Efficient isolation of specific genomic regions retaining molecular interactions by the iChIP system using recombinant exogenous DNA-binding proteins
© Fujita and Fujii; licensee BioMed Central Ltd. 2014
Received: 13 May 2014
Accepted: 12 November 2014
Published: 27 November 2014
Comprehensive understanding of mechanisms of genome functions requires identification of molecules interacting with genomic regions of interest in vivo. We previously developed the insertional chromatin immunoprecipitation (iChIP) technology to isolate specific genomic regions retaining molecular interactions and identify their associated molecules. iChIP consists of locus-tagging and affinity purification. The recognition sequences of an exogenous DNA-binding protein such as LexA are inserted into a genomic region of interest in the cell to be analyzed. The exogenous DNA-binding protein fused with a tag(s) is expressed in the cell and the target genomic region is purified with antibody against the tag(s). In this study, we developed the iChIP system using recombinant DNA-binding proteins to make iChIP more straightforward than the conventional iChIP system using expression of the exogenous DNA-binding proteins in the cells to be analyzed.
In this system, recombinant 3xFNLDD-D (r3xFNLDD-D) consisting of the 3xFLAG-tag, a nuclear localization signal (NLS), the DNA-binding domain plus the dimerization domain of the LexA protein, and the Dock-tag is used for isolation of specific genomic regions. r3xFNLDD-D was expressed using a silkworm-baculovirus expression system and purified by affinity purification. iChIP using r3xFNLDD-D could efficiently isolate the single-copy chicken Pax5 (cPax5) locus, in which LexA binding elements were inserted, with negligible contamination of other genomic regions. In addition, we could detect RNA associated with the cPax5 locus using this form of the iChIP system combined with RT-PCR.
The iChIP system using r3xFNLDD-D can isolate specific genomic regions retaining molecular interactions without expression of the exogenous DNA-binding protein in the cell to be analyzed. iChIP using r3xFNLDD-D would be more straightforward and useful for analysis of specific genomic regions to elucidate their functions as compared to the previously published iChIP protocol.
Genome functions are mediated by various molecular complexes in the context of chromatin . Comprehensive understanding of mechanisms of genome functions requires identification of molecules interacting with genomic regions of interest in vivo. To this end, we recently developed the locus-specific chromatin immunoprecipitation (ChIP) technologies consisting of insertional ChIP (iChIP) – and engineered DNA-binding molecule-mediated ChIP (enChIP) – to isolate genomic regions of interest retaining molecular interactions. The functions of the genomic regions can be comprehensively understood by analysis of DNA, RNA, proteins, or other molecules interacting with the genomic regions.
In principle, iChIP is based on locus-tagging by inserting recognition sequences of an exogenous DNA-binding protein to isolate specific genomic regions using the exogenous DNA-binding molecule. In contrast, enChIP is based on recognition of endogenous DNA sequences by engineered DNA-binding molecules such as transactivator-like (TAL) proteins and the clustered regularly interspaced short palindromic repeats (CRISPR) system. The scheme of iChIP is as follows: (i) The recognition sequences of an exogenous DNA-binding protein such as a bacterial protein, LexA, are inserted into the genomic region of interest in the cell to be analyzed. (ii) The DNA-binding domain (DB) of the exogenous DNA-binding protein is fused with a tag(s) and a nuclear localization signal(s) (NLS(s)) and expressed in the cell to be analyzed. (iii) The resultant cell is stimulated and crosslinked with formaldehyde or other crosslinkers, if necessary. (iv) The cell is lysed, and the chromatin DNA is fragmented by sonication or enzymatic digestion. (v) The complexes including the exogenous DB are immunoprecipitated with antibody (Ab) against the tag(s). (vi) The isolated complexes which retain molecular interactions are reverse crosslinked, if necessary, and subsequent purification of DNA, RNA, proteins, or other molecules allows their identification and characterization. We successfully identified proteins and RNA components of an insulator, which functions as boundaries of chromatin domains , by using iChIP combined with mass spectrometry (iChIP-MS) or RT-PCR (iChIP-RT-PCR) . iChIP has also been used for identification of proteins or DNA interacting with specific genomic regions by other researchers –. Thus, iChIP is a useful technology for elucidation of molecular mechanisms of genome functions.
In this study, we developed the iChIP system using the recombinant C-terminally Dock-tagged 3xFNLDD (r3xFNLDD-D). r3xFNLDD-D was expressed using a silkworm-baculovirus expression system and purified by affinity purification. iChIP using r3xFNLDD-D could effectively isolate the single-copy chicken Pax5 (cPax5) locus from a chicken B cell line, DT40. In addition, we could detect RNA associated with the cPax5 locus using this form of the iChIP system combined with RT-PCR. Thus, iChIP using r3xFNLDD-D would be more straightforward and useful than the conventional iChIP system using expression of the exogenous DNA-binding proteins in the cells to be analyzed to isolate specific genomic regions for their biochemical analysis.
Results and discussion
Expression and purification of r3xFNLDD-D
Efficient isolation of a target genomic region by iChIP using r3xFNLDD-D
Optimization of iChIP using r3xFNLDD-D
Isolation of RNA associated with the cPax5 locus by iChIP using r3xFNLDD-D
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 –. 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) , 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 , 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.
In this study, we established the iChIP system using r3xFNLDD-D to make the iChIP technology much more straightforward than the conventional iChIP system using expression of the exogenous DNA-binding proteins in the cells to be analyzed. Using this system, we were able to isolate target genomic regions; % of input reached more than 20% for the cPax5 1A promoter region. In addition, we could detect RNA associated with the cPax5 locus, suggesting that iChIP using r3xFNLDD-D can isolate target genomic regions retaining molecular interactions. Thus, the modified iChIP protocol described here using r3xFNLDD-D has advantages over the previously published protocol in that it is more straightforward and useful for analysis of specific genomic regions to elucidate their functions.
Expression and purification of r3xFNLDD-D
Expression of 3xFNLDD-D was performed using the silkworm-baculovirus expression system (ProCube) (Sysmex Corporation, http://procube.sysmex.co.jp/eng/) as described previously . Briefly, the coding sequence of 3xFNLDD  was inserted into the transfer vector pM31a (Sysmex Corporation) to fuse the Dock-tag at its C-terminus and co-transfected with linearized genomic DNA of ABv baculovirus (Bombyx mori nucleopolyhedrovirus; CPd strain, Sysmex Corporation) into the B. mori-derived cell line, BmN, to generate the recombinant baculovirus. The generated baculovirus was infected into a silkworm pupa to express 3xFNLDD-D. The expressed 3xFNLDD-D was purified with Dock Catch Resin (Sysmex Corporation) as described previously . The immunoblot analysis was performed with anti-Dock Ab (Sysmex Corporation).
The chicken B cell line DT40 was provided by the RIKEN BioResource Center through the National Bio-Resource Project of the Ministry of Education, Science, Sports and Culture of Japan. DT40 and DT40#205-2, in which LexA BE was inserted in the 1A promoter region of the cPax5 gene (Fujita and Fujii, manuscript submitted), were maintained in RPMI-1640 (Wako) with 4 mM glutamine, 10% (v/v) fetal bovine serum, 1% chicken serum, and 50 μM 2-mercaptoethanol at 39.5°C.
Cells (2 × 107) were fixed with 1% formaldehyde at 37°C for 5 min. The chromatin fraction was extracted and fragmented (2 kbp-long on average) by sonication as described previously  except for using 800 μl of in vitro Modified Lysis Buffer 3 (10 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) and Ultrasonic disruptor UD-201 (TOMY SEIKO). After sonication, Triton X-100 was added to final concentration at 0.1%.
iChIP using r3xFNLDD-D
Primers used in this study
Sequence (5′ → 3′)
RT-PCR in Additional file 1: Figure S2 (18S)
RT-PCR in Additional file 1: Figure S2 (18S)
Real-time PCR in Additional file 1: Figure S5 (5′HS5)
Real-time PCR in Additional file 1: Figure S5 (5′HS5)
Real-time PCR in Additional file 1: Figure S5 (IRF-1)
hIRF1-prom-R1 + 2
Real-time PCR in Additional file 1: Figure S5 (IRF-1)
Isolation of interacting RNA and RT-PCR
Chromatin preparation and iChIP using r3xFNLDD-D were performed as described above except for addition of RNasin Plus RNase Inhibitor (Promega) in all buffers. After reverse crosslinking at 65°C, RNA was isolated with Isogen II (Nippon gene) combined with Direct-zol RNA Mini Prep (Zymo Research). The purified RNA was used as template for reverse transcription with ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo). The cDNA was used as template for PCR with AmpliTaq Gold 360 Master Mix (Applied Biosystems). PCR cycles were as follows: heating at 95°C for 10 min; 40 cycles of 95°C for 30 sec, 60°C for 30 sec, 72°C for 1 min; and the final extending 72°C for 2 min. The primers used in this experiment are shown in Table 1.
We thank F. Kitaura for technical assistance. This work was supported by Takeda Science Foundation (TF), the Uehara Memorial Foundation (HF), the Kurata Memorial Hitachi Science and Technology Foundation (TF and HF), Adaptable & Seamless Technology Transfer Program through Target-driven R&D (A-STEP) by the Japan Science and Technology Agency (JST) (#AS251Z01861Q) (HF), Grant-in-Aid for Young Scientists (B) (#25830131) (TF), “Transcription Cycle” (#25118512) (HF) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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