A Potential Second Promoter in the Cd4 Gene that Functions at the DP Stage of T-cell Development
Walker Shaw and Sophia Sarafova
Biology Department, Davidson College, Davidson, NC 28036
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Background
Methods
CD4 is a protein expressed on certain cells in the immune
system, predominantly T- lymphocytes. These T-cells develop
by entering the thymus as a Double Negative T-Cell Progenitor
(no CD4 or CD8 expressed on their cell surface). In the
thymus they develop into Double Positive T- Cells (both CD4
and CD8 expressed on cell surface), before differentiating into
Single Positive CD4 (T-helper) or CD8 (T-cytotoxic) cells in the
immune system.
N
Results
Our 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.
Thymus cells
PNA Cell Panning
PNA
Reserve for
FACS staining
DN CELL
N
Cells Float
DP CELL
Cells Stick to Plate
Immunostaining
of Cells
A
Two color immunostaining
and flowcytometry of fresh
B10.A thymocytes
84.37%
6.91%
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
(occuring in intron 1). We observed 1000
900
800
both the 320bp and a larger product 700
600
500
(lanes 3 and 4 gray and black
400
arrows), indicating that an additional 300
transcription initiation site exists in 200
DP cells. The bands in lanes 1 and 100
2 represent DNA contamination.
B
Two color immunostaining
and flowcytometry of PNA-panned
B10.A thymocytes
95.58%
1.52%
C
CD8 antibody
Tagged with FITC
N
N
CD8 CELL
CD4 CELL
CD4 antibody
Tagged with PE
Throughout T-cell development, CD4 expression is
regulated by several different known transcriptional elements,
including a silencer, a promoter, a DP enhancer, a distal
enchancer, and a proximal enhancer. In DP cells, it has been
demonstrated that the DP enhancer is necessary for the
expression of CD4 on the cell membrane. We speculate that
there may be a different, unknown promoter region that is
“turned on” by this DP enhancer and other transcriptional
elements that are different from the promoter used for CD4
expression in mature T-helper cells.
…
P
Ex1
Sil
TATA
Ex2
Ex3
Ex4
Ex5
DPe
Isolate Panned Cells’ RNA with Trizol®
BEADS
Avidin-coated beads
Bind mRNA to BEADS
…
Known mRNA Product
ATG
Ex1
Ex2
Ex3
Ex4
Ex5
Potential DP mRNA Product
ATG
Ex2
Ex3
Ex4
Ex5
…
…
5’ RACE
on Beads
mRNA bound
5’ primer TTTTTTT-3’
3’-AAAAAAA-----5’
CD4
We 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.
mRNA in solution
5’-primerTTTT
3’AAAA------5’
(same products,
not attached to beads)
5’-primer TTTT------------CCC-3’
3’-GGG-capfinder
CD4
5’ RACE
In Solution
Reverse transcription of the first strand; Cs
added on 3’ end by the reverse transcriptase.
Reverse transcription of the
second strand using capfinder
5’-primer TTTT------------------CCC-3’
3’-primer AAAA -----------------GGG-capfinder-5’
Acknowledgements
1
2
3
4
Primer
dimers
Importance of Findings
5’-primer TTTTTTT
biotin
ATG
320bp
Check purity
By FACS
CD4 Locus
?
500bp
Future Work
• Investigate strategies for further purification of DP cells
cDNA products
PCR using Cd4 specific exon3 primer
exon3
5’-primer TTTT--------------------------------CCC-3’
3’-primer AAAA -------------------------------GGG-capfinder-5’
GGG-capfinder
The CD4 gene in humans has a transcriptional control region
in intron 1 that is speculated to function as a second promoter. This
second promoter is thought to be responsible for CD4 expression
in human macrophages. In mice, macrophages do not express
CD4 however, implying that a second promoter does not exist. Our
findings indicate that there is initiation of transcription starting 3’ of
the known promoter in DP cells, presumably in intron 1. We think
that this evidence supports the theory that there is a second
promoter in the murine Cd4 gene that functions to support CD4
expression specifically in DP cells. We speculate that the human
and murine second promoters may be related.
exon3----capfinder
exon3----capfinder
Analyze PCR products on agarose gel (see Results)
• 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
determine the potential alternate transcription start site(s) and help
look for a potential promoter