Knock down of SUN-1
Of the SUN-Domain Family
And it‘s influence on chromatin
tethering to the nuclear lamina
OVERARCHING GOAL
SUN-1
Of the SUN-Domain Family
Chromatin is organized in the nucleus
by anchoring specific regions of
chromatin to the inner nuclear lamina
Euchromatin (active)
Heterochromatin (silent)
OVERARCHING GOAL
If a gene is tetherd to the nuclear lamina
it gets surpressed as we see here.
SUN-1
Of the SUN-Domain Family
OVERARCHING GOAL
For example: CEC-4 is a protein anchored at
the nuclear lamina. It binds heterochromatin.
SUN-1
Of the SUN-Domain Family
But as one of the two parts of the chromatin
still binds to the lamina if CEC-4 is knocked
down, we can conclude that CEC-4 is not the
only protein that binds this part to the lamina.
OVERARCHING GOAL
So we want to find other factors that
connect chromatin to the nuclear
SUN-1
Of the SUN-Domain Family
lamina and thereby surpress genes.
For this we use RNAi to knock
down a gene of interest: SUN-1
We are doing this in C. Elegans on
Chromosom IV in embryos in an We use Nonobody staining against lmn-1::gfp.
early stage of development.
Then we use DNA FISH to determin the distance
between the lamina and parts of
the chromatin that are linked to to SUN-1.
METHODS
RNAi: RiboNucleicAcid interference SUN-1
Of the SUN-Domain Family
So the RISC complex degrades
We use siRNA that link to the these mRNA and hinders the
mRNA of SUN-1. expression of SUN-1
METHODS
SUN-1
Of the SUN-Domain Family
Nonobody staining against lmn-1::gfp
We use the NanoBody booster because
it does no have all the chains that
antibodys do have, so it is more stable,
soluable and lighter.
METHODS
SUN-1
Of the SUN-Domain Family
DNA FISH (Fluorescence In Situ Hybridisation)
We use single stranded probe DNA that is labled
with flap tails. These bind to the target repeat
regions in the after heating single stranded DNA.
When fluorescent-labeled oligos bind, the repeats
becomes visible under the microscope as dots.
OUR SPECIFIC GOAL
SUN-1
Of the SUN-Domain Family
SUN: Sad1p, UNC-84
The SUN trimer is composed of
SUN-1 Homotrimers or
SUN-1 / SUN-2 Heterotrimers SUN Domain binds Lamin and
KASH Domain (Part of the Nesprin)
PREDICTION
Direct influence of SUN-1
SUN-1
Of the SUN-Domain Family
If SUN-1 is knocked down the chromatin repeats
are still tethered to the lamina because SUN-1 is
not responsible for this tethering directly.
Known functions
Indirect influence of SUN-1 SUN-1 is bound to the LINC (Linker of
Nucleosceleton and Cytosceleton)
No mechanical force from the cytosceleton can complex, that transmitts mechanical force
influence the arrangement of the chromatin, so the from the cytosceleton to the nucleus to
chromatin will be arranged differently by default. influence the chromatin organisation.
Known from experiments: SUN-1 tethers the telomer associated
If SUN-1 is knocked down, there is no connection protein Pot-1 to the nuclear lamina as well.
between Pot-1 and the lamina, so the telomers will
not be bound to the lamina anymore.
TRUBBLESHOOTING
Because of it‘s age the embryo had many cells and
it was difficult to identify individual nuclei because
they overlaped eachother. So manual analysis was
SUN-1
Of the SUN-Domain Family
challenging.
The mask does not show many nuclei, but it was
still possible to get a few results by analyzing them
automated.
So there were too many cells for staining all the way Blue: DAPI
through the whole organism until the center, so the Red: repeats
Green: Lamina
DAPI is only visible in the outer layers of the cells.
The lamina staining did not work as well so we
substracted the DAPI signal from the lamina signal
to approximate where the nuclear lamina is. This is
not exact but the best we could do.
Nuclei mask
OUR RESULTS
MANUAL MEASURING SUN-1
Of the SUN-Domain Family
The distance between the border
of the nucleus and the SUN-1 was But the approximation manually was
X = 2.80 pixels = 0.3 μm difficult so we used another technique to
find a distance per computing.
The diameter of a nucleus in our specific
developmental stage is approximately 0.3 μm,
so it seems the gene of interest is located at the
nuclear lamina.
Blue: DAPI
Red: repeats
Cropped Measurement distance FISH - Lamina
Green: Lamina
OUR RESULTS
AUTOMATED ANALYSIS SUN-1
Of the SUN-Domain Family
Napari threshold 1300 Napari threshold 1200
Napari did not recognise the dots. If the threshhoild was set to 1200 everything was blue and if
the threshold was set to 1300 nothing was blue. It was not possible to only color the dots.
OUR RESULTS
AUTOMATED ANALYSIS SUN-1
Of the SUN-Domain Family
Control :
Most of the chromatin is with a distance
smaller than 1 μm still tethered to the
Our result:
lamina or near to it.
The distance between our chromatin
and lamina is around 0.1 μm, so it is
still bound to it.
OUR RESULTS
AUTOMATED ANALYSIS SUN-1
Of the SUN-Domain Family
P-value: 6.99 10-6
Here we see the overlap. The P-
value is smaller then 0.5 but we
have much less datapoints than
the control.
Datapoints:
Control: 300
Results: 5
CONCLUSION
SUN-1 is not a necessary factor to tether
the Chromatin IV Repeats to the lamina.
SUN-1
Of the SUN-Domain Family
We only got 5 points out of the automated So we would have to repeat the experiment
analyzation. This is neither representative with younger embryos for who the lamina
nor significant. and DAPI staining would work better to gain
more results to make a better conclusion.
The distances may not be precise as well
because we used the nuclei mask for
measurement instead of the lamina
staining which would have been more
precise.
IDEAS FOR
ADDITIONAL
EXPERIMENTS
SUN-1
Of the SUN-Domain Family
Since SUN-1 is important for the force
transmission from the externe to the
nuclei, it would be interesting to look at
the whole organisation of the
chromosoms. We could use
chromosome-painting probe to be able
to observe the whole chromosom and its
organisation without the influence of
mechanical force.
It would also be interesting to see if the
telomers would still be located to the
lamina if SUN-1 was knocked down.