I. Background and Observations

1
Identifying Transcription Factors that Bind the
Cd4 Promoter Matthew C. Surdel and Sophia D.
Sarafova Davidson College Biology Department

• I. Background and Observations
• CD4SP and CD8SP T cells are essential to immune
  system function and derive from a common
  thymocyte precursor
• Previous research has identified Th-POK, a
  transcription factor, as required and sufficient
  for CD4 lineage choice in presence of Runx3,
  however in the absence of Runx3 and Th-POK, CD4
  cells develop, indicating another signal can
  direct lineage choice (Figure 1)
• T cell development is a complicated process. The
  correct amount and timing of Cd4 expression is
  necessary for proper development and this process
  is highly controlled with a promoter, silencer,
  mature enhancer and thymocyte enhancer found to
  date
• The promoter contains four binding sites (P1-P4),
  the transcription factor that binds the P3 site
  remains unidentified (Figure 2)
• A protein-DNA complex specific to the P3 sequence
  forms with CD4 nuclear extract but is absent when
  using the D06 sequence, which differs by only
  four base pairs but reduces promoter function by
  80 (Figure 3A-B and data not shown)
• Further this protein-DNA complex does not appear
  when using CD8 nuclear extract, implying
  specificity to CD4 lineage (Figure 3C)

II. Hypothesis
The unidentified P3 transcription factor specific
to the CD4 lineage may be necessary or sufficient
for lineage commitment.
III. Approach and Results Yeast one-hybrid system
identifies proteins that bind the P3 sequence of
DNA.
A. Creating Yeast Strains
dsDNA with SalI Compatible Overhangs
5
3
5
3
Linearize pAbAi Vector with SalI
Phosphorylate of 5 Ends of dsDNA
Ligate bait sequence into open plasmid
Verifying Insert in pAbAi Plasmid (Digest plasmid
around insert, Agarose Gel Electrophoresis and
DNA Sequencing)
Linearize Plasmid using BstBI and transform into
Ura3 deficient yeast
Figure 1 (Egawa and Littman, 2008). ThPOK is
dispensable for differentiation of CD4SP
thymocytes. (a) HSA and TCRß expression in total
thymocytes (top), and CD4 and CD8 expression in
HSAlo/- TCRßhi mature thymocytes (middle) and
TCRßB220- peripheral T cells (bottom) in the
absence of ThPOK, CBFß, or both. The CD4-CD8
cells shown with asterisks in Lckcre CbfbF/F and
Lck-cre CbfbF/FZbtb7bGFP/- panels are those that
escaped Cre-mediated inactivation of Cbfb and
hence have normal Cd4 silencing. Data shown here
are representative from two independent
experiments with similar results. (b) Absolute
numbers of HAS CD69 positively selected
thymocytes and HSAlo/- mature CD4SP thymocytes in
the absence of ThPOK, CBFß, or both. Each column
shows cell numbers in a mouse with each of the
genotypes.
pBait Colonies
Uncut DNA
bp Ladder
1
2
3
4
5
6
7
8
9
10
bp
1000 -
200 -
100 -
Figure 2 (Sarafova and Siu, 2000). A schematic
diagram of the CD4 promoter and its binding
factors. P1, P2, P3 and P4 are the functionally
important binding sites. The distance between
the four binding sites is shown in base pairs.
The known binding factors for each site are
indicated.
Figure 4. After digest, pBait plasmid grown up
in E. Coli are run on an agarose gel. pBait
Colonies 2, 3, 5 and 9 have an insert of
approximately the correct size (around 102 bp)
Homologous recombination creates functional Ura3
gene and creates new yeast strain with bait
dependent AbA resistence
Figure 3 (Sarafova and Siu, 2000). Biochemical
analysis of the P3 site. (A) The P3 sequence from
the CD4 promoter is aligned with the consensus
sequences for CREB-1 and NF-1. The two half
sites of each consensus are underlined. Mutant
D06, which causes a significant decrease of
promoter activity (84), is shown, aligned to the
wild-type sequence. (B) EMSA of the P3 with
CD4CD8- D10 cell extract. Two complexes are
indicated with a filled arrow and with a thin
arrow. Non-radioactively labeled competitors are
indicated above the lanes and are used in
50-200-fold molar excess. The sequence of the
competitors is the same as in (A). (C) EMSA with
extracts from five different cell lines and the
P3 probe. The cell lines and their developmental
stage are indicated above each lane. Complexes
are labeled with arrows as in (B).
B. Creating cDNA Library and Transfroming into
Yeast
bp Ladder
LD-PCR Products
kb
4.0 -
1.0 -
IV. Future Directions
0.5 -

• Immediate Future
• Complete Y1H Screen
• Verify with previous technique of DNA Affinity
  Chromatography

Figure 5. LD-PCR products for cDNA library
before purification. Products purified to reduce
incomplete (below 400 bp) products.

• Long Term
• Explore biochemical properties of the protein
• Determine if the transcription factor is
  necessary and/or sufficient to direct CD4 lineage
  commitment

C. Screening Library
References Egawa, T., Littman, D. 2008. The
transcription factor ThPOK acts late in helper T
cell lineage specification and suppresses
Runx-mediated commitment to the cytotoxic T cell
lineage. Nat. Immunol. 9(10) 1131-1139. Gadgil,
H., Luis, J.A., Jarrett, H.W. 2001. Review DNA
Affinity Chromatography of Transcription Factors.
Analytical biochemistry. 290, 147-178 Sarafova,
S., Siu, G. 1999. Control of CD4 gene expression
connecting signals to outcomes in T cell
development. Brazilian Journal of Medical and
Biological Research. 32 785-803 Sarafova, S.,
Siu, G. 2000. Precise arrangement of
factor-binding sites is required for murine CD4
promoter function. Nucleic Acids Research. Vol.
28 No. 14 2664-2671 Wada, T., Watanabe, H.,
Kawaguchi, H. 1995. DNA Affinity Chromatography.
Methods in Enzymology. 254 595-604
To grow yeast cell must have - pAbAi vector with
bait sequence in genome (SD/-Ura selection) -
Contain pGADT7 plasmid with a cDNA insert, each
yeast cell should contain a different gene
(SD/-Leu selection) - Express correct protein
from cDNA insert to bind bait sequence (AbA
resistance)
Acknowledgements This research was made possible
by the Davidson Research Initiative (Mimms
Biochemistry Fellowship), Merck/AAAS Biochemistry
Internship Program, Sigma Xi Grants-in-Aid of
Research, and the Davidson Biology Department.
We thank Karen Bernd, Karen Bohn, Zach Carico,
Doug Golann, Cindy Hauser, Karmella Haynes, Chris
Healey, Barbara Lom, Jeffrey Myers, Walker Shaw,
Darina Spasova, Erland Stevens, Gary Surdel, and
Peter Surdel.