A Potential Second Promoter in the Cd4 Gene that Functions at the DP Stage of T-cell Development

1
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
Results
Background
Methods
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. 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 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.
A
Two color immunostaining and flowcytometry of
fresh B10.A thymocytes

• 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.
• 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.
  

84.37
Thymus cells
6.91
B
PNA Cell Panning
Two color immunostaining and flowcytometry of
PNA-panned B10.A thymocytes
PNA
Reserve for FACS staining
95.58
Cells Float
Cells Stick to Plate
1.52
Immunostaining of Cells
C
RT on Beads
Liquid RT
CD8 antibody Tagged with FITC
500bp
320bp
CD4 antibody Tagged with PE
Primer dimers
1 2 3 4
Check purity By FACS
Importance of Findings
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.
Isolate Panned Cells RNA with Trizol
BEADS
Avidin-coated beads
Bind mRNA to BEADS
TTTTTTT
5-primer
CD4 Locus
biotin
?


mRNA bound
mRNA in solution
5 RACE on Beads
5 RACE In Solution
TATA
Sil
DPe
Known mRNA Product
TTTTTTT-3
5 primer
5-primerTTTT
3-AAAAAAA-----5
3AAAA------5

Reverse transcription of the first strand
Cs added on 3 end by the reverse transcriptase.
Potential DP mRNA Product
(same products, not attached to beads)
5-primer TTTT------------CCC-3
Future Work
3-GGG-capfinder

Reverse transcription of the second strand
using capfinder

• Investigate strategies for further purification
  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

5-primer TTTT------------------CCC-3
cDNA products
-----------------GGG-capfinder-5
3-primer AAAA
Acknowledgements
PCR using Cd4 specific exon3 primer
exon3
5-primer TTTT--------------------------------CCC-
3
exon3----capfinder
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.
-------------------------------GGG-capfinder-5
3-primer AAAA
exon3----capfinder
GGG-capfinder
Analyze PCR products on agarose gel (see Results)