Jorge Santamaría
Mentor: Marianna Feretzaki
Institution: ISREC, EPFL
Identify the transcription factors of telomere long non-coding RNAs
CTCF and SP1 as two possible transcription factors of the telomere.
INTRODUCTION
The DNA is constantly transcribing into RNA in our organism. It’s a common
and necessary function of our bodies, which need the RNA to realize a lot of
different functions inside our cells. The transcription factors are proteins, which
are essential for the activation of the process of transcription.
However, the region of the telomere is also transcribed but in a different type of
RNA. The long non-coding RNA (IncRNA) is a type of RNA which isn’t
translated into proteins and which function is unknown. If we identify all the
transcription factors of the telomere, we would be able to know the function of
the IncRNA. By removing all the transcription factors we could see which
function gets deactivated or reduced in the cells.
With our project we try to verify that two proteins (CTCF and SP1) act as
transcription factors that bind in the telomere and control the transcription of the
IncRNA.
METHODS
1. Chromatin Immunoprecipitation (ChIP):
Several parts compose this process.
First we did a crosslinking of the proteins on DNA by using formaldehyde, which
produces a strong binding between proteins and DNA.
In second place we expose the DNA and the proteins to a sonication. The
sonication is a method, which fragmentizes the DNA in smaller fragments.
Right after that we did an immunoprecipitation. We added beads-attached to
antibodies to pull down target proteins bound on DNA.
Next step was the reverse crosslinking. We used it to produce the unbinding of
the proteins from the DNA obtaining the parts of the DNA, in which we were
interested.
Last step was the qPCR. Using this method we duplicated the specific parts of
the DNA we had obtained and when we reached a specific amount of them, we
could detect the target sequence we were looking for.
2. Western Blooting
Several parts compose this process.
-Gel electrophoresis: We ran the proteins in a special gel by electrophoresis to
separate them.
-Gel transfer: The proteins were transferred to a membrane.
-Blocking: We used a cow serum to block all the proteins, so in the next step the
antibodies won’t bind unspecific proteins.
-Detection of proteins: By using specific primary antibodies we detect the target
proteins we were looking for. The antibody is capable of destroy the binding
between the target protein and the serum allowing its detection.
-Chemiluminescence: The addition of a secondary antibody that detects the
primary antibody produces this specific reaction. The secondary antibody has
an enzyme (HRP) that with luminol produces emits energy in form of photons
when the secondary antibody detects the primary antibody.
Detection and chemiluminescence
Protein Primary antibody Secondary antibody*
*Chemiluminescence produced by the enzyme HRP
RESULTS
Results: DNA Sonication
HeLa cells were isolated from liquid cultures and diluted in 1%
formaldehyde to crosslink the proteins on the DNA. Following
crosslinking the cells were sonicated to for 10 minutes. Two
samples of sonicated DNA were subjected to agarose
electrophoresis. After electrophoresis we observed that most DNA
fragments range between 200 – 700 base pairs.
Results: Detection of the proteins
Proteins were seperated in a gel with electrophoresis and transferred to a
membrane. We detected the Ctcf, Sp1 and the loading control protein
hnRNPA1 using the appropriate primary antibodies. Comparing the proteins
before and after sonication, we found that sonication broke a portion of the Ctcf
and Sp1 transcription factors.
Results: ChIP  Transcription Factors Bind the Telomeric Region
With chromatin immunopercipitation we showed that Trf2, which is a protein that
protects the end of the chromosome, binds at the telomere. We also found that
the transcription factors Ctcf and Sp1 bind the subtelomere, a region that
contains a promoter adjacent to the telomere. These transcription factors may
regulate the transcription of the telomeric long non-coding RNA.
CONCLUSION & DISCUSSION
In our study we found that both proteins, CTCF and Sp1, are transcription factors
that bind the telomere. By using ChIP and qPCR we observed that the percentage
of the proteins on the telomeric sequence is very high in multiple parts of the
telomere, which shows that they bind frequently in the telomere. TRF2 is another
protein that protects the telomere and we used it as a positive control to compare
the results of CTCF and SP1. We also did qPCR from an IgG control antibody to
demonstrate that the antibodies do not interact with the DNA. The IgG antibody did
not bind on the DNA, which shows that the results of CTCF and SP1 are accurate.
The binding of CTCF and SP1 was higher in some parts than TRF2.This final result
demonstrates definitely that CTCF and SP1 are transcription factors that bind the
telomere.
ACKNOWLEDGMENTS
I would like to thank Marianna Feretzaki for guiding me through this amazing project
and Dr. Joachim Lingner for his hospitality. I am grateful to Alice Emery-Goodman
for organizing the science week.