この文書は、(C. フリッチュ) に助成金 T32ES019851 からの資金によって可能になった、R01AG054641 と遠く若手研究者は (M. Vermulst) に付与します。

<ol>
	<li>Kireeva, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+reversal+of+RNA+polymerase+II+active+site+closing+controls+fidelity+of+transcription+elongation.">Transient reversal of RNA polymerase II active site closing controls fidelity of transcription elongation.</a> Molecular Cell. 30, 557-566 (2008).</li><li>Walmacq, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rpb9+subunit+controls+transcription+fidelity+by+delaying+NTP+sequestration+in+RNA+polymerase+II.">Rpb9 subunit controls transcription fidelity by delaying NTP sequestration in RNA polymerase II.</a> The Journal of Biological Chemistry. 284, 19601-19612 (2009).</li><li>Strathern, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fidelity+of+transcription:+RPB1+(RPO21)+mutations+that+increase+transcriptional+slippage+in+S.+cerevisiae.">The fidelity of transcription: RPB1 (RPO21) mutations that increase transcriptional slippage in S. cerevisiae.</a> The Journal of Biological Chemistry. 288, 2689-2699 (2013).</li><li>Irvin, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+assay+for+transcription+errors+reveals+multilayer+control+of+RNA+polymerase+II+fidelity.">A genetic assay for transcription errors reveals multilayer control of RNA polymerase II fidelity.</a> PLoS Genetics. 10, e1004532 (2014).</li><li>Gout, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+landscape+of+transcription+errors+in+eukaryotic+cells.">The landscape of transcription errors in eukaryotic cells.</a> Science Advances. 3, e1701484 (2017).</li><li>Gout, J. F., Thomas, W. K., Smith, Z., Okamoto, K., Lynch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+detection+of+in+vivo+transcription+errors.">Large-scale detection of in vivo transcription errors.</a> Proceedings of the National Academy of Science of the United States of America. 110, 18584-18589 (2013).</li><li>Viswanathan, A., You, H. J., Doetsch, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+change+caused+by+transcriptional+bypass+of+uracil+in+nondividing+cells.">Phenotypic change caused by transcriptional bypass of uracil in nondividing cells.</a> Science. 284, 159-162 (1999).</li><li>Bregeon, D., Doddridge, Z. A., You, H. J., Weiss, B., Doetsch, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+mutagenesis+induced+by+uracil+and+8-oxoguanine+in+Escherichia+coli.">Transcriptional mutagenesis induced by uracil and 8-oxoguanine in Escherichia coli.</a> Molecular Cell. 12, 959-970 (2003).</li><li>Saxowsky, T. T., Doetsch, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+polymerase+encounters+with+DNA+damage:+transcription-coupled+repair+or+transcriptional+mutagenesis.">RNA polymerase encounters with DNA damage: transcription-coupled repair or transcriptional mutagenesis.</a> Chemical Reviews. 106, 474-488 (2006).</li><li>Saxowsky, T. T., Meadows, K. L., Klungland, A., Doetsch, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=8-Oxoguanine-mediated+transcriptional+mutagenesis+causes+Ras+activation+in+mammalian+cells.">8-Oxoguanine-mediated transcriptional mutagenesis causes Ras activation in mammalian cells.</a> Proceedings of the National Academy of Science of the United States of America. 105, 18877-18882 (2008).</li><li>Vermulst, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+errors+induce+proteotoxic+stress+and+shorten+cellular+lifespan.">Transcription errors induce proteotoxic stress and shorten cellular lifespan.</a> Nature Communications. 6, 8065 (2015).</li><li>van Leeuwen, F. W., Burbach, J. P., Hol, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+RNA:+a+first+example+of+molecular+misreading+in+Alzheimer's+disease.">Mutations in RNA: a first example of molecular misreading in Alzheimer's disease.</a> Trends in Neurosciences. 21, 331-335 (1998).</li><li>van Leeuwen, F. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frameshift+mutants+of+beta+amyloid+precursor+protein+and+ubiquitin-B+in+Alzheimer's+and+Down+patients.">Frameshift mutants of beta amyloid precursor protein and ubiquitin-B in Alzheimer's and Down patients.</a> Science. 279, 242-247 (1998).</li><li>Morreall, J. F., Petrova, L., Doetsch, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+mutagenesis+and+its+potential+roles+in+the+etiology+of+cancer+and+bacterial+antibiotic+resistance.">Transcriptional mutagenesis and its potential roles in the etiology of cancer and bacterial antibiotic resistance.</a> Journal of Cellular Physiology. 228, 2257-2261 (2013).</li><li>Strathern, J. N., Jin, D. J., Court, D. L., Kashlev, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+transcription+fidelity+mutants.">Isolation and characterization of transcription fidelity mutants.</a> Biochimica et Biophysica Acta. 1819, 694-699 (2012).</li><li>Acevedo, A., Andino, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Library+preparation+for+highly+accurate+population+sequencing+of+RNA+viruses.">Library preparation for highly accurate population sequencing of RNA viruses.</a> Nature Protocols. 9, 1760-1769 (2014).</li><li>Traverse, C. C., Ochman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-Wide+Spectra+of+Transcription+Insertions+and+Deletions+Reveal+That+Slippage+Depends+on+RNA:DNA+Hybrid+Complementarity.">Genome-Wide Spectra of Transcription Insertions and Deletions Reveal That Slippage Depends on RNA:DNA Hybrid Complementarity.</a> mBio. 8, (2017).</li><li>Martin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutadapt+Removes+Adapter+Sequences+From+High-Throughput+Sequencing+Reads.">Cutadapt Removes Adapter Sequences From High-Throughput Sequencing Reads.</a> EMBnet.journal. 17 (1), 10-12 (2011).</li><li>Kent, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BLAT--the+BLAST-like+alignment+tool.">BLAT--the BLAST-like alignment tool.</a> Genome Research. 12, 656-664 (2002).</li><li>Kim, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TopHat2:+accurate+alignment+of+transcriptomes+in+the+presence+of+insertions,+deletions+and+gene+fusions.">TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions.</a> Genome Biology. 14, R36 (2013).</li><li>Zhou, Y. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+RNA+polymerase+rpoB+mutations+that+alter+transcription+slippage+during+elongation+in+Escherichia+coli.">Isolation and characterization of RNA polymerase rpoB mutations that alter transcription slippage during elongation in Escherichia coli.</a> The Journal of Biological Chemistry. 288, 2700-2710 (2013).</li><li>Reid-Bayliss, K. S., Loeb, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+RNA+consensus+sequencing+for+high-fidelity+detection+of+transcriptional+mutagenesis-induced+epimutations.">Accurate RNA consensus sequencing for high-fidelity detection of transcriptional mutagenesis-induced epimutations.</a> Proceedings of the National Academy of Science of the United States of America. 114, 9415-9420 (2017).</li></ol>

著者が明らかに何もありません。

ここでは、酵母、ショウジョウバエ、線虫で転写エラーの検出のための C seq ライブラリの準備のための最適化されたプロトコルについて述べる。このプロトコルには、代替技術と同様に、既存のプロトコルでは、多くの利点があります。
過去 15 年間、レポーターの多数のシステム開発されているルシフェラーゼ7,<sup class="xr...

ゲノムは生命の正確な生物の設計図を提供します。この青写真を正しく実装するには、素晴らしい精度で転写されるゲノムのために重要です。ただし、転写は、エラーが発生する可能性がありますはありません。たとえば、RNA ポリメラーゼがエラーを起こしやすい体外1、2、長い知られている、最近エラー体内だけでなく、3、5,6、犯すことが示されました。特に、DNA 損傷7,8,9,10に直面しています。一緒に取られて、これらの観察は、転写エラー発生する継続的にすべての生きた細胞でタンパク質構造変異の強力なソース可能性があることを示唆していることを示します。
転写変異と呼ばれるこのプロセスは、2 つの方法で古典的な変異によって異なります。最初に、彼らは DNA の複製に依存しない、遺伝子の突然変異とは対照的転写エラーは分裂期と分裂後の細胞を影響します。転写の忠実度に影響を与えるメカニズムを勉強して、したがって、分裂、分裂後の細胞の突然変異荷重に対して貴重な洞察を提供します。興味深いことに、転写エラーが最近タンパク質凝集11,12,13のプロモーションに関与しているし、10両の発癌に寄与するように仮定されたが、細菌14の抗生の抵抗の開発。
第二に、遺伝子の突然変異と対照をなして転写エラー、自然で一時的です。転写エラーを検出するため極めて困難になりますので、彼らの一時的な存在は特に挑戦的です。たとえば、いくつかのラボは、転写の突然変異誘発の研究のための貴重な記者アッセイを考案した、一方これらの試、コンテキストとモデル生物4,15の限られた数の転写エラーを測定することができるのみ。これらの制限を克服するために多くの研究者は、理論により、任意の種のトランスクリプトームを通して記録される転写エラー RNA 塩基配列決定技術 (RNA シーケンス) になっています。ただし、これらの研究は、逆のトランスクリプション エラー、PCR 増幅エラー シーケンス自体のエラーを起こしやすい性質など、図書館建設の成果物によって簡単に混同されます。たとえば、逆転写酵素約 1 つのエラーすべて 〜 20,000 拠点間コミット RNA ポリメラーゼ (RNAPs) は、1 つだけエラーをすべて 300,000 拠点5,6にすると予想されます。従来の RNA シーケンス データ (ライブラリ準備で起因するアーチファクトと真転写エラーを区別することは事実上不可能じゃないので単独で逆のトランスクリプションの誤り率を小さく細胞内 RNA ポリメラーゼのエラー率、図 1 a)。
この問題を解決するために試金5,16円シーケンス (Cirseq、または今後 C seq) の最適化されたバージョンを行った。このアッセイでは、トランスクリプトーム5で RNA の転写エラー、その他の稀な変形を検出することができます。円形配列アッセイは、この試金の重要なステップは、環状 RNA を中心に展開するためにこの名前を運ぶ。RNA ターゲットが円形、一度彼らは同じ RNA テンプレートの数多くのコピーを含む線形 cDNA 分子を生成する、ローリング サークル ファッションを転写したリバースされます。エラーは、これらのテンプレートの 1 つには、このエラーは cDNA 分子内に含まれるすべての単一の繰り返しで現在もでしょう。対照的に、逆のトランスクリプション、PCR 増幅、またはシーケンスによって導入されたエラーはランダムに発生する傾向がある、こうして繰り返されるがのみ 1 つまたは 2 つがあります。したがって、各 cDNA 分子コンセンサス シーケンスを生成してランダム エラーを区別するすべての繰り返しで発生するエラーで、ライブラリ構築アーティファクト効果的にから分離できる真の転写エラー (図 1 b)。
任意の種のトランスクリプトームで RNA の塩基置換、挿入、および削除の速度を正確に検出 C seq 試金を使用することができます適切に使用する場合 (たとえば、トラバース ・ オフマン17を参照)。たとえば、我々 は単一の基本解像度は、5 と酵母、ショウジョウバエ、線虫で転写の誤り率のゲノム全体の測定値を提供するために C seq 試金を使用しています。(未発表の観察)。最初に正確に RNA ウイルス集団をシーケンスに使用、C seq 試金のこの最適化されたバージョンがライブラリ構築アーティファクトに貢献するライブラリの準備中に過酷な条件を最小限に抑えるために合理化されています。さらに、市販のキットの数を使用して、アッセイのスループットが大幅に向上、その使いやすさだけでなく、適切に使用する場合、この試金は正確にあたり、大幅改善前の研究6転写エラーの何千もを検出できます。全体的にみて、このメソッドは転写変異を研究する強力なツールを提供、幅広い生物の転写の忠実度を制御するメカニズムに新しい洞察力を得るためにユーザーになります。

<table><tbody><tr><td>RiboPure RNA purification kit</td><td>ThermoFisher</td><td>AM1926</td><td>Total RNA purification</td></tr><tr><td>Genelute mRNA purification kit</td><td>Sigma-Aldrich</td><td>MRN70-1KT</td><td>mRNA purification</td></tr><tr><td>Nuclease-free Water</td><td>Ambion</td><td>AM9937</td><td>Elution and dilution</td></tr><tr><td>Ambion RNase III</td><td>ThermoFisher</td><td>AM2290</td><td>RNA fragmentation</td></tr><tr><td>T4 RNA Ligase 1 (ssRNA Ligase)</td><td>New England Biolabs</td><td>M0204S</td><td>RNA circularization</td></tr><tr><td>Ribolock</td><td>ThermoFisher</td><td>EO0381</td><td>RNase inhibitor</td></tr><tr><td>SuperScript III Reverse Transcriptase</td><td>ThermoFisher</td><td>18080044</td><td>Rolling circle reverse transcription</td></tr><tr><td>10 mM dNTP mix</td><td>ThermoFisher</td><td>18427013</td><td>Rolling circle reverse transcription</td></tr><tr><td>Random hexamers (50 ng/&amp;micro;L)</td><td>ThermoFisher</td><td>N8080127</td><td>Rolling circle reverse transcription</td></tr><tr><td>NEB Second Strand Synthesis Module</td><td>New England Biolabs</td><td>E6111S</td><td>Second Strand Synthesis</td></tr><tr><td>NEBNext Ultra DNA Library Prep Kit for Illumina</td><td>New England Biolabs</td><td>E7370S</td><td>cDNA library preparation</td></tr><tr><td>NEB Next index primers</td><td>NEB</td><td>E7335S</td><td>Multiplex PCR primers</td></tr><tr><td>Oligo Clean &amp;amp; Concentrator</td><td>Zymo Research</td><td>D4061</td><td>Clean up of RNA and DNA samples</td></tr><tr><td>DynaMag-2 Magnet</td><td>ThermoFisher</td><td>12321D</td><td>Magnetic bead purification</td></tr><tr><td>AMPure XP beads</td><td>Beckman Coulter</td><td>A63881</td><td>Magnetic bead purification</td></tr><tr><td>Eppendorf 5424 Microcentrifuge</td><td>FisherScientific</td><td>05-403-93</td><td>centrifugation</td></tr><tr><td>INCU-Shaker 10 L</td><td>Benchmark Scientific</td><td>H1010</td><td>Cell culture</td></tr><tr><td>T100 Thermal Cycler</td><td>BIO RAD</td><td>1861096</td><td>Medium to High temperature cycling conditions</td></tr><tr><td>PTC-200 Thermal Cycler</td><td>GMI</td><td>8252-30-0001&amp;nbsp;</td><td>Low temperature cycling conditions</td></tr><tr><td>RNase Away</td><td>Molecular Bioproducts</td><td>700S-11</td><td>Sterilization</td></tr><tr><td>50 mL Centrifuge Tube</td><td>Corning</td><td>430290</td><td>Nuclease-free</td></tr><tr><td>15 mL Centrifuge Tube</td><td>Corning</td><td>430052</td><td>Nuclease-free</td></tr><tr><td>Eppendorf tubes</td><td>USA Scientific</td><td>1615-5500</td><td>Nuclease-free</td></tr><tr><td>4200 Tapestation System</td><td>Agilent</td><td>G2991AA</td><td>Nucleotide analysis instrument for quality control of RNA and single stranded DNA samples</td></tr><tr><td>High Sensitivity RNA Screen Tape</td><td>Agilent</td><td>5067-5579</td><td>Quality control of RNA and single stranded DNA samples</td></tr><tr><td>RNA ScreenTape Sample Buffer</td><td>Agilent</td><td>5067-5577</td><td>Quality control of RNA and single stranded DNA samples</td></tr><tr><td>RNA ScreenTape Ladder</td><td>Agilent</td><td>5067-5578</td><td>Quality control of RNA and single stranded DNA samples</td></tr><tr><td>2100 Bioanalyzer Instrument</td><td>Agilent</td><td>G2939BA</td><td>Double stranded DNA quality control</td></tr><tr><td>High Sensitivity DNA Kit</td><td>Agilent</td><td>5067-4626</td><td>Quality control for double stranded cDNA samples</td></tr><tr><td>Water Bath</td><td>VWR</td><td>462-0244</td><td>Incubation</td></tr><tr><td>NanoDrop 2000/2000C Spectrophotometer</td><td>ThermoFisher</td><td>ND-2000C</td><td>Determination of RNA concentration</td></tr></tbody></table>

genome-wide surveillance of transcription errors in eukaryotic organisms

正確な表記は、遺伝情報の忠実な表現に必要です。しかし意外にも、少しは転写の忠実度を制御するメカニズムについて知られています。科学の知識で、このギャップを埋めるためには、我々 は最近線虫、ショウジョウバエ、酵母のトランスクリプトームを通して転写エラーを検出するサークル シーケンス分析を最適化線虫。このプロトコルは、前例のない詳細の転写の忠実度を制御するメカニズムを解明することができますので、真核細胞での転写エラーの風景にマップする強力な新しいツールを使って研究を提供します。

大量並列シーケンスのアプローチのすべてのような各 C seq 実験は扱いにくく、大規模なデータセットを生成します。初めてユーザーのためそれはこれらのデータセットを処理することは困難することができます。したがって、実験前に経験豊富なバイオ informatician をすべてのユーザーに連絡をお勧めします。平均では、期待は、ユーザーあたりのほとんどの大量並列...

Watch this Scientific Journal Video about Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms at JoVE.com

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

このプロトコルは、複数のモデル生物における転写の忠実度を監視する新しいツールと研究者を提供します。

真核生物の転写エラーのゲノム全体の監視

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

genome-wide-surveillance-transcription-errors-eukaryotic

Research

JoVE Journal

Genetics

9.4K ビュー.  Children's Hospital of Philadelphia. このプロトコルは、複数のモデル生物における転写の忠実度を監視する新しいツールと研究者を提供します。

ビデオ: Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on (TRO) Approach

This manuscript describes a protocol for detecting transcription termination defect in vivo. The strand-specific TRO protocol using BrUTP described here is a powerful experimental approach for analyzing the transcription termination defect under physiological conditions. Like the traditional TRO assay, it relies on the presence of a transcriptionally active polymerase beyond the 3&#8242; end of the gene as an indicator of a transcription termination defect1. It overcomes two major problems encountered with the traditional TRO assay. First, it can detect if the polymerase reading through the termination signal is the one that initiated transcription from the promoter-proximal region, or if it is simply representing a pervasively transcribing polymerase that initiated non-specifically from somewhere in the body or the 3&#8242; end of the gene. Secondly, it can distinguish if the transcriptionally active polymerase signal beyond the terminator region is truly the readthrough sense mRNA transcribing polymerase or a terminator-initiated non-coding anti-sense RNA signal. Briefly, the protocol involves permeabilizing the exponentially growing yeast cells, allowing the transcripts that initiated in vivo to elongate in the presence of the BrUTP nucleotide, purifying BrUTP-labelled RNA by the affinity approach, reverse transcribing the purified nascent RNA and amplifying the cDNA using strand-specific primers flanking the promoter and the terminator regions of the gene2.

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on TRO Approach

We describe a basic experimental approach for analysis of termination of transcription by RNA polymerase II in vivo using BrUTP by the strand-specific transcription run-on (TRO) approach in budding yeast. This protocol can be extended to study transcription termination by other RNA polymerases both in yeast and higher eukaryotes.

BrUTP本鎖特異的転写ランオン（TRO）のアプローチを使用した転写の終結の分析

This manuscript describes a protocol for detecting transcription termination defect in vivo. The strand-specific TRO protocol using BrUTP described here ...

遺伝学

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

ChEC-seqとのタンパク質 - DNA相互作用のゲノムワイドマッピング<em&gt; Saccharomyces cerevisiae</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

転写因子の結合、細胞活性中に事象を表示する転写、翻訳、回転率のリアルタイム解析

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break.
The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. 
Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

強化されたゲノム細胞や生物の多様な Cas9 リボ核蛋白質と編集

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

哺乳類細胞における微小コッカルヌクレアーゼを用いてクロマチン免疫沈殿アッセイ

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using &#967;CRAC

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be bound by many different RBPs that will regulate its production, stability, activity, and degradation. As such, much has been done to understand the dynamics that exist between these two types of molecules. A particularly important breakthrough came with the emergence of &#8216;cross-linking and immunoprecipitation&#8217; (CLIP). This technique allowed stringent investigation into which RNAs are bound by a particular RBP. In short, the protein of interest is UV cross-linked to its RNA substrates in vivo, purified under highly stringent conditions, and then the RNAs covalently cross-linked to the protein are converted into cDNA libraries and sequenced. Since its conception, many derivative techniques have been developed in order to make CLIP amenable to particular fields of study. However, cross-linking using ultraviolet light is notoriously inefficient. This results in extended exposure times that make the temporal study of RBP-RNA interactions impossible. To overcome this issue, we recently designed and built much-improved UV irradiation and cell harvesting devices. Using these new tools, we developed a protocol for time-resolved analyses of RBP-RNA interactions in living cells at high temporal resolution: Kinetic CRoss-linking and Analysis of cDNAs (&#967;CRAC). We recently used this technique to study the role of yeast RBPs in nutrient stress adaptation. This manuscript provides a detailed overview of the &#967;CRAC method and presents recent results obtained with the Nrd1 RBP.

Kinetic cross-linking and analysis of cDNA is a method that allows investigation of the dynamics of protein-RNA interactions in living cells at high temporal resolution. Here the protocol is described in detail, including the growth of yeast cells, UV cross-linking, harvesting, protein purification, and next generation sequencing library preparation steps.

χCRACを用いたin vivoでのタンパク質-RNA相互作用ダイナミクスの高時間分解能モニタリング

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be ...

生化学

Proximity Ligation Assay to Study Oncogene-Derived Transcription-Replication Conflicts

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. Conflicts between the replication and transcription machinery, termed transcription-replication conflicts (TRCs), pose a considerable risk to genome stability, a critical factor in cancer development. While several factors regulating these collisions have been identified, pinpointing primary causes remains difficult due to limited tools for direct visualization and clear interpretation. In this study, we directly visualize TRCs using a proximity ligation assay (PLA), leveraging antibodies specific to PCNA and phosphorylated CTD of RNA polymerase II. This approach allows precise measurement of TRCs between replication and transcription processes mediated by RNA polymerase II. The method is further enhanced through DNA primers conjugated covalently to these antibodies, coupled with PCR amplification using fluorescent probes, providing a highly sensitive and specific means of detecting endogenous TRCs. Fluorescence microscopy enables the visualization of these conflicts, offering a powerful tool to study genome instability mechanisms associated with cancer. This technique addresses the gap in direct TRC visualization, allowing for a more comprehensive analysis and understanding of the underlying processes driving genome instability in cells.

Here, we present a sensitive and specific method to detect oncogene-induced transcription-replication conflicts (TRCs) using the proximity ligation assay (PLA). This approach leverages specific antibodies targeting PCNA and phospho-CTD RNAPII, enabling the assessment of TRC prevalence between RNA polymerase II transcription and DNA replication machinery.

がん遺伝子由来の転写-複製コンフリクトを研究するための近接ライゲーションアッセイ

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. ...

がん研究

Capturing Common Fragile Site Breaks by Native &#947;H2A.X ChIP

Replication stress induced by exposure to extrinsic agents can lead to DNA breaks at common fragile sites, which are regions in the genome known to be prone to structural instability. The &#947;H2A.X chromatin immunoprecipitation (ChIP) assay serves as a powerful tool in genotoxicity studies, as &#947;H2A.X phosphorylation is a well-established marker for DNA double-strand breaks. Traditional &#947;H2A.X ChIP assays, however, are often labor-intensive and involve multiple, time-consuming steps. In this study, we present a simplified yet effective method that combines subcellular fractionation with native ChIP to isolate &#947;H2A.X-associated complexes. This approach is particularly suitable for analyzing &#947;H2A.X-chromatin interactions with enhanced specificity and efficiency. Using subcellular fractionation, chromatin-unbound materials are effectively removed, resulting in a purified chromatin fraction. Subsequent micrococcal nuclease (MNase) digestion under mild conditions allows chromatin fragmentation while preserving physiological interactions between &#947;H2A.X and its associated protein complexes. This preservation is essential for studying native interaction partners involved in DNA damage response pathways. This optimized native ChIP protocol substantially reduces the time and labor associated with conventional &#947;H2A.X ChIP assays. The streamlined procedure not only simplifies the workflow but also yields highly reproducible results, making it particularly advantageous in settings where high-throughput processing of multiple samples is required. This method has broad applicability in studies focused on genome stability, DNA repair, and chromatin biology, where accurate and efficient detection of DNA damage sites is critical. By employing optimized protocols and streamlined steps, this method enables the detection of DNA damage at fragile sites with improved sensitivity and minimal sample handling, making it a valuable tool for studies on genome stability and DNA damage response.

We present a rapid and efficient method to detect common fragile site breaks through native &#947;H2A.X chromatin immunoprecipitation (ChIP). This approach significantly reduces both the time and labor associated with traditional &#947;H2A.X ChIP assays while maintaining high reproducibility and reliability of results.

ネイティブγH2Aによる一般的な脆弱サイトブレークのキャプチャ。X ChIPの

Replication stress induced by exposure to extrinsic agents can lead to DNA breaks at common fragile sites, which are regions in the genome known to be ...

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene expression. Along with a better understanding of the involved molecular mechanisms, precise measurements of the underlying kinetics have become increasingly important. Here, these powerful methodologies face major limitations due to intrinsic properties of the template samples they study, i.e. total cellular RNA. In many cases changes in total cellular RNA occur either too slowly or too quickly to represent the underlying molecular events and their kinetics with sufficient resolution. In addition, the contribution of alterations in RNA synthesis, processing, and decay are not readily differentiated.
We recently developed high-resolution gene expression profiling to overcome these limitations. Our approach is based on metabolic labeling of newly transcribed RNA with 4-thiouridine (thus also referred to as 4sU-tagging) followed by rigorous purification of newly transcribed RNA using thiol-specific biotinylation and streptavidin-coated magnetic beads. It is applicable to a broad range of organisms including vertebrates, Drosophila, and yeast. We successfully applied 4sU-tagging to study real-time kinetics of transcription factor activities, provide precise measurements of RNA half-lives, and obtain novel insights into the kinetics of RNA processing. Finally, computational modeling can be employed to generate an integrated, comprehensive analysis of the underlying molecular mechanisms.

Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations.

細胞培養におけるRNA合成、処理およびディケイの高解像度遺伝子発現プロファイリングのための新規転写されたRNAの代謝標識

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene ...

生物学

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

機能電気泳動移動度シフトアッセイ（EMSA）を用いた非コーディング遺伝的変異体およびDNA親和性沈殿アッセイ（DAPA）のスクリーニング

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical ...