विशिष्ट जीनोमिक क्षेत्र और enChIP द्वारा एसोसिएटेड अणु की पहचान का अलगाव
Summary
The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.
Abstract
The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.
Introduction
हित के विशिष्ट जीनोमिक क्षेत्रों के साथ जुड़े अणुओं की पहचान ऐसे प्रतिलेखन और epigenetic विनियमन के रूप में जीनोमिक कार्यों के विनियमन के तंत्र को समझने के लिए आवश्यक है। कई तकनीकों विशिष्ट जीनोमिक क्षेत्रों 1-7 के जैव रासायनिक विश्लेषण के लिए विकसित किया गया है, क्योंकि वे ऐसी सीमित आवेदन (जैसे, केवल दोहराता के साथ उच्च प्रतिलिपि संख्या लोकी या लोकी के लिए) और के रूप में उनके आंतरिक समस्याओं के इस स्तर पर व्यापक रूप से इस्तेमाल नहीं कर रहे हैं बहुत अधिक समय और प्रयास की आवश्यकता है।
आसानी से विशिष्ट जीनोमिक क्षेत्रों की जैव रासायनिक विश्लेषण प्रदर्शन करने के क्रम में, हम 14-17 दो ठिकाना विशिष्ट chromatin immunoprecipitation (चिप) प्रौद्योगिकी, अर्थात् इन्सर्शनल चिप (iChIP) 8-13 और इंजीनियर डीएनए बाध्यकारी अणु की मध्यस्थता चिप (enChIP) विकसित किया है । IChIP में, ब्याज की एक ठिकाना ऐसे लेक्स के रूप में एक exogenous डीएनए बाध्यकारी प्रोटीन की डालने मान्यता दृश्यों द्वारा चिह्नित किया जाता हैए ठिकाना तो टैग किया डीएनए बाध्यकारी प्रोटीन का उपयोग आत्मीयता शुद्धि से अलग है। EnChIP में, इस तरह जस्ता उंगली प्रोटीन, प्रतिलेखन उत्प्रेरक की तरह (ताल) प्रोटीन के रूप में डीएनए बाध्यकारी अणुओं, इंजीनियर, और क्लस्टर नियमित रूप से interspaced कम मुरजबंध संबंधी दोहराता (CRISPR) परिसरों, चित्रा (1) ब्याज की एक ठिकाना टैग करने के लिए इस्तेमाल कर रहे हैं। बाद में, जीनोमिक क्षेत्र गईं डीएनए बाध्यकारी अणुओं की आत्मीयता शुद्धि से अलग है।
IChIP खत्म enChIP के लाभ में से एक एक exogenous डीएनए बाध्यकारी प्रोटीन की मान्यता दृश्यों की प्रविष्टि के लिए आवश्यक नहीं है कि है। Cas9 (dCas9) के एक catalytically निष्क्रिय रूप से मिलकर CRISPR परिसरों और एक गाइड आरएनए (gRNA) का उपयोग लोकी को निशाना ताल और जस्ता उंगली प्रोटीन का उपयोग iChIP या enChIP द्वारा इन क्षेत्रों के लक्षित कर की तुलना में ज्यादा आसान है। यहाँ, हम मास स्पेक्ट्रोमेट्री और आरएनए अनुक्रमण के साथ संयुक्त enChIP के लिए एक कदम-दर-कदम प्रोटोकॉल का वर्णन (आरएनए seq) ठिकाना संघों की पहचान करने के लिएटेड प्रोटीन और आरएनए, क्रमशः।
Protocol
Representative Results
Discussion
Here, we describe the purification of specific genomic regions using engineered DNA-binding molecules such as the CRISPR system and TAL proteins, and the identification of proteins and RNAs bound to these genomic regions. Binding of engineered DNA-binding molecules to the genome may affect chromatin structure, including nucleosome positioning, and may abrogate genomic functions, as described in CRISPR interference experiments21. To avoid these potential aberrant effects, we propose specific guidelines for choosing target genomic regions. First, to avoid potential inhibition of the recruitment of RNA polymerases and transcription factors, as well as disruption of nucleosome positioning around the transcription start site, the target regions for analyses of promoter regions should be several hundred base pairs upstream of (5′ to) the transcription start site. By contrast, when analyzing genomic regions with distinct boundaries, such as enhancers and silencers, genomic regions that are directly juxtaposed to these regions can be targeted because it is less likely that the binding of engineered DNA-binding molecules will affect their functions. Furthermore, it is best to avoid using target regions that are conserved among different species, because important DNA-binding molecules often bind to evolutionarily conserved regions and inhibition of their binding might disrupt the functions of the target genomic regions. In this regard, it is always necessary to check that the function of the target genomic region is maintained in the established cells used for enChIP analyses. Because multiple gRNAs can be tested easily and it is tedious and expensive to generate multiple versions of TAL or zinc-finger proteins recognizing different target genomic regions, enChIP using CRISPR is more advantageous than enChIP using other proteins.
It has been shown that dCas9 binds to off-target sites although affinity to those sites might be weaker than that to the target sites22-25. There are several ways to manage contamination of molecules bound to those off-target sites. First, the use of several, at least two, different gRNAs would be recommended. Those molecules commonly observed in enChIP using distinct gRNAs would be true positives. Second, comparison of different conditions for enChIP would be effective in cancelling contamination of non-specific molecules and molecules bound to off-target sites. Examples of those comparison sets would be (i) stimulation (-) and (+), or (ii) different cell types such as T cells vs. B cells. Finally, quantitative analysis of binding of candidate molecules should be performed to confirm their specific binding to the target sites. It is preferable to prepare cells expressing only dCas9 but not gRNA as a negative control.
Using enChIP analyses, we were able to successfully identify a number of known and novel molecules interacting with specific genomic regions (Tables 1-3)14-17. However, this technique failed to detect some other known proteins interacting with these regions. For example, STAT1 reportedly associates with the IRF-1 promoter upon IFNγ stimulation8, but our enChIP-SILAC analysis did not detect STAT1 as a protein induced to interact with this genomic region16. In addition, in the enChIP-MS analysis of telomeres, we did not detect shelterin proteins consisting of TRF-1 and TRF-215, which have been shown to interact with telomeres26. There are a few potential reasons for these discrepancies. First, the stoichiometry of binding of Stat1 to the IRF-1 promoter might be very low. It is reasonable that enChIP-MS, including enChIP-SILAC, detects proteins that are more abundantly associated with target genomic regions; hence, the analysis of more cells might be necessary to detect these proteins. Increases in the sensitivities of MS instruments would also contribute to the efficient detection of proteins with low stoichiometric binding. Second, some proteins, possibly including Stat1 and shelterins, might be difficult targets for MS analyses. Third, in our analysis of telomere-binding proteins15, the 3×FLAG-TAL proteins recognizing telomeres (3×FN-Tel-TAL) might have blocked the binding of shelterins to telomeres in a competitive fashion.
In contrast to the relative difficulty of detecting transcription factors binding to specific genomic regions using enChIP, we successfully identified epigenetic regulators such as histone modification enzymes using enChIP analyses. The success of this technique may be due to the fact that epigenetic regulators bind to a broad range of genomic regions; hence, more proteins per genomic region are available for MS. Because epigenetic regulators are increasingly recognized as important targets for drugs against intractable diseases such as cancer, enChIP would be a useful tool for the identification of epigenetic drug targets.
Disclosures
The authors have nothing to disclose.
Acknowledgements
इस काम ताकेदा विज्ञान फाउंडेशन (टीएफ) द्वारा समर्थित किया गया था; असाही ग्लास फाउंडेशन; Uehara मेमोरियल फाउंडेशन (एचएफ); Kurata मेमोरियल हिताची विज्ञान और प्रौद्योगिकी फाउंडेशन (टीएफ और एचएफ); सहायता अनुदान में युवा वैज्ञानिकों के लिए (बी) (# 25,830,131), अनुदान सहायता में साइंटिफिक रिसर्च (सी) (# 15K06895) (टीएफ) के लिए; और एक अनुदान सहायता में अभिनव क्षेत्रों 'ट्रांसक्रिप्शन साइकिल' पर वैज्ञानिक अनुसंधान के लिए (# 25118512 & # 15H01354), अनुदान सहायता में साइंटिफिक रिसर्च (बी) (# 15H04329) के लिए, अनुदान सहायता में खोजपूर्ण अनुसंधान के लिए ( # 26650059) और जापान के शिक्षा मंत्रालय, संस्कृति, खेल, विज्ञान और प्रौद्योगिकी से 'जीनोम समर्थन' (# 221S0002) (एचएफ)।
Materials
| gBlock synthesis service | Life Technologies | Gene Synthesis by GeneArt | |
| gBlock synthesis service | IDT (Integrated DNA Technologies) | gBlocks Gene Fragments | |
| pSIR-neo | Addgene | 51128 | |
| pSIR-GFP | Addgene | 51134 | |
| pSIR-DsRed-Express2 | Addgene | 51135 | |
| pSIR-hCD2 | Addgene | 51143 | |
| TAL synthesis service | Life Technologies | GeneArt Precision TALs | |
| 3xFLAG-dCas9/pCMV-7.1 | Addgene | 47948 | |
| 3×FLAG-dCas9/pMXs-puro | Addgene | 51240 | |
| 3×FLAG-dCas9/pMXs-IG | Addgene | 51258 | |
| 3×FLAG-dCas9/pMXs-I2 | Addgene | 51259 | |
| 3×FLAG-dCas9/pMXs-neo | Addgene | 51260 | |
| anti-FLAG M2 Ab | Sigma-Aldrich | F1804 | |
| FITC-conjugated anti-FLAG M2 | Sigma-Aldrich | F4049 | |
| DMEM medium for SILAC | Life Technologies | 89985 | Other medium can be purchased from Life Technologies |
| Dialyzed FBS for SILAC | Life Technologies | 89986 | |
| L-Lysine-2HCl for SILAC | Life Technologies | 89987 | for Light medium |
| L-Arginine-HCl for SILAC | Life Technologies | 89989 | for Light medium |
| L-Lysine-2HCl, 13C6 for SILAC | Life Technologies | 89988 | For Heavy medium |
| L-Arginine-HCl, 13C6, 15N4 for SILAC | Life Technologies | 89990 | For Heavy medium |
| Complete, mini, EDTA-free | Roche Diagnostics | 4693159 | |
| Ultrasonic Disruptor UD-201 | Tomy Seiko | ||
| ChIP DNA Clean & Concentrator | Zymo Research | D5205 | |
| Dynabeads-Protein G | Life Technologies | DB10004 | |
| RNasin Plus RNase Inhibitor | Promega | N2611 | |
| Isogen II | Nippon Gene | 311-07361 | |
| Direct-zol RNA Miniprep kit | Zymo Research | R2050 | |
| LTQ Orbitrap Velos | Thermo Fisher Scientific | a component of a nanoLC-MS/MS system for MS analysis | |
| nanoLC | Advance, Michrom Bioresources | a component of a nanoLC-MS/MS system for MS analysis | |
| HTC-PAL autosampler | CTC Analytics | a component of a nanoLC-MS/MS system for MS analysis |
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