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 α (hERα), one of the best studied transcription factors. We found that expression of Dam-hERα fusion proteins in MCF-7 cells introduces adenosine methylation near a series of known direct hERα binding sites. Specific methylation tags are also found at indirect hERα 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.

Human MAF1 targets and represses active RNA polymerase III genes by preventing recruitment rather than inducing long-term transcriptional arrest

“…For the digestion assay, 200 ng of gDNA was digested with 5 U of either DpnII (NEB, R0543S) or Sau3A (NEB, R0169L), purified on column (MN, 740609), and analyzed by qPCR with primers listed in Supplemental Table S4. DamIP-seq was as previously described (Xiao et al 2010(Xiao et al , 2012 with modifications. In brief, 10 µg of gDNA was sheared to 100-300 bp with a Covaris S220 focusedultrasonicator in 6 × 16 mm µTUBE (Covaris, catalog 520045).…”

DNA Methylation

“…Immunoprecipitation is used to isolate DNA fragments that are methylated which can then be used for high-throughput sequencing, microarrays, etc. [258]. …”

CATaDa reveals global remodelling of chromatin accessibility during stem cell differentiation in vivo

“…CATaDa is limited by its resolution, which is restricted by the frequency of GATC sites in the genome (median spacing of ~200 bp in Drosophila). However, this can be increased by using a modified Dam in conjunction with immunoprecipitation (Dam-IP) [35]. Due to the dependence of Dam for methylation of GATC sequences, biases may be observed at loci which are depleted for GATC.…”