Epitope tagging of endogenous proteins for genome-wide ChIP-chip studies

Abstract

We developed a strategy to introduce epitope tag-encoding DNA into endogenous loci by homologous recombination-mediated 'knock-in'. The tagging method is straightforward, can be applied to many loci and several human somatic cell lines, and can facilitate many functional analyses including western blot, immunoprecipitation, immunofluorescence and chromatin immunoprecipitation-microarray (ChIP-chip). The knock-in approach provides a general solution for the study of proteins to which antibodies are substandard or not available.For ease in constructing directed knock-in of Flag-epitope tags, we developed a universal knock-in vector. The vector contains two multiple cloning sites, sequences that encode a triple Flag epitope tag (3×Flag), a neomycin gene flanked by loxP sites and two inverted terminal repeats (Fig. 1). For targeted knock-in of 3×Flag, we inserted sequences homologous to 5′ and 3′ regions flanking the target locus into the two respective cloning sites, and packaged the resulting vector into recombinant adeno-associated virus (rAAV). Then we infected cells with targeting virus and selected neomycin-resistant clones. We identified correctly targeted clones by genomic PCR and then excised the neomycin gene by infection with adenovirus expressing Cre recombinase. As detailed in Supplementary Methods online, we successfully used this strategy to Flag-tag the C terminus of the following five proteins in three different colorectal cancer cell (CRC) lines: STAT3 (signal transducer and activator of transcription 3; Fig. 1c Table 1). The resultant epitope tagged proteins were readily detectable by western blot, immunoprecipitation and immunofluorescence, using commercially available anti-Flag ( Fig. 2 and Supplementary Fig. 1). Moreover, Flag-STAT3 retained the ability to activate the expression of a target gene 1 , and Flag-MRE11 remained associated with known interacting proteins 2 , suggesting that the presence of the 3×Flag tag is not likely to interfere with targeted protein function ( Supplementary Fig. 2 online). These data indicate that the rAAV-mediated targeting method is feasible for multiple loci across several different CRC cell lines and that 3×Flag can serve as a universal epitope for several antibody-based applications. NIH Public AccessTo test whether the tagging method can be used for global chromatin immunoprecipitation (ChIP) analyses, which require antibodies with very high specificity, we performed ChIP-chip analyses on homozygously tagged STAT3 and hemizygously tagged CHD7 loci. We performed ChIP using antibodies either to the Flag tag, or to native STAT3 or CHD7 proteins. Hybridizations were carried out on tiled microarrays that span the Encyclopedia of DNA Elements (ENCODE) regions 3 . A histogram displaying the intensity ratios of oligonucleotide probes after hybridization showed a skewing of the data in the positive direction, consistent with enrichment of DNA fragments captured by ChIP with Flag antibody (Fig. 3a and data not shown). We also plotted intensity ratios by thei...

Acetylated STAT3 is crucial for methylation of tumor-suppressor gene promoters and inhibition by resveratrol results in demethylation

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LSm14A is a processing body-associated sensor of viral nucleic acids that initiates cellular antiviral response in the early phase of viral infection

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