DNA-bound transcription factor complexes analysed by mass-spectrometry: binding of novel proteins to the human c-fos SRE and related sequences

Abstract

Transcription factors control eukaryotic polymerase II function by influencing the recruitment of multiprotein complexes to promoters and their subsequent integrated function. The complexity of the functional 'transcriptosome' has necessitated biochemical fractionation and subsequent protein sequencing on a grand scale to identify individual components. As a consequence, much is now known of the basal transcription complex. In contrast, less is known about the complexes formed at distal promoter elements. The c-fos SRE, for example, is known to bind Serum Response Factor (SRF) and ternary complex factors such as Elk-1. Their interaction with other factors at the SRE is implied but, to date, none have been identified. Here we describe the use of mass-spectrometric sequencing to identify six proteins, SRF, Elk-1 and four novel proteins, captured on SRE duplexes linked to magnetic beads. This approach is generally applicable to the characterisation of nucleic acid-bound protein complexes and the post-translational modification of their components.

Figure 1

Elk-1 and SRF binding to the SRE. A biotinylated SRE duplex bound to streptavidin-conjugated magnetic beads was incubated with increasing amounts of ³⁵S-labelled Elk-1 in the presence (+) or absence (–) of bacterially-expressed core^SRF. Beads were collected, washed and Elk-1 binding was monitored by SDS–PAGE and autoradiography.

Figure 2

SRE complexes isolated from HEK 293 cell lysates. (A) Oligonucleotide sequences used in this study. Inverted arrows indicate the palindromic sequences while ets binding sites and CArG boxes are bracketed. Asterisks above the PalXE sequence indicate CArG box mutations. (B) Proteins isolated by scrambled (lanes 1 and 2), SRE (lanes 3 and 4), PalRE (lanes 5 and 6) and PalSE (lanes 7 and 8) duplexes from serum-starved (–) or EGF-treated (+) HEK 293 cells resolved by SDS–PAGE and stained with SYPRO ruby. Numbered arrows indicate the proteins analysed in this work. The asterisk between lanes 2 and 3 indicates the 80 kDa protein referred to in the text. (C) Proteins isolated by scrambled (lanes 1 and 2), PalXE (lanes 3 and 4), ScraPal (lanes 5 and 6) and ScraPalΔ (lanes 7 and 8) duplexes from serum-starved (–) or EGF-treated (+) HEK 293 cells. Labelling as in (B).

Figure 2

SRE complexes isolated from HEK 293 cell lysates. (A) Oligonucleotide sequences used in this study. Inverted arrows indicate the palindromic sequences while ets binding sites and CArG boxes are bracketed. Asterisks above the PalXE sequence indicate CArG box mutations. (B) Proteins isolated by scrambled (lanes 1 and 2), SRE (lanes 3 and 4), PalRE (lanes 5 and 6) and PalSE (lanes 7 and 8) duplexes from serum-starved (–) or EGF-treated (+) HEK 293 cells resolved by SDS–PAGE and stained with SYPRO ruby. Numbered arrows indicate the proteins analysed in this work. The asterisk between lanes 2 and 3 indicates the 80 kDa protein referred to in the text. (C) Proteins isolated by scrambled (lanes 1 and 2), PalXE (lanes 3 and 4), ScraPal (lanes 5 and 6) and ScraPalΔ (lanes 7 and 8) duplexes from serum-starved (–) or EGF-treated (+) HEK 293 cells. Labelling as in (B).

Figure 3

MS-derived sequence for BSA, SRF and Elk-1. The spectra show the derivative ions from doubly charged ions derived from (A) sample 2 (m/z = 464.24), (B) sample 3 (m/z = 525.76) and (C) sample 4 (m/z = 650.30) (see Fig. 2). Peptide sequences are generated from Y ions whereby Y1 corresponds to the C-terminal amino acid, in each case an arginine, consistent with their origin from tryptic digestion. In the case of SRF, the peptide shown differs from the retrieved sequence by one amino acid (asterisk). This difference is interpreted as a deamidation of N47.

Figure 4

Co-purification of an 80 kDa protein with SRF. Lysates prepared from untransfected COS1 cells (C) or cells expressing a C-terminal his-tagged version of SRF (WT) or SRF-M9 (M9), were incubated with nickel-agarose beads. Bound proteins were labelled with biotin, eluted and visualised with peroxide-coupled streptavidin after SDS–PAGE and electrotransfer to nitrocellulose. Arrows indicate SRF and the co-purified 80 kDa protein.

Figure 5

MS-derived sequences of DDX1, p55.2 and CG1-99. The spectra show derivative ions from doubly charged ions derived from (A) sample 1 (m/z = 636.82), (B) sample 5 (m/z = 610.84) and (C) sample 6 (m/z =906.96) (see Fig. 2). Peptide sequences are generated from Y ions whereby Y1 corresponds to the C-terminal amino acid, in each case a lysine, consistent with their origin from tryptic digestion.

Figure 6

S1 nuclease sensitivity of SRE duplexes. (A) DNA duplexes ³²P-labelled at one end were incubated with S1 nuclease and the digestion products resolved on a 15% sequencing gel. Markers correspond to two oligonucleotides of 35 and 17 nt similarly end-labelled. Arrows indicate S1 cleavage within the A/T-rich CArG box. (B) Full-length duplexes before (–) and after (+) S1 digestion.