3 LC Concepts of Molecular Biology 06.10.2022
Transcription and Regulation:
3D genome organization, loop extrusion, enhancer promoter communication
In the nucleus we have roughly 2 meters of DNA folded up into a 0.2 micrometers; that means that if the nucleus is a soccer ball,
then DNA is 80 km long. The cell regulates the folding and the way it is organized fairly tight but not too much.
Nucleus has different compartments where the dna is more tighly packaged, and other areas where there is still dna but less
packaged more tight packaged heterochromatin and more loosely tight euchromatin.
It has been realized that these two compartments need to be tighly organized to bring dna either into certain parts of the
nucleus, for example to the nuclea lamina where we normally have heterochromatine really attached to it and repressed; other
parts of the nucleus, like the nucleolus itself, is a little bit dense and it’s where massive amount of transcription going on.
Between the nucleolus
and the nuclear lamina there is the labeled TS A compartment where euchromatin is kept amnd
where the actual transcription of mRNA are happening.
HiC mappings (Chromatine conformation capture)
Method to interrogate the folding of DNA, crosslinking dna fragments with each other. You can digest the dna using for ex.
restriction enzymes or Mna micrococconucleases and cutting surrounding proteins that keeps this in place originating two
restriction site that can be ligated to each other.
Once they’re ligated to each other you can look at that either by q – pcr, designing primers left in front of it and asking do I get
more of this interaction under certain circumstances, or NGS next generation sequences again reading out an identifier of where
these interactions are happening.
The idea is that once we have this new interactions, or these new borders between these 2 different dna fragments , that’s
where they have been in proximity to each other.
This has been done recently with different restriction enzymes sites, using in diffirent ways in small little difference in techniques
getting afterwards a map like the one in BCD.
in BCD
- You take all the reads that you get, you align them across the chromosomes finding out where is the other piece coming
from; when you ligated two fragments that shouldn’t have been together but are now in proximity, it is possible to
identify the border in between. it is possible to get an idea of what is actually in close proximity in the 3D genome
when it’s folded up in the cells.
o We have 2 pieces that in 2d are far away; in 3d they much be in close proximity, we ligate them to each other
and then we read out the connection between them. Then we identify If they have been in close proximity and
since this is a semi-quantitative assay we can say how often they have been in contact.
This allows us to ideintify how is the genome organize within the nucleus and how the cell is able to
achieve putting two meters of dna in such a small nucleus.
Lessons learned from the first HiC mappings:
- Between different cells the overall massive organization is very similar, so if you for ex. compare mesodermal lineages,
endodermal and ectodermal lineages between different cell lines that have been isolated and then ask how are these
organization looking like. They all maintain some connections between dna that are steady and it is possible to find
them in all different cell types. This happens even across species such es human and mouse taking very similar
organization suggesting that what we see in the 3D genome is conserved across different species.
, 3 LC Concepts of Molecular Biology 06.10.2022
2 different compartments in the cells: A and B
We can identify two different compartments within the nucleus, we can have an A compartment and a B compartment, that are
identified by taking a far way lookout from the outside and find regions that within the bigger chromosomes are coming into
close proximity. that doesn’t mean that they’re directly interacting to each other but they’re often times more closer to each
other then other regions within the chromosomes.
This ones are associated with either heterochromatin and euchromatin:
- Compartment A: is normally more often transcribed, you can find active chromatin more often closer to each other.
- Compartment B
o DAPI PICTURE:
EUCRHOMATINE (active transcription) is a more open chromatin and thereby more closer
HETEROCHROMATINE (more compact and histones) is organized at the limina and other fossa within
the genome much more closer together in more closer proximity
You can also show that by doing Fish experiments (Fluorescence in Cito-
Hybridization) you label three different dna fragments with each other at equal
distance and two of them are in compartment A and the other ones in
compartment B.
You then ask what is the distance between them; if you look at the ones that are in
one compartment are generally close to each other (A) where as others that are in
compartment B are not as close to each other.
If you compare the distance from L3 that is in comp B and L2, even though is
closer to it is even further from it (2D vs 3D?).
The main explanation face operation
For ex. we have H3K9me3 that leads to recruitments to HP1 which then leads to
forming face that might be more closer and closer associated with each other
excluding other parts and thereby overall collaps on top of each other ???
Euchromatin very similarly seems to exclude from heterochromatin but also there
are transcription factories, regions where we have very highly active transcription
that might come closer into proximity to each other.
The first big lesson is therefore that we have two different compartments; but
then it is possible to step a little bit in with Topologically Associated Domains:
TADs.
Transcription and Regulation:
3D genome organization, loop extrusion, enhancer promoter communication
In the nucleus we have roughly 2 meters of DNA folded up into a 0.2 micrometers; that means that if the nucleus is a soccer ball,
then DNA is 80 km long. The cell regulates the folding and the way it is organized fairly tight but not too much.
Nucleus has different compartments where the dna is more tighly packaged, and other areas where there is still dna but less
packaged more tight packaged heterochromatin and more loosely tight euchromatin.
It has been realized that these two compartments need to be tighly organized to bring dna either into certain parts of the
nucleus, for example to the nuclea lamina where we normally have heterochromatine really attached to it and repressed; other
parts of the nucleus, like the nucleolus itself, is a little bit dense and it’s where massive amount of transcription going on.
Between the nucleolus
and the nuclear lamina there is the labeled TS A compartment where euchromatin is kept amnd
where the actual transcription of mRNA are happening.
HiC mappings (Chromatine conformation capture)
Method to interrogate the folding of DNA, crosslinking dna fragments with each other. You can digest the dna using for ex.
restriction enzymes or Mna micrococconucleases and cutting surrounding proteins that keeps this in place originating two
restriction site that can be ligated to each other.
Once they’re ligated to each other you can look at that either by q – pcr, designing primers left in front of it and asking do I get
more of this interaction under certain circumstances, or NGS next generation sequences again reading out an identifier of where
these interactions are happening.
The idea is that once we have this new interactions, or these new borders between these 2 different dna fragments , that’s
where they have been in proximity to each other.
This has been done recently with different restriction enzymes sites, using in diffirent ways in small little difference in techniques
getting afterwards a map like the one in BCD.
in BCD
- You take all the reads that you get, you align them across the chromosomes finding out where is the other piece coming
from; when you ligated two fragments that shouldn’t have been together but are now in proximity, it is possible to
identify the border in between. it is possible to get an idea of what is actually in close proximity in the 3D genome
when it’s folded up in the cells.
o We have 2 pieces that in 2d are far away; in 3d they much be in close proximity, we ligate them to each other
and then we read out the connection between them. Then we identify If they have been in close proximity and
since this is a semi-quantitative assay we can say how often they have been in contact.
This allows us to ideintify how is the genome organize within the nucleus and how the cell is able to
achieve putting two meters of dna in such a small nucleus.
Lessons learned from the first HiC mappings:
- Between different cells the overall massive organization is very similar, so if you for ex. compare mesodermal lineages,
endodermal and ectodermal lineages between different cell lines that have been isolated and then ask how are these
organization looking like. They all maintain some connections between dna that are steady and it is possible to find
them in all different cell types. This happens even across species such es human and mouse taking very similar
organization suggesting that what we see in the 3D genome is conserved across different species.
, 3 LC Concepts of Molecular Biology 06.10.2022
2 different compartments in the cells: A and B
We can identify two different compartments within the nucleus, we can have an A compartment and a B compartment, that are
identified by taking a far way lookout from the outside and find regions that within the bigger chromosomes are coming into
close proximity. that doesn’t mean that they’re directly interacting to each other but they’re often times more closer to each
other then other regions within the chromosomes.
This ones are associated with either heterochromatin and euchromatin:
- Compartment A: is normally more often transcribed, you can find active chromatin more often closer to each other.
- Compartment B
o DAPI PICTURE:
EUCRHOMATINE (active transcription) is a more open chromatin and thereby more closer
HETEROCHROMATINE (more compact and histones) is organized at the limina and other fossa within
the genome much more closer together in more closer proximity
You can also show that by doing Fish experiments (Fluorescence in Cito-
Hybridization) you label three different dna fragments with each other at equal
distance and two of them are in compartment A and the other ones in
compartment B.
You then ask what is the distance between them; if you look at the ones that are in
one compartment are generally close to each other (A) where as others that are in
compartment B are not as close to each other.
If you compare the distance from L3 that is in comp B and L2, even though is
closer to it is even further from it (2D vs 3D?).
The main explanation face operation
For ex. we have H3K9me3 that leads to recruitments to HP1 which then leads to
forming face that might be more closer and closer associated with each other
excluding other parts and thereby overall collaps on top of each other ???
Euchromatin very similarly seems to exclude from heterochromatin but also there
are transcription factories, regions where we have very highly active transcription
that might come closer into proximity to each other.
The first big lesson is therefore that we have two different compartments; but
then it is possible to step a little bit in with Topologically Associated Domains:
TADs.
