DamIP: a novel method to identify DNA binding sites in vivo

Abstract

Identifying binding sites and target genes of transcription factors is a major biologic problem. The most commonly used current technique, chromatin immunoprecipitation (ChIP), is dependent on a high quality antibody for each protein of interest, which is not always available, and is also cumbersome, involving sequential cross-linking and reversal of cross-linking. We have developed a novel strategy to study protein DNA binding sites in vivo, which we term DamIP. By tethering a mutant form of E. coli DNA adenine methyltransferase to the target protein, the fusion protein introduces N-6-adenosine methylation to sequences proximal to the protein binding sites. DNA fragments with this modification, which is absent in eukaryotes, are detected using an antibody directed against methylated adenosine. For an initial test of the method we used human estrogen receptor alpha (hERalpha), one of the best studied transcription factors. We found that expression of Dam-hERalpha fusion proteins in MCF-7 cells introduces adenosine methylation near a series of known direct hERalpha binding sites. Specific methylation tags are also found at indirect hERalpha binding sites, including both primary binding sites for the ER interactors AP-1 and SP1, and promoters that are activated by upstream ER bound enhancers. DamIP provides a new tool for the study of DNA interacting protein function in vivo.

Figure 1. Expression and transcriptional activities of DamK9A-hERα fusion protein.

(A). HeLa cells were transfected with various plasmids as indicated and cell lysate was analyzed by Western blot. MCF-7 and untransfected HeLa cell lysate were loaded as positive and negative control, respectively. The same blot was detected with anti-β-actin antibody as loading control. Molecular weights (Mw) are indicated as kilodaltons (kDa). (B). Fusion protein transcriptional activities in HeLa cells. Cells were co-transfected with various plasmids and luciferase reporter vectors. Luciferase activities were measured 24 hours after ligand treatment. Data are presented as mean +/- SEM.

Figure 2. Illustration of the procedure of DamIP.

DamK9A or DamK9A fusion proteins are expressed in cells. Genomic DNA is purified, sonicated and denatured before mixed with anti-N-6-methyladenine antibodies. Methylated DNA recognized by the antibody is enriched and analyzed by various methods, e.g. qPCR, microarray and next-generation sequencing. Details are described in the Methods section.

Figure 3. RT-qPCR analysis of DamIP.

Primers were designed to amplify genomic regions with or without known ERE. DNA enriched with DamIP from MCF-7 cells transfected with DamK9A-hER (Dam-ER) or Dam alone was analyzed by qPCR. Data are presented as mean +/- SEM.

Figure 4. DamIP has great resolution.

A series of primers were designed to amplify the regions upstream of the FSIP locus that contains known ERE. These targeted regions are about 500bp from each other and designated as -2k, -1.5k, -1k, -0.5k, respectively. The specific modification introduced by DamK9A-hER is only observed at the FSIP locus.

Figure 5. DamIP identifies indirect regulation by ER.

ER can regulate gene expression through long range chromosome looping (A, B) or binding with other transcriptional factors (C). Carbonic anhydrase XII (C12) and cathepsin D (CTSD) enhancers contain functional EREs but not promoter regions, however, DamIP indicates ER communicates with the ERE-less promoter regions during transcription. ER regulation through interaction with AP1 (C, left panel) or Sp1 (C, right panel) can also be detected by DamIP.