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.

Figure 1

Schematic diagram of tagging endogenous protein with 3×Flag. (a) Targeting (NEO-loxP-3×Flag) vector. L-ITR and R-ITR, left and right inverted terminal repeats, respectively; MCS, multiple cloning site; CMV, cytomegalovirus promoter; NEO, neomycin resistance gene. (b)Diagram of knock-in strategy. rAAV targeting vectors contain a left and right ‘arm’ homologous to sequences in the target gene, flanking a NEO-loxP-3×Flag cassette. Clones are then screened by genomic PCR with primers complementary to the neomycin resistance gene and upstream of the left (P1 and NR) or downstream of the right (NF and P2) homologous arms. The neomycin gene cassette is excised with Cre recombinase, and genomic PCR using primers P3 and P4 identifies clones with the correct excision. (c) Genomic PCR of parental (P) and STAT3 3×Flag knock-in cells (clones 1 and 2). WT, wild-type STAT3; Flag, Flag-tagged STAT3. Arrow indicates the targeted allele.

Figure 2

3×Flag tagged proteins are detectable by western blot, immunoprecipitation and immunofluorescence. (a) Western blots of wild-type and 3×Flag-tagged STAT3 in DLD1 cells with either anti-STAT3 or anti-Flag. (b) Cell lysates of STAT3 3×Flag knock-in cells immunoprecipitated with anti-Flag and western blotted with anti-STAT3. (c) Parental and STAT3 3×Flag tagged cells were treated with or without IL-6 for 30 min and fixed. Immunofluorescence staining was performed with a rabbit anti-STAT3 and a mouse monoclonal anti-Flag. Note that similar to wild-type STAT3, Flag-tagged STAT3 translocates to the nucleus upon IL-6 stimulation. Scale bar, 10 μm.

Figure 3

ChIP analysis of wild-type and Flag-tagged STAT3. (a) Histogram of mean signal ratios of Flag-STAT3 chromatin-immunoprecipitated DNA versus random-sheared total genomic DNA. The distinct tail at the right-hand end corresponds to DNA fragments enriched by Flag-STAT3 ChIP (see insert). Tiled oligos that displayed the top 0.25% ratios are located to the right of the red bar. (b) STAT3 binding profiles from a 500-kb region on chromosome 21. Normalized raw ratio data from the indicated ChIP-chip experiments are plotted. The top 0.25% is displayed as a dotted horizontal line. An expanded view of a positive signal from the left is shown on the right. (c)The maximum signal intensity ratios for each STAT3-occupied site are plotted on the x and y axes and correspond to the filled circles (n = 214). For comparison, 15 randomly selected regions are plotted as open circles. (R = 0.73, P < 2.2 × 10⁻¹⁶). (d) Signal intensities of STAT3 bound regions are plotted as a heatmap. Note the high degree of overlap of STAT3-bound sites found in all three experiments. For comparison, 15 randomly selected sites were included (bracket on the right).