A Potential Second Promoter in the Cd4 Gene that Functions at the Double Positive Stage of Development
Walker Shaw and Sophia Sarafova
Biology Department, Davidson College, Davidson, NC 28036
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Abstract
Methods
Cd4 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.
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
Reserve for
FACS staining
Cells Stick to Plate
Immunostaining
of Cells
CD8 antibody
Tagged with FITC
CD4 Locus
…
?
P
Ex1
Sil
TATA
ATG
Ex2
Ex3
Ex4
Ex5
DPe
…
Known mRNA Product
ATG
Ex1
Ex2
Ex3
Ex4
Ex5
Potential DP mRNA Product
ATG
Ex2
Ex3
Ex4
Ex5
…
…
CD4 antibody
Tagged with PE
Check purity
By FACS
CD4
Two color immunostaining
and flowcytometry of PNA-panned
B10.A thymocytes
95.58%
1.52%
500bp
320bp
1
2
3
4
Primer
dimers
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
BEADS
Avidin-coated beads
Bind mRNA to BEADS
5’-primer TTTTTTT
biotin
mRNA bound
5’ primer TTTTTTT-3’
3’-AAAAAAA-----5’
mRNA in solution
5’ RACE
In Solution
5’-primerTTTT
3’AAAA------5’
Reverse transcription of the first strand; Cs
added on 3’ end by the reverse transcriptase.
(same products,
not attached to beads)
5’-primer TTTT------------CCC-3’
3’-GGG-capfinder
CD4
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 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.
Conclusions and Future Work
Isolate Panned Cells’ RNA with Trizol®
5’ RACE
on Beads
6.91%
C
Background Information
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.
84.37%
B
PNA
Cells Float
A
Two color immunostaining
and flowcytometry of fresh
B10.A thymocytes
• 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 determine
the 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.
Reverse transcription of the
second strand using capfinder
5’-primer TTTT------------------CCC-3’
3’-primer AAAA -----------------GGG-capfinder-5’
cDNA products
PCR using Cd4 specific exon3 primer
exon3
5’-primer TTTT--------------------------------CCC-3’
3’-primer AAAA -------------------------------GGG-capfinder-5’
GGG-capfinder
exon3----capfinder
exon3----capfinder
Analyze PCR products on agarose gel (see Results)
Acknowledgements
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.