عزل المناطق الجينوم محددة وتحديد أسوشيتد الجزيئات التي enChIP
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
مطلوب تحديد الجزيئات المرتبطة مناطق الجينوم محددة من الفائدة لفهم آليات تنظيم وظائف الجينوم مثل النسخ وتنظيم جينية. على الرغم من أن وضعت العديد من التقنيات لتحليل الكيمياء الحيوية من مناطق الجينوم محددة 1-7، لا يتم استخدامها على نطاق واسع في هذه المرحلة بسبب المشاكل الجوهرية من قبيل التطبيق المحدود (على سبيل المثال، فقط من أجل عالية نسخ مواضع عدد أو مواضع حيث تتكرر) و الكثير من الوقت والجهود المطلوبة.
من أجل إجراء تحليل كيميائي حيوي من مناطق الجينوم معينة بسهولة، وضعنا اثنين من موضع معين لونين مناعي (رقاقة) التكنولوجيات، وهي إقحامي رقاقة (iChIP) 13/08 وهندستها بوساطة جزيء رقاقة الحمض النووي ملزم (enChIP) 14-17 . في iChIP، ووضع علامة على موضع الاهتمام عن طريق إدراج تسلسل الاعتراف بروتين ملزمة الحمض النووي الخارجية مثل ليكسثم يتم عزل A. موضع كتبها تنقية تقارب باستخدام بروتين ملزمة DNA الموسومة. في enChIP وهندستها جزيئات الحمض النووي ملزم، مثل البروتينات والزنك الاصبع، النسخ الشبيهة المنشط (TAL) البروتينات، وتتجمع interspaced بانتظام يكرر المتناوب قصيرة (كريسبر) المجمعات، وتستخدم لوضع علامة على موضع الاهتمام (الشكل 1). وفي وقت لاحق، تم عزل المنطقة الجينومية التي كتبها تنقية تقارب من جزيئات الحمض النووي ملزم المعلمة.
واحدة من مزايا enChIP على iChIP هو أن إدراج تسلسل الاعتراف بروتين ملزمة الحمض النووي الخارجية ليست ضرورية. استهداف مواضع باستخدام المجمعات كريسبر يتكون من شكل غير نشط حفاز من Cas9 (dCas9) ودليل الحمض النووي الريبي (gRNA) هو أسهل بكثير من استهداف هذه المناطق من قبل iChIP أو enChIP باستخدام TAL والزنك والبروتينات الإصبع. هنا، نحن تصف بروتوكول خطوة بخطوة لenChIP جنبا إلى جنب مع مطياف الكتلة وRNA تسلسل (RNA تسلسل) لتحديد مكان-لجمعياتالبروتينات تيد والرنا، على التوالي.
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.
Divulgations
The authors have nothing to disclose.
Acknowledgements
وأيد هذا العمل من قبل مؤسسة العلوم تاكيدا (TF)؛ مؤسسة الزجاج أساهي؛ مؤسسة ميموريال أوهارا (HF)؛ وكوراتا التذكارية هيتاشي علوم والتكنولوجيا (TF وHF)؛ منح في المعونة للعلماء الشباب (B) (# 25830131)، منحة في والمعونة للبحوث العلمية (C) (# 15K06895) (TF)؛ ومنحة في والمعونة للبحوث العلمية في "النسخ الدراجات المجالات المبتكرة (# 25118512 & # 15H01354)، منحة في والمعونة للبحوث العلمية (B) (# 15H04329)، منحة في والمعونة للبحوث الاستكشافية ( # 26650059) و "دعم الجينوم '(# 221S0002) (HF) من وزارة التربية والتعليم والثقافة والرياضة والعلوم والتكنولوجيا في اليابان.
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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