Transcript Determining the Existence of a Second Promoter in the CD4

Determining the Existence of a Second Promoter in the CD4
Gene With Chromatin Immunoprecipitation
Louisa Wall and Sophia Sarafova
Biology Department, Davidson College, Davidson, NC 28036
Methods
Background
The T cells of the immune system become either helper T cells,
which express a glycoprotein called CD4 on their surface, or killer T
cells, which express CD8. The T cell lineage commitment process
begins when the T cell precursor migrates to the thymus as a
double-negative cell, lacking expression of CD4 and CD8. In the
thymus, the cell becomes double positive, which means that it
produces both CD4 and CD8. The cell must then undergo positive
selection, a complex progression involving enhancers, promoters,
silencers, and transcription factors, in order to develop into a
functional CD4 SP T cell. The region of the gene sequence that
controls CD4 expression during the entirety of the process has been
located between LAG-3 and triosephosphate isomerase I gene. A
single promoter (P1) has been characterized on the mouse (Mus
musculus) CD4 locus. In humans, a second promoter (P2) has been
discovered, which suggests that a second promoter may also exist
in mice.
DoubleNegative
Extract thymus cells from mouse
The CD8 antibody
(tagged with FITC)
Some saved for immunostaining
The CD4 antibody
(tagged with PE)
Isolate the DP cells with PNA panning
(The DP cells stick to the PNA
At the bottom of the plate)
= PNA
75%
Stained cells before
PNA panning
6%
0.2%
81%
Immunostaining of cells
2%
Stained cells
after
PNA panning
Step1. Crosslink proteins to the DNA in each DP cell
with formaldehyde
Step 2. Lyse cells and shear the DNA
with Ultrasonic cleaner
=proteins
Step 4. Reverse the cross linking
and save the supernatant.
DoublePositive
CD4
1%
(save a portion of the sheared DNA to compare
To chIP samples)
(save)
(dispose)
CD8
TBP
TBP
The TATA binding protein initiates transcription by binding to promoter
regions and bending the double helix DNA so that the two strands can
be split. Therefore, if the second promoter exists in the region of DNA
that expresses CD4, TBP will bind to it.
bead
bead
Step 3. Add pre-blocked beads
and TBP antibody. The antibodies
will bind to TBP and to the beads.
The weight of the beads will drag
the DNA fragments with attached
TBP to the bottom of the tube.
Remove the other fragments
of DNA.
Step 5. qPCR: compare DNA from input sample (from step 2)
to chIP samples and negative controls (antibody isotype IgG, no antibody, and silencer primer set)
Experimental primers: 3 sets of overlapping primers in the supposed promoter 2
Region
Exon 2
DNA region of
Promoter 2
= primer set 1, 365 bp
= primer set 2, 406 bp
= primer set 3, 384 bp
product
product
product
(note: these primers
Did not work in PCR
Testing, so they were
Not used in qPCR)
input
TBP
ChIP samples, negative control
Results
Our first TBP antibody, provided with our Diagenode chIP kit produced no qPCR
results. It was only proven to react with human cells. Although the similarities in
the TBP protein sequences in human and mouse suggested that the antibody
would work, our western blot showed the it did not (A).
A)
Molecular Weight Marker
Future Work
High concentration human protein
1. Find an TBP antibody that works for mouse.
Low concentration human protein
2.Troubleshoot the chIP experimental process so that a significant amount of
DNA can be obtained from the immunoprecipitation step.
10^6 mouse cells
10^5 mouse cells
With our second antibody, the DNA concentration decreased significantly after the
immunoprecipitation step and the negative control contained more DNA than either
of the chIP samples (B), which suggests that the second antibody was not effective
on mice
B)
Input
Concentration
57.8 ng/L
chIP sample 1
Concentration
4.8 ng/L
chIP sample 2
Concentration
3.4 ng/L
No Antibody
Control
Concentration
4.5 ng/L
3. If promoter 2 does exist, investigate the relative activity of the supposed
promoter 2 by comparing the results of double-positive cells with singlepositive CD4 cells.
Acknowledgements
We would like to thank Amy Becton for taking car of our mice and Chris Van Rooyen for
Maintaining our lab equipment. We would also like to thank Qing Yu for her promoter 1 and
Silencer primer sets.