Cross-linking using BrdU oligonucleotides

5.2.1.2

5.2.1.2

5.2.1.2

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Whole cell HeLa extract was used in conjunction with each of the 32P-labelled BrdU oligonucleotides in cross-linking assays to investigate the binding parameters of individual proteins within the intervening sequence. Figure 5.3B demonstrates that each BrdU oligonucleotide is capable of forming the same core DNA-protein complexes (illustrated with

> in lane 2), one approximately 105 kDa, another approximately 90 kDa and the third approximately 85 kDa in size. In other experiments where the resolution was better, the 105 kDa complex actually separated into two bands of similar size (data not shown), however this is not clear in figure 5.3.

Lane 1) 0.5 ng ICB1wt/GC1wt only

Lane 2) 0.5 ng ICB1wt/GC1wt + 5 µg HeLa extract Lane 3) 0.5 ng ICB1mt/GC1wt + 5 µg HeLa extract Lane 4) 0.5 ng ICB1mt/GC1wt BrdU1+ 5 µg HeLa extract Lane 5) 0.5 ng ICB1mt/GC1wt BrdU2+ 5 µg HeLa extract Lane 6) 0.5 ng ICB1mt/GC1wt BrdU3+ 5 µg HeLa extract Lane 7) 0.5 ng ICB1mt/GC1wt BrdU4+ 5 µg HeLa extract Lane 8) 0.5 ng ICB1mt/GC1wt BrdU5+ 5 µg HeLa extract Lane 9) 0.5 ng ICB1mt/GC1wt BrdUF+ 5 µg HeLa extract Lane 10) 0.5 ng ICB1mt/GC1wt BrdUall+ 5 µg HeLa extract

Testing different BrdU oligonucleotides using EMSA

1 2 3 4 5 6 7 8 9 10 * * * Unbound oligonucleotide GC1 ICB1 Sp1 1 NF-Y 2 3 Sp3 4 GC1 ICB1

x

Figure 5.3: Different BrdU oligonucleotides used in cross-linking assays.

(B) 2 ng of each BrdU 32P-oligonucleotide was mixed with 5 µg whole cell HeLa extract, then either

cross-linked using UVB or kept at room temperature for 30 minutes. Samples were denatured and SDS- PAGE carried out using an 8% polyacrylamide gel. The gel was coomassie stained then dried onto DE-81

paper and exposed to X-ray film for about 6 hours at -80oC. > denotes the protein-DNA complexes that

appear to be common with all the BrdU oligonucleotides, > denotes other protein-DNA complexes and

approximate sizes of these bands are as annotated. Protein size standard is visualised using fluorescent

paint. These results are representative of triplicate experiments. (A) Is a representation of a Coomassie

blue stained SDS-PAGE of whole cell HeLa extract, to demonstrate the protein complexity of this extract.

Based on the molecular weight of some of the bands seen in figure 5.3, it could be speculated that the 105 kDa complex is composed of Sp3-oligonucleotide, and that the 90 kDa complex is made up of Sp1 associating with the oligonucleotide. In other cross-linking assays there is

250 150 100 75 50 37 25 kDa (A) HeLa extract

only Lane 1) 2 ng BrdUall only

Lane 2&9) 2 ng BrdU1 + 5 µg whole cell HeLa extract Lane 3&10) 2 ng BrdU2 + 5 µg L whole cell HeLa extract Lane 4&11) 2 ng BrdU3 + 5 µg whole cell HeLa extract Lane 5&12) 2 ng BrdU4 + 5 µg whole cell HeLa extract Lane 6&13) 2 ng BrdU5 + 5 µg whole cell HeLa extract Lane 7&14) 2 ng BrdUF + 5 µg whole cell HeLa extract Lane 8&15) 2 ng BrdUall + 5 µg whole cell HeLa extract

M) Protein size standard

SDS-PAGE of cross-linking experiment using different BrdU oligonucleotides

UV treated Not UV treated

1 M 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M 105> 90> <85 <145 80> 130> 70> <250 200> (B) Unbound oligonucleotide GC1 ICB1

x

occasionally the appearance of a 95 kDa band (data not shown) which could be the phosphorylated version of Sp1. However, it should not be forgotten that there are no “T” bases (therefore no incorporated BrdU) in the GC element to which both Sp1 and Sp3 are found to bind. This does not eliminate the possibility that Sp1 or Sp3 could be recruited to the intervening sequence through an association with proteins bound nearby. In addition, non-BrdU oligonucleotides will still cross-link proteins to a certain degree (data not shown) therefore it is possible that Sp1 and Sp3 may be cross-linked to the oligonucleotide.

When examining lanes 2 through to 6 there appears to be an increasing number of protein- DNA complexes forming (shown with >), suggesting that the position where BrdU is incorporated may play a role in the cross-linking of different protein components. There is an 80 kDa protein-DNA complex which only appears in lanes 5-8, in these lanes BrdU is incorporated in positions close to the GC1 element. Therefore it is possible a particular protein component is preferentially binding to this region within the intervening sequence. In other lanes there is a larger complex approximately 145 kDa in size that only appears with BrdU3 (lane 4), BrdUF (lane 7) and BrdUall (lane 8). This 145 kDa complex also appears with weak affinity for the other BrdU oligonucleotides when more oligonucleotide is used (data not shown), suggesting this band is not just an artefact of BrdU3, BrdUF and BrdUall. However, this 145 kDa (>) complex is readily formed when BrdU is in position 3, than any of the other locations. This may demonstrate that a particular protein component is binding directly at position 3 in the intervening sequence between ICB1 and GC1. With the oligonucleotides that have BrdU incorporated at all positions (lanes 7 and 8) there is an additional band approximately 70 kDa in size which can also resolve as two bands close together when separation is better (data not shown). These 70 kDa bands are usually quite faint, suggesting that the particular protein(s) involved in the formation of these complexes are either in low abundance or that the protein-DNA interactions are fragile or possibly even transient.

There are two high molecular weight complexes, approximately 250 kDa and 200 kDa in size (lane 8). The 200 kDa complex is often observed with the other BrdU oligonucleotides (data not shown) when the oligonucleotide is used in great excess and cross-linking has worked well, but the 250 kDa complex is only observed with BrdUF and BrdUall. It is possible these larger complexes are made up of multiple proteins associating with the oligonucleotide at different regions of the intervening sequence as both these oligonucleotides have BrdU

incorporated at multiple sites, although this is considered unlikely to occur due to the low frequency of cross-linking events. There is also a 130 kDa complex that appears only when the oligonucleotides have BrdU incorporated at all possible locations (lane 7 and 8 figure 5.3). Overall, the oligonucleotide that exhibits the greatest number of protein-DNA complexes was BrdUall. This is most likely due to the greatest amount of BrdU being incorporated into this oligonucleotide therefore more overall cross-linking would be expected, possibly even at multiple sites. However, it was also the BrdUall oligonucleotide that gave questionable mobility for complex 3 in EMSA (figure 5.2, lane 10) as the bands did not clearly resolve, therefore cross-linking results obtained using this particular oligonucleotide were treated tentatively.

Lane 1 was included as a control to demonstrate that in the absence of protein no bands are observed. The BrdU oligonucleotides are not capable of forming complexes on their own. In the absence of exposure to UV light (lanes 9-15) no protein-DNA complexes can be seen, indicating that these protein-DNA complexes are dependent on the successful cross-linking of protein to 32P-labelled oligonucleotide. Generally, whole cell HeLa extract is a complicated mixture of proteins (figure 5.3A). The fact that so few proteins from this mixture were found to cross-link to the BrdU oligonucleotides provides an indication of how specific these protein-DNA interactions are and also that they are in low abundance, as would be expected for transcription factors.