Lesson 13

Question 1

what is epigenetics specifically relating to nucleotides?

Answer 1

how cells interpret what is written on DNA → not the sequence of the nucleotides but the information deposited on top of them

Question 2

what the most modern way to define epigenetic?

Answer 2

any process that changes the activity of a gene without altering the nucleotide sequence of composition → alteration must be transferred to daughter cells

Question 3

what two mechanisms are involved in epigenetic?

Answer 3

DNA methylation and histone composition

Question 4

the number of genes in humans is almost the same as in Drosophila however we are much more complex - how is this possible?

Answer 4

our genes have much more regulation

Question 5

what is a third mechanism that some consider to be part of epigenetics, but Prof. Landsberger does not consider to be a main factor?

Answer 5

non-coding RNAs

Question 6

in terms of the complexity of genome regulation, what is the most important function in gene regulation ?

Answer 6

silencing genes seems more important than activating (ex: improper activation of proliferation genes leading to tumor development)

Question 7

what mechanisms are in place in order to better control gene expression?

Answer 7

presence of the nucleus and chromatin

Question 8

describe the idea of the nucleus being a checkpoint for mRNA:

Answer 8

if a gene must be translated, its mRNA will pass through the nuclear
pore and go to the cytoplasm where it will be translated, but if a gene must not be translated its mRNA is degraded in the nucleus before going into the cytoplasm

Question 9

what has been discovered for sure in research about mRNA and the nucleus?

Answer 9

evidence that there are big differences (in particular for some disorders) between the mRNA in the nucleus and the ones in the cytoplasm

Question 10

how much does chromatin structure repress gene expression?

Answer 10

by 50x

Question 11

describe transcription in relation to chromatin in prokaryotic cells:

Answer 11

most of the transcription is a basal P transcription: the DNA is almost naked and therefore transcription depends only on RNA Polymerase and on the sequence of the promoter (while only sometimes there’s the regulation of gene expression by activators or repressors)

Question 12

describe transcription in relation to chromatin in eukaryotes:

Answer 12

the promoter (TATA box and the core promoter) is assembled into nucleosomes, so TBP is unable to bind the TATA box, meaning that there’s no transcription going on

Question 13

what is a major difference between chromatin interaction and gene expression between prokaryotes and eukaryotes?

Answer 13

all of our DNA is repressed and the cell activates only what it needs

Question 14

what does epigenetics allow us to do?

Answer 14

permits us to fine tune our gene expression and it mainly introduces repressing mechanisms to repress what is not needed

Question 15

describe the number of genomes compared to epigenomes:

Answer 15

1 genome, many epigenomes

Question 16

what is epigenetics?

Answer 16

a code that is dynamic and flexible → it is affected by the environment

Question 17

in the first experiment studying epigenetics, describe the two types of mouse mothers:

Answer 17

high LG (licking and grooming) and low LG

Question 18

what were the results of the first mouse experiment in terms of the type of mother the baby became and how well it handled future stress?

Answer 18

the babies from good mothers grew up less sensitive to stress and became good mothers ; the babies from low LG mothers were more sensitive to stress and more likely to develop cardiovascular issues, also bad mothers to their babies

Question 19

what did they discover biologically to be the difference between high LG and low LG babies?

Answer 19

if the mother was high LG, the gene promotors were not methylated, if the mother was low LG, the same promotor was methylated

Question 20

when does the methylation of the specific promotor occur in the mouse study?

Answer 20

within the first 6 days of life → correlates to the first 3 years in humans

Question 21

brain samples from patients with different childhoods and background were observed, and what was found?

Answer 21

those with trauma did have differences from the rest of the population

Question 22

at the end of the primary mouse experiment, they treated the animals with some drugs that effect epigenetics: what was the result?

Answer 22

they could revert the phenotype in some cases, proving that the role of epigenetics in the development of a phenotype

Question 23

what was the discovery of the second experiment studying monozygotic twins?

Answer 23

the epigenetic patterns can change affecting also the sensitivity to some diseases → If two monozygotic twins are divided, they have a different
epigenetic pattern compared to a couple of monozygotic twins that has not been divided.

Question 24

in the third study which analyzed two twins working at NASA (one went into space and the other remained on earth), what did they discover?

Answer 24

the epigenetics changes depending on the two situations → the one who went in space came back to the Earth, most of the epigenetics and gene expression quickly went back to the normal level

Question 25

what was the main takeaway from the third experiment dealing with the NASA twins?

Answer 25

environment is affecting but we are also very much dynamic

Question 26

what are the three main players in epigenetics?

Answer 26

writers, readers, and erasers

Question 27

what are writers?

Answer 27

enzymes that modify the histones or DNA

Question 28

what are readers?

Answer 28

protein that binds to a histone or nucleotide only because it has a specific modification, and after this binding they exert their function

Question 29

since epigenetics is not stable, what else does it have besides readers and writers?

Answer 29

erasers → can change our epigenetic modifications in order to adapt to new situation

Question 30

what percentage of epigenetics is transferred from one cell to the daughter cells?

Answer 30

85-90%

Question 31

what are histones important for?

Answer 31

nearly every fundamental process in an organism

Question 32

what are the 4 main functions of chromatin?

Answer 32

-Helps during cell division by decompressing DNA that can be divided into two new cells (permits a correct inheritance of genomic content in each cell division)
-Packing 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 (regulates gene expression)
-DNA protection from degradation/breakage/somatic recombination

Question 33

what is the structure of chromatin?

Answer 33

made of repeating units made up of 147 bp of DNA that make 1.7 turn around the core of histone proteins. The histone core is an octamer composed of 2 copies of each of the four core histones H2A, H2B, H3, and H4

Question 34

what occurs if chromatin structure is very tight?

Answer 34

some transcription factors cannot interact with specific DNA regions because DNA is not accessible at this point

Question 35

even if the binding sites for transcription factors are on the outside of the DNA-chromatin structure, what can occur?

Answer 35

they still cannot interact due to the difference in conformation

Question 36

why is chromatin structure very repressive?

Answer 36

due to all of the wrapping around the histones → if the transcription factor has to insert in between this region, the two coils block this process, thus the transcription factor might not be able to insert

Question 37

what is the core histone domain composed of?

Answer 37

made by 3 ⍺-helices and an N-terminal tail that has no structure

Question 38

when experiments are done to understand histone function, what area is cloned?

Answer 38

the histone proteins that do not have a tail

Question 39

what can the first level of chromatin structure (the first 10nm fibers) be formed by?

Answer 39

only using the histone fold domain / core histones

Question 40

what are the histone fold domains / core histones?

Answer 40

a protein-protein interaction domain that stabilizes the interaction between histones → also called the N-shape domain which contains a long alpha helix in the middle and two sides

Question 41

how can you prove that DNA/proteins are on the inside or outside of a structure?

Answer 41

You treat it with hydrolytic enzymes, such as DNAse1 or protease1 → If you add a little amount of these enzymes, they will digest it into smaller, more accessible parts

Question 42

what are histone tails important for?

Answer 42

the subsequent levels of chromatin organization

Question 43

what are histone tails the most important sites for?

Answer 43

post-translational modification

Question 44

How can you prove that nucleosomes are inhibitory for binding of transcription factors?

Answer 44

using electrophoretic mobility shift assay (EMSA)

Question 45

in an EMSA, what is the shift in mobility indicative of?

Answer 45

the binding of your protein of interest

Question 46

what can most translation factors not bind to?

Answer 46

nucleosomal DNA

Question 47

what is Pu1?

Answer 47

central transcription factor for hematopoietic differentiation

Question 48

experimentally Pu1 is associated with about 60% of the total possible consensus sites, but the other 40% are not bound - why?

Answer 48

they are assembled into the nucleosome → Pu1 binding is limited and controlled by the nucleosome = because of steric hindrance nucleosomes are avoiding the binding of TF to sequences that they are not supposed to bind to

Question 49

what is another in vitro technique that can be used to determine if a protein is bound to DNA?

Answer 49

footprinting

Question 50

In vivo, what technique is used to prove that a protein is bound to a specific gene sequence?

Answer 50

chromatin immunoprecipitation (ChIP)

Question 51

what is ChIP?

Answer 51

ChIP involves cross-linking of the chromatin-bound proteins by formaldehyde (you do so in order to precipitate DNA as well, not only proteins), followed by sonification or nuclease treatment to obtain small DNA fragments

Question 52

what question does ChIP seek to answer?

Answer 52

is the protein binding to a specific promotor?

Question 53

what question does ChIP-Seq answer?

Answer 53

where does the protein bind all over the genome

Question 54

after the cross-linking of chromatin-bound proteins occurs in ChIP, what happens?

Answer 54

immunoprecipitaion is carried out using specific antibodies to the DNA binding protein of interest → DNA is then released from the proteins and analyzed

Question 55

describe promotor regions in relation to nucleosomes:

Answer 55

they can have precisely positioned nucleosomes or be devoid of them

Question 56

what is the PHO5 promotor?

Answer 56

promotor from yeast that is sensitive to phosphates

Question 57

how does PHO5 react to phosphates?

Answer 57

when phosphate is high the promotor doesn’t transcribe, when its low it does transcribe

Question 58

what PHO TF has to bind in order to activate transcription?

Answer 58

PHO4

Question 59

how is PHO4 regulated?

Answer 59

the PHO4 protein is regulated by phosphate itself - when this protein is not phosphorylated it binds, instead when it is phosphorylated it doesn’t bind

Question 60

Nucleosomes are distributed in specific positions, in order to have a binding-site for Pho4 that is maintained free (naked), in the linker DNA. On the other hand, the second site is occluded by nucleosomes - why?

Answer 60

When we are in the presence of little phosphate, everything is opened up. No more nucleosomes are there, and TFIIB can bind. When there is no phosphate, PHO4 is active, binds a protein that recruit chromatin remodelling complexes that modify the chromatin structure, and eventually, chromatin structure opens up

Question 61

If we look at PHO5 expression in a cell that is depleted of nucleosomes, you will find out that the gene is transcribed even when phosphate is high - why?

Answer 61

That’s because the TATA box is free and TFIIB can bind it → also, because there is a consensus site that is very similar to the consensus site of a house-keeping transcription factor