Download RNA Polymerase II analysis in Drosophila Melanogaster

Lola Rouvinez
Mentor : Michael Frochaux
RNA Polymerase II analysis in Drosophila Melanogaster
Aim
Most of the differences in nucleotides between organisms are situated in noncoding DNA
regions. These non coding regions affect the expression levels of genes thus making
phenotypes depending more on differential expression rather than genes mutation.
This project aim is to study the behaviour of RNA polymerase II in different Drosophila
melanogaster strain.
RNA Polymerase II role
The RNA polymerase II (RNA Pol II) is the enzymes that transcribes the DNA into
messenger RNA (mRNA). The binding of the RNA Pol II to the gene, is dependant on other
proteins called transcription of a gene by helping the polymerase to bind. By modulating the
binding events, these factors modulate the expression of the gene where the Polymerase can
start to transcribe the gene.
Methods
1) ChIP
The principle is that DNA-binding proteins in cells are cross-linked to the DNA that they are
binding. By using a specific antibody, we can immunoprecipitate the protein–DNA complex.
After the crosslinking, the cells are bursted and the DNA is broken into pieces by sonication.
The DNA is purified with an antibody -pol II, then they are heated to reverse the crosslinking, and the DNA is separated from the proteins. We then verify the enrichment in the
region, that we are interested in is targeted by a qPCR.
2) Bioinformatic
The analysis pipeline is as follow
a)
b)
c)
d)
Alignement to reference genome with bowtie2
Peak Calling with HOMER
Peak profiling with HOMER
Visualisation with UCSC genome browser
Setup : the analysis was performed on Anti-RNA Pol II sample.
Results
RNA Pol II ChIP-seq analysis
1234
Agarose gel of the sonicated chromatin
The smear of the chromatin extend from 200bp to 2000bp indicating a successful
sonication. The target fragment length for library preparation is 200 bp, thus the
amount of DNA to be extracted must be higher to compensate for the loss due to
high length fragment.
Legend :
1. anti-Cad chromatin
2. anti-Pol II chromatin
3. anti-Rel chroatin
4. Ladder
Quantification of the DNA extracted
140317 Concentration
unit
ng/mL
Pol II IP
Pol II 1% input
Pol II chrom 1
Pol II chrom 2
dilution
0,64
2,22
6,46
74,3
Concentration
Volume
ng/uL
uL
103
0,06592
104
0,23088
105
0,6783
106
7,8758
Total amount
ng
52
3,42784
53
12,23664
54
36,6282
55
433,169
Quantification of the DNA extracted
The amount yielded is sufficient for library preparation although we have to take into account
that some of this DNA is too big to be sequenced.
Enrichment
Sample
Primer
140317 Pol II Fer1_0
140317 Pol II Fer1_1
140317 Pol II ChIP-Neg
Adjsuted
Pull down
Enrichment
Input
over CN
18,441452 1,9165892606 4,2074248401
17,776681 1,1996492777 0,2827835297
11,2934685 0,4555254897
Enrichment of Fer1HCH genes show enrichment in Pol II binding region versus non binding
region ChIP-Neg.
Analysis of RNA Pol II binding sites
Tags distribution near Pol II peaks
This figure shows the distribution of tags near the RNA Pol II separated by their presence on
either the positive strand and the negative strand. Combining the tags and looking at both the
control and the IP experiment, we obtain the following figure.
RNA Pol II peak profile
Visualisation of Pol II ChIP-seq in UCSC genome browser
Fer1HCH, used to measure the enrichment, show a highly enriched profile througout the
gene, hinting that the gene was completely expressed
Per exhibit a peak at the beginning of the coding region (TSS) but not afterwards. The gene
expression is probably temporary posed.
Discussion
The enrichment and the profiling of RNA Pol II ChiP-seq are satisfying although the
sonication leads to a large array of fragments. It could be useful to test different sonication
protocol to optimize the process.
The peak profiling of the RNA Pol II shows the expected pattern, showing that the data can be
trusted.
The visualisation in the genome browser is interesting to get some information, but it is less
than sufficient. Moreover, the data only show a snapshot of the regulation and more
experiment are needed to obtain a significant depth for the analysis.
Conclusion
Although the information provided by the RNA Pol II ChIP-seq is good, to obtain a better
understanding of the regulation of genes, other experiments such as RNA-seq and chromatin
mark ChIP-seq would complement perfectly this analysis.
Finally I am grateful to the foundation « Schweizer Jugend Forscht » for this amazing
opportunity and also to my mentor who helped me a lot to do this project.