CD4 is a transmembrane glycoprotein expressed on the cell surface of thymocytes and mature T- lymphocytes with helper function. T-cells develop in the thymus and their stages of development are defined by the level of CD4 and CD8 expression. Briefly, T-cell progenitors enter the thymus as DN cells, expressing no CD4 or CD8 on their cell surface. Upon successful rearrangement of TCR they develop into DP thymocytes, which express both CD4 and CD8. Finally, positively selected DP thymocytes differentiate into CD4 or CD8 single positive cells.

The DP enhancer is necessary for the expression of CD4 protein in DP cells. We speculate that there could be a different, unknown promoter region that is “turned on” by this DP enhancer instead of or in addition to the promoter used for CD4 expression in mature T-helper cells. The diagram below shows the Cd4 gene and the transcriptional elements that regulate CD4 expression. It also shows how a potential secondary promoter could express the same protein.

A Potential Second Promoter in the Cd4 Gene that Functions at the Double Positive Stage of DevelopmentWalker Shaw and Sophia Sarafova

Biology Department, Davidson College, Davidson, NC 28036

Background Information

We identified a second transcription start site for the murine Cd4 gene, located in the first intron, that functions at the DP stage of T cell development . We think it may represent a second promoter in the Cd4 gene, similar to the one described in humans. To confirm the presence, identify, and understand the function of this transcriptional control element we will • Repeat current work with a FACS sorted pure population of DP cells.• Repeat the analysis using CD4 SP cells to determine if the alternate transcription start site is unique to DP cells. • Purify and sequence the DNA segments from the 5’ RACE to determinethe potential alternate transcription start site(s) and help look for a potential promoter.•Identify the putative second promoter of the Cd4 gene by transient transfection into AKR1G1 double positive cell line of constructs containing DNA from upstream of the newly identified transcription start site.

ResultsOur FACS stain data showed

that PNA panning of thymocytes decreased the CD4 cell contamination by 78% (compare A and B) yielding a 95% pure DP cell population (B), which was used for RNA isolation and 5’ RACE.

Cd4 gene transcription products were visualized by RT followed by Rapid Amplification of cDNA 5’ Ends (5’ RACE) and agarose gel electrophoresis (C). Normally, a 320bp product is expected if the known Cd4 promoter is used in the DP cells. Products of larger size are unexpected and can be explained only if Cd4 transcription is initiated from a different promoter in DP cells (occurring in intron 1). We observed both the 320bp and a larger product (lanes 3 and 4 gray and black arrows), indicating that an additional transcription initiation site exists in DP cells. The bands in lanes 1 and 2 represent DNA contamination.

Conclusions and Future Work

84.37%

6.91%

95.58%

1.52%

NO cDNA

(Contro

l)NO R

T

(Contro

l)

Liquid

RT

RT on

Beads

320bp

Methods

Thymus cells

CD8 antibodyTagged with FITC

CD4 antibodyTagged with PE

TTTTTTT-3’3’-AAAAAAA-----5’

5’-primerTTTT3’AAAA------5’

5’-primer TTTT------------CCC-3’

PNA Cell Panning

Cells Float Cells Stick to Plate

Immunostaining of Cells

Check purityBy FACS

Isolate Panned Cells’ RNA with Trizol®

Reserve for FACS staining

mRNA bound mRNA in solution

Bind mRNA to BEADS

5’ RACEon Beads

5’ RACEIn Solution

CD4 Locus

…Known mRNA Product

Ex1 Sil Ex2

ATG

Ex3 Ex4 Ex5

?TATA

Ex1 Ex2

ATG

Ex3 Ex4 Ex5

Potential DP mRNA Product

Ex2

ATG

Ex3 Ex4 Ex5

CD4

CD4

…

…

…

P DPe

5’-primer TTTT------------------CCC-3’

exon3----capfinderexon3----capfinder

3’-GGG-capfinder

5’ primer

TTTTTTT5’-primer

BEADS

Avidin-coated beads

biotin

3’-primer AAAA -----------------GGG-capfinder-5’

Reverse transcription of the first strand; Csadded on 3’ end by the reverse transcriptase.

Analyze PCR products on agarose gel (see Results)

(same products, not attached to beads)

Reverse transcription of the second strand using capfinder

cDNA products

PNA

5’-primer TTTT--------------------------------CCC-3’ 3’-primer AAAA -------------------------------GGG-capfinder-5’

GGG-capfinder

exon3 PCR using Cd4 specific exon3 primer

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

A

B

Two color immunostaining and flowcytometry of fresh B10.A thymocytes

Two color immunostaining and flowcytometry of PNA-panned B10.A thymocytes

C

500bp

1 2 3 4Primerdimers

100

200

3004005006007008009001000

AbstractCd4 gene expression is regulated by several different known

transcriptional elements, including a silencer, a promoter, an immature enhancer, a distal enhancer, and a proximal enhancer. Different combinations of these elements are employed to regulate Cd4 expression in mature CD4 cells versus immature double positive (DP) cells. In humans an additional transcriptional control element has been described that serves as a second promoter for Cd4 and is proposed to be responsible for Cd4 expression in macrophages and some dendritic cells. Since mice do not have cell surface expression of CD4 in the macrophage, the presence of a second promoter and its potential function during T cell development has not been investigated so far. To determine if a second promoter is present in the first intron of the Cd4 gene and utilized during the DP stage of T cell development, we looked for new transcription start sites in RNA from murine DP cells by 5’RACE. In addition to the expected 320bp band that indicates transcription from the known promoter, we observed a strong 500bp band, indicating that there is another transcription start site in the Cd4 gene. We are in the process of cloning and sequencing all the Cd4 transcription products and identifying the second promoter. Whether the putative second promoter is used in mature CD4 cells remains to be determined.

AcknowledgementsWe would like to thank Amy Becton for maintaining our mouse colony, Susan Sharrow (NCI, EIB) for antibodies and FACS advice, and Terry Guinter (NCI, EIB) for help with the PNA panning protocol.