Expression and purification of r3xFNLDD-D
For preparation of the purified r3xFNLDD-D, we utilized a silkworm-baculovirus expression system [14]. In this system, r3xFNLDD-D was expressed in a silkworm pupa by infection of baculoviruses expressing r3xFNLDD-D. The expressed protein was purified from the pupal homogenates using Dock Catch Resin, which specifically binds to Dock-tag in a calcium-dependent manner [14]. As shown in Figure 2A, SDS-PAGE followed by Coomassie Brilliant Blue (CBB) staining detected a single protein band at 35 kDa in the elution fraction. This protein was confirmed as r3xFNLDD-D by immunoblot analysis with anti-Dock Ab (Figure 2B). Thus, r3xFNLDD-D could be expressed in a silkworm pupa and purified without visible degradation.
Efficient isolation of a target genomic region by iChIP using r3xFNLDD-D
Next, we examined whether the purified r3xFNLDD-D could be utilized for isolation of genomic regions of interest from vertebrate cells. To this end, we used the chicken DT40-derived cell line, DT40#205-2, in which 8 × repeats of LexA BE were inserted 0.3 kbp upstream of the exon 1A of the single-copy endogenous cPax5 gene [15] (Figure 3A). The crosslinked chromatin prepared from the cell line was subjected to iChIP using r3xFNLDD-D as shown in Figure 1. After purification of the immunoprecipitated genomic DNA, the yield of the cPax5 1A promoter region was evaluated by detection of the LexA BE site (LexA BE) and the region 0.2 kbp upstream of LexA BE (i.e., 0.7 kbp upstream of the transcription start site (TSS) of cPax5 exon 1A) (−0.7 k) by real-time PCR (Figure 3A). As shown in Figure 3B, the yields of LexA BE and −0.7 k were more than 20% and 5% of input, respectively, when 10 μg of each r3xFNLDD-D and anti-FLAG Ab were used. In contrast, the yield of the genomic region 10 kbp upstream of the TSS of the exon 1A (−10 k) was less than 0.01%. These results suggested that r3xFNLDD-D can bind to LexA BE even in the crosslinked chromatin and iChIP using r3xFNLDD-D is able to specifically purify target genomic regions. The specific isolation of the cPax5 1A promoter region was completely blocked when we inhibited binding of r3xFNLDD-D to anti-FLAG Ab with excessive amounts of 3xFLAG peptide (Figure 3C). The cPax5 1A promoter region was not isolated when parental DT40 was used instead of DT40#205-2 (Figure 3C). These results clearly demonstrated that isolation of target genomic regions is mediated by binding of r3xFNLDD-D to LexA BE. The yield of the target genomic region by the modified iChIP system was comparable with that of the previously reported iChIP protocol [4].
Optimization of iChIP using r3xFNLDD-D
Next, we titrated amounts of r3xFNLDD-D and anti-FLAG Ab to optimize the system (Figure 4A). The yield of the LexA BE site in the cPax5 1A promoter was comparable when 0.5 - 10 μg of each r3xFNLDD-D and anti-FLAG Ab was used with chromatin prepared from 1 × 107 of DT40#205-2 cells. In contrast, use of 0.01 - 0.1 μg of each protein showed lower yield, suggesting that 0.5 μg of each r3xFNLDD-D and anti-FLAG Ab are sufficient for 1 × 107 cells. The yield of iChIP using 0.5 μg of r3xFNLDD-D was 20% of input for LexA BE and less than 0.01% for −10 k, which is comparable with that using 10 μg of r3xFNLDD-D (Figures 3B and 4B). In this regard, we observed the yield of 15% of input for the same locus when 3xFLNDD was expressed in DT40#205-2 and the conventional iChIP protocol was used (T.F., H.F., unpublished observation). These results showed that the iChIP using r3xFNLDD-D could purify the target region with efficiency comparable to the conventional iChIP. We also examined whether it would be possible to purify the cPax5 1A promoter region using r3xFNLDD-D with Dock Catch Resin, which binds to the C-terminal Dock-tag of r3xFNLDD-D. As shown in Additional file 1: Figure S1, 2% of input of the LexA BE site could be isolated with negligible contamination of −10 k, indicating that the C-terminal Dock-tag of r3xFNLDD-D can also be utilized for iChIP, although the yield was much lower than that using anti-FLAG Ab.
Isolation of RNA associated with the cPax5 locus by iChIP using r3xFNLDD-D
Next, we examined whether iChIP using r3xFNLDD-D could be utilized to isolate genomic regions of interest and identify molecules interacting with those genomic regions in cells. To this end, we attempted to isolate the cPax5 locus including the exon 1A region by iChIP using r3xFNLDD-D and detect the nascent RNA transcribed from the TSS of the cPax5 exon 1A by RT-PCR (Figure 5A). Transcription from the cPax5 exon 1A was not disrupted by the presence of LexA BE inserted in the 1A promoter region (Additional file 1: Figure S2). As shown in Figure 5B, iChIP using r3xFNLDD-D isolated the cPax5 exon 1A region but not the exon 3 region of the irrelevant cAID gene, which encodes an enzyme essential for B cell-specific immunoglobulin somatic hypermutation and class switch recombination [16]. After purification of the associated RNA, RT-PCR analysis detected RNA transcribed from the exon 1A of the cPax5 gene but not that from the cAID gene in the iChIP sample (Figure 5C) (the full-length images with size markers are shown in Additional file 1: Figure S3). These results suggested that iChIP using r3xFNLDD-D is able to isolate specific genomic regions retaining molecules interacting with the genomic regions.
Feasibility of enChIP using recombinant engineered DNA-binding molecules
Lastly, we examined whether enrichment of specific genomic regions is feasible by enChIP using recombinant TAL proteins (Additional file 1: Figure S4). enChIP uses engineered DNA-binding molecules such as TAL proteins or the CRISPR system consisting of a catalytically inactive form of Cas9 (dCas9) and guide RNA (gRNA) for locus-tagging and affinity purification of the target loci [6]–[8]. We generated a construct expressing a fusion protein, r3xFN-5′HS5-TAL-G, consisting of 3xFLAG-tag, an NLS, a recombinant TAL protein recognizing human 5′HS5 region, which functions as an insulator to regulate transcription of the β-globin genes (Additional file 1: Figure S5A) [17], and the glutathione S-transferase (GST)-tag, and prepared the recombinant protein by using the silkworm-baculovirus expression system (Additional file 1: Figure S5B and C). We used the recombinant protein for enChIP analysis of the 5′HS5 locus. As shown in Additional file 1: Figure S5D, the 5′HS5 site was enriched several-fold compared to the irrelevant interferon regulatory factor 1 (IRF-1) promoter region when non-crosslinked native chromatin prepared from the 293T cell line was used. These results suggest that enChIP using recombinant TAL proteins is feasible. However, we found that the r3xFN-5′HS5-TAL-G (ca. 160 kDa) showed massive degradation (Additional file 1: Figure S5B and C). In addition, we failed to detect enrichment of the target 5′HS5 site when we used crosslinked chromatin (data not shown). These results also suggest that improvement in production of recombinant TAL proteins and their access to target loci might be required for efficient isolation of target regions and identification of associated molecules.
Applications and advantages of iChIP using r3xFNLDD-D
In this study, we applied RT-PCR to detection of RNA interacting with a genomic region of interest in cells. Next-generation sequencing or microarray analysis can be combined with iChIP using r3xFNLDD-D for non-biased identification of interacting RNA as well as DNA. Moreover, mass spectrometry can be combined for non-biased identification of interacting proteins.
Because iChIP using r3xFNLDD-D does not require expression of 3xFNLDD in cells, it is of great use in the iChIP analysis of primary cells isolated from organisms, especially higher eukaryotes such as mice. In the case of application of the standard iChIP technology to mice, it is time-consuming to establish mouse lines expressing 3xFNLDD in the cells to be analyzed as well as those possessing LexA BE in specific genomic regions. In this regard, iChIP using r3xFNLDD-D is able to skip the mating steps between mice expressing 3xFNLDD and those possessing LexA BE, substantially accelerating iChIP analysis using organisms.
Compared to enChIP or proteomics of isolated chromatin (PICh), which uses specific biotinylated nucleic acid probes such as locked nucleic acids (LNAs) that hybridize target genomic regions for their isolation [18], iChIP requires insertion of LexA BE, which takes time and effort. However, recent advancement of genome editing technologies using TALEN and CRISPR makes insertion of exogenous sequences in target loci much more easily. In addition, insertion of such exogenous sequences may abrogate function of genomic regions through changes in nucleosome positioning or other mechanisms. Therefore, it is necessary to confirm that the insertion of LexA BE does not abrogate function of genomic regions before isolating the genomic regions by iChIP. On the other hand, the locus-tagging system used in iChIP can be used for isolation of a specific target allele such as a maternal or paternal allele. Feasibility of such allele-specific analysis is one of advantages of iChIP over enChIP and PICh when allele-specific analysis is required, for example, in the analysis of genome imprinting.