A SILAC-based DNA protein interaction screen that identifies candidate binding proteins to functional DNA elements

Abstract

Determining the underlying logic that governs the networks of gene expression in higher eukaryotes is an important task in the post-genome era. Sequence-specific transcription factors (TFs) that can read the genetic regulatory information and proteins that interpret the information provided by CpG methylation are crucial components of the system that controls the transcription of protein-coding genes by RNA polymerase II. We have previously described Stable Isotope Labeling by Amino acids in Cell culture (SILAC) for the quantitative comparison of proteomes and the determination of protein-protein interactions. Here, we report a generic and scalable strategy to uncover such DNA protein interactions by SILAC that uses a fast and simple one-step affinity capture of TFs from crude nuclear extracts. Employing mutated or nonmethylated control oligonucleotides, specific TFs binding to their wild-type or methyl-CpG bait are distinguished from the vast excess of copurifying background proteins by their peptide isotope ratios that are determined by mass spectrometry. Our proof of principle screen identifies several proteins that have not been previously reported to be present on the fully methylated CpG island upstream of the human metastasis associated 1 family, member 2 gene promoter. The approach is robust, sensitive, and specific and offers the potential for high-throughput determination of TF binding profiles.

Figure 1.

Principle of the DNA–protein interaction screen. Proteomes are metabolically labeled with ²H₄-lysine (Lysine-d4) to allow discrimination based on differences in peptide mass (4 Da). Biotinylated (Bio) DNA molecules bearing functional elements (e.g., potential TF binding sites or methylated CpGs) are synthesized and immobilized on streptavidin magnetic beads. Control columns are designed to lack the functional element of interest (e.g., point mutations in cis-elements or nonmethylated CpGs). Nuclear extracts from unlabeled (Lysine-d0) and labeled cells are prepared and subjected to DNA affinity chromatography, followed by the release of DNA–protein complexes by restriction enzyme digestion. After in-gel digestion with trypsin, differentially labeled forms of lysine containing tryptic peptides are detected by mass spectrometry (MS). Peptides originating from proteins specifically recognizing functional elements will have a larger peak intensity of the lysine-d4 (Kd4) form. Unspecific interaction partners will have 1:1 ratios between both isotopic forms.

Figure 2.

Properties of the DNA–protein interaction screen exemplified by a functional element harboring a binding site for the TF AP2. (A) The sequence of the DNA baits used for the experiment. The AP2 binding site is shown in boldface type, whereas the point mutations designed to disrupt DNA binding are highlighted in gray. (B–D) SILAC is essential for the identification of specific binders. (B) Specific interaction partners cannot be identified by one-dimensional SDS-PAGE and silver staining of the eluted material from the AP2 wild-type (WT) and mutant (MT) columns. (C) Western blot analysis of selected specific interaction partners recapitulates results from the SILAC analysis. Equal amounts (10% of total) of input (IN) and flow-through (FT) as well as eluted (Elution) material (50% of total) from the AP2 wild-type (WT) and mutant (MT) columns, respectively, were analyzed by immunoblotting with antibodies directed against TFAP2A, the POLR2A subunit of RNA polymerase II, and PURA. POLR2A serves as a control for equalized total protein amounts and unspecific binding. (D) SILAC analysis discriminates specific from unspecific binders. Proteins containing peptides with lysine-d4 to lysine-d0 ratios of greater than 3:1 are considered to be specific.

Figure 3.

The orphan nuclear receptor ESRRA (ERRalpha) exhibits specific binding to its bioinformatically predicted binding sequence. (A) Western blot analysis (same conditions as in Fig. 2C) confirms the specific interaction of ESRRA as revealed by the SILAC experiment (B–D) (Table 1). Sequences of the DNA baits employed are depicted. The predicted ESRRA binding site derived from an autoregulatory motif of the ESRRA gene (Mootha et al. 2004) is shown in boldface type whereas the point mutation for the control column is highlighted in gray. (B) Representative peptide mass spectrum demonstrating the specific binding of ESRRA. The MS spectrum of a quadruply charged labeled (monoisotopic peak marked with solid circle) and unlabeled (monoisotopic peak marked with open circle) tryptic ESRRA peptide acquired in the ICR cell of the LTQ-FT (0.3 ppm mass deviation after recalibration) is shown. (C) MS/MS (MS2) fragmentation spectrum that identifies the peptide shown in B. The precursor ion (m/z 684.81) was fragmented in the linear ion trap (LTQ part) of the mass spectrometer to obtain sequence information. (D) MS/MS/MS (MS3) spectrum of the y ₁₅(2+) ion present in the MS2 experiment (C) further confirming the identification of the peptide shown in B.

Figure 4.

Methyl-CpG specific binding partners can be discriminated by SILAC analysis. (A) Scheme of the CpG island in the upstream region of the human MTA2 gene that is located at position −623 to −601 relative to the transcription start site (arrowhead). The cytosine residues highlighted in gray were either fully methylated (MeCpG) or not methylated (CpG) in the SILAC experiment. The structure of 5-methyl cytosine is shown. (B) Representative peptide mass spectrum demonstrating the preferential binding of the methyl-CpG binding protein ZBTB33 (Kaiso), which was known to interact with the fully methylated MTA2 CpG island. The MS spectrum of a labeled (monoisotopic peak marked with filled circle) and unlabeled (monoisotopic peak marked with open circle) tryptic ZBTB33 peptide acquired is shown. (C) Western blot analysis (same conditions as in Fig. 2C) of selected specific interaction partners recapitulates results from the SILAC analysis (Table 2).