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

1
Determining the Existence of a Second Promoter in
the CD4 Gene With Chromatin Immunoprecipitation
Louisa Wall and Sophia SarafovaBiology
Department, Davidson College, Davidson, NC 28036
Methods
Background
Extract thymus cells from mouse
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.
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
Stained cells before PNA panning
75
1
6
81
0.2
Immunostaining of cells
Stained cells after PNA panning
2
Step1. Crosslink proteins to the DNA in each DP
cell with formaldehyde
Double- Negative
Step 2. Lyse cells and shear the DNA with
Ultrasonic cleaner
Step 4. Reverse the cross linking and save the
supernatant.
(save a portion of the sheared DNA to compare To
chIP samples)
Double- Positive
(save)
(dispose)
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.
CD4
CD8
TBP
TBP
bead
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
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
TBP
DNA region of Promoter 2
primer set 1, 365 bp product
primer set 2, 406 bp product
primer set 3, 384 bp product (note these
primers Did not work in PCR Testing, so they
were Not used in qPCR)
input
Results
ChIP samples, negative control

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)
Future Work
Molecular Weight Marker
High concentration human protein

• 1. Find an TBP antibody that works for mouse.
• 2.Troubleshoot the chIP experimental process so
  that a significant amount of DNA can be obtained
  from the immunoprecipitation step.
• 3. If promoter 2 does exist, investigate the
  relative activity of the supposed promoter 2 by
  comparing the results of double-positive cells
  with single-positive CD4 cells.

Low concentration human protein
106 mouse cells
105 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
Acknowledgements
Input Concentration chIP sample 1 Concentration chIP sample 2 Concentration No Antibody Control Concentration
57.8 ng/?L 4.8 ng/?L 3.4 ng/?L 4.5 ng/?L
B)
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.