA will migrate depending on MW while the binding of SP1 to the specific DNA will slow the speed of the mobility of the DNA
shift is indicative of the binding of the protein of interest
https://www.youtube.com/watch?v=sPPvxyA74_g
What has been found in some experiments in regards to EMSA?
most TF cannot bind to nucleosomal DNA
List a specific example of an experiment where TF could not bind to nucleosomal DNA:
Pu1 TF
Pu1 is the central TF for hematopoietic differentiation
40% of Pu1 is not bound despite have many sites all over the genome
Why? BECAUSE THEY ARE ASSEMBLED INTO NUCLEOSOMES
Pu1 binding is limited and controlled by nucleosomes, and steric hinderance causes nucleosomes to avoid the binding of TF they are supposed to bind to!!
What is another technique that can be used to determine if a protein is bound to DNA?
!!
footprinting
In vivo, what technique is used to prove that a protein is bound to a specific gene?
Chromatin precipitation (ChIP) NOT ChIP Seq
What does ChIP do?
allows us to know if a protein is binding to a specific promoter
How does ChIP work?
!!
involves the cross-linking of the chromatin-bound proteins (or DNA) by formaldehyde
sonification or nuclease treatment follows to obtain small DNA fragments
immunoprecipitation is then carried out using specific antibodies to the DNA-binding site of interest
DNA is then released from the proteins and analyzed using various methods like PCR
What are some advantages regarding ChIP?
takes a week to run and costs about 100 euros
How can we see if our specific promoter is in the sequence after running ChIP?
use a Southern Blot (no longer used) or rtPCR
PHO5 promoter
yeast promoter
study was done to see how nucleosomes would distribute themselves on promoter regulatory sequences
*promoter is sensitive to phosphate
In the PHO5 promoter, when phosphate levels are high, ____. When the phosphate levels are low, ____.
promoter does NOT transcribe
promoter transcribes
What are the 2 regulatory elements for transcribing in PHO5?
PHO4 binding sites
What is PHO4?
TF that has to bind to activate transcription
regulated by phosphate
not phosphorylated = binds
phosphorylated = not bound
Why would the TATA box be recruited by nucleosomes when they are distributed on the promoter?
so that TFIIB cannot bind and the gene promoter can be repressed
Why are nucleosomes distributed in specific positions?
to have a binding site for Pho4 that is free, in the linker DNA
In the presence of a phosphate, ____
everything is open, no nucleosomes are present and TFIIB can bind.
In the absence of phosphate, ____
Pho4 is active, protein is bound that recruits chromatin remodeling complexes so that chromatin eventually opens up
Why is the gene transcribed in Pho5 even when it is depleted of nucleosomes?
the TATA box is free and TFIIB can bind and because the consensus site is similar to the consensus site of housekeeping TF
NUCLEOSOMES ARE CRUCIAL FOR REGULATING GENE EXPRESSION
