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

MethodsBackgroundThe 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 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.

Some saved for immunostainingThe CD8 antibody (tagged with FITC)

The CD4 antibody (tagged with PE)

Step1. Crosslink proteins to the DNA in each DP cellwith formaldehyde

=proteins

Step 2. Lyse cells and shear the DNA with Ultrasonic cleaner

(save a portion of the sheared DNA to compareTo chIP samples)

Step 4. Reverse the cross linking and save the supernatant.

bead

TBP

(save)

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 fragmentsof DNA.

(dispose)

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 2Region

DNA region ofPromoter 2

= primer set 1, 365 bpproduct

= primer set 2, 406 bpproduct

= primer set 3, 384 bpproduct(note: these primers Did not work in PCRTesting, so they wereNot used in qPCR)

Double- Negative

CD4

Double-Positive

CD8

Extract thymus cells from mouse

Isolate the DP cells with PNA panning

= PNA

TBP

(The DP cells stick to the PNAAt the bottom of the plate)

Immunostaining of cells

Stained cells before PNA panning

Stained cells afterPNA panning

TBP

Future Work1. 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.

Exon 2

ResultsOur 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).

Molecular Weight Marker

High concentration human protein

Low concentration human protein

10^6 mouse cells

10^5 mouse cells

75%1%

6%

81%0.2%

2%

input

ChIP samples, negative control

A)

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

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) AcknowledgementsWe 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.