Download Isolating and Identifying Transcription Factors

Isolating and Identifying Transcription Factors that
Bind the Cd4 Promoter
Matthew C. Surdel and Sophia D. Sarafova
Davidson College Biology Department
I. Background and Observations
II. Goal
Isolate and identify the putative transcription
factor that binds to the promoter region of the
Cd4 locus at the P3 site.
CD4 and CD8 cells are essential in immune system function. Both are classified as Tcells and derive from a common precursor, but only CD4 cells express the Cd4 gene. CD4 is a
transmembrane glycoprotein that modulates the T-cell receptor signal and therefore the
magnitude of the immune response. Having the correct amount and timing of Cd4 expression
is essential for the proper development and function of the CD4 cells. Not surprisingly, Cd4
B. DNA Affinity Chromatography and SDS-PAGE
expression is a highly controlled process; regulatory elements found to date include a promoter
(containing four binding sites – P1-P4), a silencer, a mature enhancer, and a thymocyte
Wash beads with 1 X G/B
To purify the protein of interest, we used the known
enhancer. In the promoter region, the proteins that bind to the P1, P2, and P4 regions have
Binding Buffer I
region from the Cd4 locus (P3) and a mutant form of that
been identified, while P3 remains unknown as shown in Figure 1.
1
Previous research has shown a protein-DNA complex formation specific to the P3
region (D06) that does not bind the protein we are looking for.
Add AKR1G1 nuclear extract solution sequence that does not form with the D06 sequence, which differs by only four base pairs from
containing unlabeled P3 or D06 as
the P3 site but reduces promoter function by 80% (Figure 2 and data not shown). Furthermore
A. Preparing DNA on Streptavidin Coated Beads
it has been shown that this protein-DNA complex does not appear when using CD8 nuclear
specific competitor, salmon sperm as
5’
extract (Figure 2C), implying that CD8 cells may not contain this protein.
non-specific competitor, spermidine,
3’ ss DNA
5’
3’
To further understand the control of Cd4 expression we wish to isolate and identify
3’
buffer, and water
5’
5’
transcription factors that bind to the P3 site of the Cd4 gene promoter region. DNA affinity
3’
Annealing DNA Strands Proteins attach to DNA
2
chromatography and SDS polyacrylamide gel electrophoresis (SDS-PAGE) were used to
Heat to 95° C and gradually
identify proteins that bound to the P3 DNA sequence, but not the D06 sequence. To ensure
cool to 4° C, Store at -20°
Spin beads to from pellet, remove
5’
specificity of the transcription factor of interest we used the D06 mutant sequence as a specific
3’ C 5’
3’
supernatant containing proteins that
competitor to P3 and vice versa. This protein would be the second transcription factor specific
5’
5’
3’
3’
did not bind or that bound to the
to CD4 cells identified to date.
Phosphorylation of 5’ Ends
specific and non-specific competitors
of ds DNA using T4
Figure 1 (Sarafova and Siu, 2000). A schematic
Polynucleotide Kinase
diagram of the CD4 promoter and its binding factors.
3
P1, P2, P3 and P4 are the functionally important
III. Approach
binding sites. The distance between the four binding
sites is shown in base pairs. The known binding
factors for each site are indicated.
Figure 2 (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 CD4+CD8- 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).
Wash with 1X G/B Binding Buffer I
and then with 1X G/B Binding
Buffer I with 0.25M NaCl to remove
any loose proteins
5’ overhangs are complimentary,
allowing for ligation to occur
Mix and ligate biotinylated pieces
with non-biotinylated pieces using
T4 DNA Ligase
4
Elute proteins with 2 mM (E2), 4
mM (E4), and 8 mM (E8) biotin
Separate beads by centrifugation
Run proteins on SDS Gel
+
IV. Results
Attach DNA to streptavidin
coated beads by streptavidin
and biotin interaction
For results, see Figure 3
C. Southwestern Blot
1) Run protein from DNA affinity chromatography on SDS gel
2) Transfer protein to PVDF membrane
3) Renature proteins
4) Incubate with biotinylated P3 or D06 sequence
5) Incubate with avidin-horseradish peroxidase (HRP)
6) Develop
KDa
After successful completion of DNA affinity chromatography and
MW
P3
D06
E4
E8 E2
E4
E8
Marker E2
SDS-PAGE, one distinct band was seen (Figure 3). This band represents
proteins that were pulled down using DNA affinity chromatography with
the P3 sequence that were absent when D06 was used indicating
specificity for the P3 sequence (shown in Figure 3).
This band was analyzed by liquid chromatography tandem mass
92.5 spectrometry (LC-MS/MS) at Duke Proteomics. The data was viewed in
Scaffold (Figure 4). A transcription factor, upstream binding factor
69 (UBF), was present in the P3 E2 and E4 bands and was of the correct
molecular weight. UBF however does not meet the requirements to be
Figure 3. One specific band of 90 kDa appear in the presence
the transcription factor of interest - it is slightly smaller, does not
of P3 but not D06 DNA
specifically bind DNA, and recruits RNA polymerase I and not RNA
polymerase II (Figure 5).
To determine if our DNA affinity chromatography did in fact purify
a protein specific for the P3 sequence, a southwestern blot was
performed. Results show a band present in nuclear extract and purified
protein using the P3 sequence and DNA affinity chromatography above
100 kDa that was probed with the P3 sequence (Figure 6). This band is
of a different molecular weight that previously thought, but is specific to
the P3 sequence and is present in previous SDS-PAGE gels.
Immediate Future - Two Options
 Analyze band above 100 kDa found in Southwestern blot
 Further purify extract prior to using DNA affinity
chromatography
 Set up a yeast one-hybrid system to identify the protein of interest
Long Term
 Explore the biochemical properties of the protein
 Confirm binding to the P3 region of the Cd4 promoter
 Explore the secondary and tertiary structures of the protein
B
Figure 4. Representation of data analyzed in Scaffold.
(A) Results from LC-MS/MS shows UBF as the only
protein present in both bands (P3 E2 and E4) with the
correct molecular weight. (B) Scaffold viewer
showing specific amino acid sequences that were
found in LC-MS/MS, verifying presence of UBF in
the samples.
Figure 5. UniProtKB/Swiss-Prot database entry for UBF.
UBF helps recruit RNA polymerase I, promoting rRNA
transcription.
P3
MW NE E2
Figure 6. Southwestern blot of
unpurified nuclear extract (NE)
and protein purified through DNA
affinity chromatography (P3 E2)
probed with P3 sequence. A band
92.5 is seen at above 100 kDa in both
69 NE and P3 E2 that is specific for
46 the
P3
sequence.
30 -
KDa
V. Future Directions
A
References
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 factorbinding sites is required for murine CD4 promoter
function. Nucleic Acids Research. Vol. 28 No. 14 2664Acknowledgements
This research was made possible by Merck/AAAS Biochemistry Internship Program, Sigma Xi Grants-in-Aid 2671
Wada, T., Watanabe, H., Kawaguchi, H. 1995. DNA Affinity
of Research, and the Davidson Biology Department. We thank Karen Bernd, Karen Bohn, Doug Golann,
Cindy Hauser, Karmella Haynes, Barbara Lom, Jeffrey Myers, Erland Stevens, Gary Surdel, and Peter Surdel. Chromatography. Methods in Enzymology. 254: 595-604