私たちはKaryn Esser、Koike Nobuya、Park Noheon Parkに助けになることに感謝します。この研究は、NIH / NIGMS（R01GM114424）がS.-HYに、またRobert A. Welch Foundation（AU-1731）とNIH / NIA（R01AG045828）がZC

動物のケアは、施設の動物のケアおよび使用委員会（IACUC）ガイドラインの下で実施され、手順はヒューストンのテキサス大学健康科学センターによって承認された動物のプロトコルに従って行われた。 1.架橋骨格筋からの核の単離<ol><li>前に詳述したように、氷冷リン酸緩衝生理食塩水（PBS）中、約20週齢のC57BL / 6雄マウスから単離した後肢骨格筋を秤量して切り刻む。 注：我々は通常、1つのマウスから1.0〜1.5 gの骨格筋を得る。 <ol><li>氷上で10mLのPBSを含む50mLコニカルチューブに細かい骨格筋組織を置く。 </li></ol></li><li> 4℃で300xgで5分間遠心分離する。 </li><li>慎重に吸引によりPBS上清を除去する。 </li><li> 1、2、3、および4 mLの水を含む参照チューブを使用して、各50 mLチューブのペレット容量を見積もります。 </li><li> 7巻追加PBS中の氷冷した1％ホルムアルデヒドで洗浄し、プローブ組織ホモジナイザー（ 表を参照）を用いて氷上でサンプルをホモジナイズする。 注：オプション：ホモジナイゼーション手順は、低温室で行うことができます。 </li><li>室温で10分間インキュベートすることによって試料を架橋させる。 </li><li> 1Mグリシンを添加して架橋反応を停止させ、0.125Mの最終濃度とし、室温で5分間インキュベートする。 </li><li>サンプルを3,000 xgで5分間4℃で遠心分離する。吸引により上清を除去する。 </li><li>新たに加えたプロテアーゼインヒビター（0.2mMのフェニルメチルスルホニルフルオライド（PMSF））を含有する10mLの氷冷塩基緩衝液（pH7.3,10mM EDTA、10mM KCl、5mM MgCl 2、0.5mM DTTの10mM HEPES-KOH） 、及び10μg/ mLのロイペプチン）を含む。 3,000 gで5分間4℃で遠心分離し、上清を慎重に吸引除去する。 </li><li>ペレットを6mLの溶解緩衝液（10mM HEPES-KOH、p（0.2mMのPMSF、および10μg/ mLのロイペプチン）を含有する10mMのHEPES緩衝液（pH7.5,10mM EDTA、10mM KCl、5mM MgCl 2、0.5mM DTT、1％Triton X- </li><li>試料を予め冷却した15mL Dounceホモジナイザーに移し、氷上で10分間インキュベートする。ゆるやかな乳棒を用いて15回のゆっくりとしたストロークで各試料を氷上でホモジナイズし、密栓した15ストロークで核を放出させる。 </li><li>ホモジネート（6mL）を細胞ストレーナー（孔径：100μm）で濾過し、4mLの溶解緩衝液ですすぐ。 4℃で1,000 xgで10分間遠心します。 5mLの氷冷した塩基緩衝液に再懸濁し、密な乳棒で10回叩いて核を放出させる。 <ol><li>分光光度計（OD260 / OD280）でDNA濃度を測定するために20μLのアリコートを取り出し、必要に応じてトリパンブルーで顕微鏡で観察します（下図参照）（ 図 1A ）。 </li></ol></li><li>サスペンションをフィルタリングする細胞ストレーナ（細孔サイズ：70μm）を用いて、チューブをすすぎ、上記のように2mLの塩基緩衝液で再度濾過する。 </li><li>徐々に細孔サイズ（40μm、30μm、20μmおよび10μm）が減少した細胞ストレーナーで、ステップ1.13と同様にろ過を繰り返します。 注：合計で、6つの孔サイズのストレーナーがステップ1.12-1.14で使用された。 </li><li> 4℃、1,000gで10分間遠心分離する。上清を除去し、ペレットを500μLの塩基緩衝液に再懸濁する。 </li><li>上記のようにDNA濃度を測定する。 注：約200μgの核酸DNAが1匹の動物からの両方の後肢を組み合わせて得られた。必要に応じて、トリパンブルー染色（ストック濃度0.4％、1：5希釈）による顕微鏡観察のために20μLを保存する。核が青く染色される（ 図 1B ）。 </li><li> 4℃、1000gで10分間遠心分離し、上清を捨てる。ペレットを-80℃で保存する。 </li></ol> 2.超音波 &lt;ol&gt; <li>サンプルを500μLのSDS溶解バッファーで解凍し、穏やかなピペッティングで再懸濁します。 </li><li>核のDNA懸濁液を氷上のガラスバイアルに移す。 </li><li>皿状のトランスデューサからの超音波音響エネルギーの集中したバーストで超音波処理を実行します。装置の詳細については、 表を参照してください。次の設定を使用します。強度：5;デューティサイクル：20;温度4〜6℃; 30秒オン/オフ。 注：音波処理の有効性を評価するための例を図2に示します 。 </li><li>超音波処理したクロマチンを1.5mLチューブに移す。 12,000 xgで15分間遠心分離し、上清を新しいチューブに移す。 65℃で一晩逆架橋させるために、超音波処理されたサンプル（〜7-10μg/筋肉サンプル）50μLを新しい1.5mLチューブに移す。残りのサンプルを-80℃で凍結させる。 </li></ol> 3.超音波と定量の評価<ol><li> 1.0を追加保存した50μLの超音波処理されたサンプルに、RNase A（500U / mL RNase A：20,000U / mL RNase T1）を加え、37℃で30分間インキュベートする。 </li><li> 8μLのプロテアーゼK（10mg / mL）を添加し、55℃で30分間インキュベートする。 </li><li> DNA抽出キットを使用してDNAを収穫する。 <ol><li> 5倍量の結合緩衝液を添加し、スピンカラムにかけ、4,000xg、10分間遠心する。 </li><li>パススルーを同じカラムにかけ、再度遠心します。 </li><li>洗浄バッファーで13,000 xg、1分間で2回洗浄する。 </li><li>カラムを乾燥させるために13,000 xgで1分間遠心分離します。 </li><li> 50μLのH 2 Oを加え、13,000×g、1分間遠心分離してDNAを溶出する。 </li><li>パススルーを同じカラムにかけ、再度遠心します。 </li></ol></li><li>分光光度計（OD260 / OD280）でDNAの量を定量する。 0.8％ゲルでサンプルを実行して超音波処理された製品のサイズおよび量（1μg）を評価する（ 図2 ）。 注：平均クロマチン収率は約20％です。 </li></ol> 4.チップ<ol><li>以前保存したクロマチンを-80°Cで解凍し、クロマチンをチューブあたり約100〜120μgに等分します。放射免疫沈降アッセイ（RIPA）緩衝液（20mM Tris-HCl（pH7.4）、150mM NaCl、1mM EDTA、1mM NaF、1mM Na 3 VO 4、1mM PMSF、1×プロテアーゼ阻害剤カクテル（PIC）、0.1％ w / v）、1％トリトン（Triton）X-100）。 <ol><li>各グループに複数のサンプルがある場合は、同量のクロマチンDNAを最終量約100〜120μg/サンプルに合わせます。入力として10％を保存します。 </li></ol></li><li> BSAプレブロック（〜2時間、4℃）プロテインA / Gアガロース（非鶏抗体）またはIgYビーズ（鶏抗体）の40μL（RIPAバッファー中50％v / vスラリー）を添加し、 4℃で3時間回転させた。 </li><li> 1,000 xgで10分間遠心分離し、上清を注意深く新しいチューブに移す。 </li><li>抗体を1μg/25〜100μgのクロマチンDNA。 </li><li>約15 rpmで一晩4℃で静かに回転させます。 </li><li> 10μLのBSAプレブロック（〜2時間、4℃）プロテインA / Gアガロース（非鶏抗体）またはIgYビーズ（鶏抗体）を加え、冷室で2時間回転させます。 </li><li>ビーズを以下のように洗浄する。 RIPA緩衝液1mLで3分間2回洗浄し、1mLの高塩緩衝液（20mM Tris-HCl（pH7.4）、500mM NaCl、2mM EDTA、1mM PMSF、1％Triton X-100）で3分間2回洗浄する。 1mLのLiCl緩衝液（20mM Tris-HCl（pH7.4）、250mM LiCl、2mM EDTA、1mM PMSF、1％Na-デオキシコール酸塩、0.5％NP40）で3分間2回洗浄する。 <ol><li> 1mLのTris-EDTA（TE）緩衝液（10mM Tris-HCl（pH7.4）、1mM EDTA）で3分間1回洗浄する。次いで、溶出緩衝液（20mM Tris-HCl、5mM EDTAおよび0.5％SDS）でDNAを溶出する。 </li></ol></li><li> 1,000gで1分間遠心分離し、上清を慎重に除去する。 </li><li>溶出バッファー50μLでビーズを再懸濁する。 </li><li> 10日間インキュベートする分で65℃で保存した。 </li><li> 12,000 gで5分間遠心分離する。 </li><li>上清を新しいチューブに移し、さらに50μLを加え、12,000gで5分間遠心分離する。最終溶出量は100μLになります。 </li><li>逆架橋およびDNA溶出。 <ol><li>溶出したクロマチンを一晩65℃でインキュベートする。 </li><li> 1.0μLのRNase Aを添加し、37℃で30分間インキュベートする。 </li><li> 8μLのプロテアーゼK（10mg / mL）を添加し、55℃で30分間インキュベートする。 </li><li>製造者のプロトコールに従って、50μLの溶出容量を有するPCRクリーンアップキットを用いてDNAを収穫する。 </li></ol></li><li>先に説明したようにリアルタイムqPCRによるプロファイルを分析する（ 25,26） 。 注：プライマー配列は図3の 凡例にリストされています 。データは、平均±SEMとして示す （ 図3 ）。 </li></ol>

<ol>
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A., Schibler, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhythmic+CLOCK-BMAL1+binding+to+multiple+E-box+motifs+drives+circadian+Dbp+transcription+and+chromatin+transitions.">Rhythmic CLOCK-BMAL1 binding to multiple E-box motifs drives circadian Dbp transcription and chromatin transitions.</a> Nat Genet. 38 (3), 369-374 (2006).</li><li>Laplante, 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=DEPTOR+cell-autonomously+promotes+adipogenesis,+and+its+expression+is+associated+with+obesity.">DEPTOR cell-autonomously promotes adipogenesis, and its expression is associated with obesity.</a> Cell Metab. 16 (2), 202-212 (2012).</li><li>Blechman, J., Amir-Zilberstein, L., Gutnick, A., Ben-Dor, S., Levkowitz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metabolic+regulator+PGC-1alpha+directly+controls+the+expression+of+the+hypothalamic+neuropeptide+oxytocin.">The metabolic regulator PGC-1alpha directly controls the expression of the hypothalamic neuropeptide oxytocin.</a> J Neurosci. 31 (42), 14835-14840 (2011).</li></ol>

著者は何も開示することはない。

ここでは、架橋された骨格筋組織を用いて高品質の核を単離する堅牢な方法について説明します。塵から核を効果的に分離するために逐次濾過を行い、皿状トランスデューサからの超音波エネルギーがChIP分析のためにクロマチンを剪断した。この結果は、標的プロモーターにBMAL1の概日特異的結合を示した。 ChIPは、架橋が起こるときにゲノムDNA上のリアルタイムタンパ...

骨格筋は生理および行動において重要な役割を果たす。多核筋繊維は、アクチンとミオシンが収縮力を発生させる筋節と呼ばれる機能単位を形成する筋原繊維からなる。骨格筋は体内で最大の代謝臓器でもあり、食後のグルコース摂取量が80％を超え、インスリン応答と代謝ホメオスタシスを調節します1,2 。筋肉の生理および代謝は、内在的な時計である内在的な生物学的タイマー3,4,5,6によって厳密に規制されている。例えば、概日の主要な時計成分の1つであるBmal1の骨格筋特異的欠損は、インスリン抵抗性をもたらし、骨格筋におけるグルコース取り込みを減少させ、動物は2型糖尿病を発症することが判明した7 。私さらに、骨格筋も内分泌臓器8として評価され、全身の代謝および生理機能を調節するためにミオシンを分泌する。骨格筋におけるこれらの調節機能を完全に理解するためには、機構的研究が必要である。 ChIPは、DNA結合タンパク質のプロモーター補充を描写するための強力なアプローチである。 ChIPは、最初にクロマチンDNA 9,10上のヌクレオソーム構成を同定するために開発された。ホルムアルデヒド、硫酸ジメチルまたは紫外線照射（UV） 11,12を使用してタンパク質およびクロマチンDNAを架橋する様々な方法が報告されている。ホルムアルデヒド架橋は、最も一般的に使用され、クロマチン構造およびDNA-タンパク質相互作用を保存している9,13,14。架橋クロマトグラフィーinを音波処理により細断し、目的の特定のDNA結合タンパク質15,16に対する抗体で免疫沈降させる。近年、ChIPとNGSを組み合わせたChIP配列決定法（ChIP-seq）は、ゲノム全体の転写因子結合を調べるために開発されており、場合によっては時間経過に伴う動的変化をモニターするために開発されている18,19,20 。例えば、概日ChIP-seq研究は、24時間の概日周期18にわたって時間的に正確な遺伝子発現を駆動する概日クロック成分およびヒストンマーカーのゲノム結合の高度に調整された配列を明らかにした。 最も入手可能なChIPプロトコルは、軟組織（ 例えば、肝臓、脳など ）用に設計されており、骨格タルマシン。繊維が豊富な骨格筋を均質化し、高品質の核21を分離することは技術的に困難であり、特に架橋を必要とするChIP実験の場合には困難である。最近の筋肉ChIP研究22では、衛星細胞を筋繊維から分離し、核を両方の細胞型から、組織消化を含む長期の手順によって調製した。ホルムアルデヒド架橋が単離された核の上で行われる前に、全工程が完了するまでに約3時間を要した。この手順は、筋肉組織をより効率的な均質化に対して不応性にし、高品質の核を生成することができた筋繊維の架橋を回避したが、組織の収集から核の架橋への有意なタイムラグは、 - タンパク質相互作用。対照的に、ほとんどの研究は、リアルタイムDNA-プロテイムを保存するために、実験的処置または組織回収の直後に架橋を行ったn結合12 。架橋前の核単離の第2の欠点は、典型的には3〜4時間間隔で生じる概日検体採取のような時間依存性の適用を排除することである。核を架橋させることなく、解離の直後に単離を進行させる必要があるのに対し、架橋した試料は時間経過全体が完了した後に一緒に処理することができ、実験の一貫性を保証する。 未架橋骨格筋からの核の単離のための他のプロトコールも報告されている。 2つの研究では、勾配超遠心分離を使用して残りの筋原線維および細胞破片から核を分離することが記載されている23,24 。スクロースまたはコロイド勾配超遠心分離が未架橋筋肉組織に有効である一方で、本発明者らの実験は、架橋後、勾配超遠心分離が細胞dから核を分離することができないことを明らかにしたエブリスはグラデーションに乗っています。 したがって、我々は、架橋マウス骨格筋組織を用いて高品質の核を単離するための手順を開発した。グラジエント超遠心分離ではなく、連続したろ過法を考案し、核を残骸から効果的に分離しました。超音波処理後、クロマチンサンプルは、標的プロモーターに結合するBMAL1タンパク質の概日パターンを示すChIP研究に首尾よく適用された。我々の方法は、筋肉組織の様々な機械的研究に広く適用することができる。

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Formaldehyde solution</td><td>Sigma Aldrich</td><td>F8775&amp;nbsp;</td><td></td></tr><tr><td>Glycine</td><td>Fisher Scientific</td><td>BP381-5</td><td></td></tr><tr><td>Trypan Blue solution (0.4%)</td><td>Fisher Scientific</td><td>15250061</td><td></td></tr><tr><td>RNase A</td><td>Sigma Aldrich</td><td>10109142001</td><td></td></tr><tr><td>Protease K</td><td>Sigma Aldrich</td><td>3115887001</td><td></td></tr><tr><td>Chicken Anti-BMAL1 antibody</td><td></td><td></td><td>Generated in chicken (Cocalico Biologicals) against antigen aa 318 - 579, and IgY was affinity purified using the same antigen.&amp;nbsp;</td></tr><tr><td>Chicken IgY Precipitating Resin</td><td>GenScript</td><td>L00405</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>KINEMATICA&amp;nbsp;Polytron PT2100 Benchtop Homogenizer</td><td>Fisher Scientific</td><td>08-451-178</td><td></td></tr><tr><td>15 mL Dounce tissue grinder</td><td>Whearton</td><td>357544</td><td></td></tr><tr><td>Falcon Cell Strainers 100 &amp;micro;m</td><td>Fisher Scientific</td><td>08-771-19</td><td></td></tr><tr><td>Falcon Cell Strainers 70 &amp;micro;m</td><td>Fisher Scientific</td><td>08-771-1</td><td></td></tr><tr><td>Falcon Cell Strainers 40 &amp;micro;m</td><td>Fisher Scientific</td><td>08-771-2</td><td></td></tr><tr><td>pluriStrainer 30 &amp;micro;m</td><td>PluriSelect</td><td>43-50030-03</td><td></td></tr><tr><td>pluriStrainer 20 &amp;micro;m</td><td>PluriSelect</td><td>43-50020-03</td><td></td></tr><tr><td>pluriStrainer 10 &amp;micro;m</td><td>PluriSelect</td><td>43-50010-03</td><td></td></tr><tr><td>Covaris S2 Focused-ultrasonicator</td><td>Covaris</td><td>Model S2</td><td></td></tr><tr><td>Labquake&amp;nbsp;</td><td>Thermo Scientific</td><td>C415110</td><td></td></tr><tr><td>CCD Microscope Camera</td><td>&amp;nbsp;Leica Microsystems</td><td>DFC3000 G</td><td></td></tr><tr><td>&lt;strong&gt;Reagent Kit&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DNA extraction kit</td><td>Thermo Scientific</td><td>K0691</td><td></td></tr><tr><td>&lt;strong&gt;Buffers&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>All buffer components are discribed in the protocol. Each component was purchased from Sigma Aldrich</td><td></td><td></td><td></td></tr></tbody></table>

a filtration-based method of preparing high-quality nuclei from cross-linked skeletal muscle for chromatin immunoprecipitation

クロマチン免疫沈降（ChIP）は、クロマチンDNAへのタンパク質結合を決定する強力な方法です。しかしながら、繊維が豊富な骨格筋は、筋原繊維の汚染を最小限に抑えた高品質の核の単離における技術的困難のために、ChIPの課題であった。以前のプロトコールでは、架橋前に核を精製しようと試みたが、これは核形成の延長過程でDNA-タンパク質相互作用の変化のリスクを招く。現在のプロトコールでは、まずマウスから採取した骨格筋組織を架橋し、組織を細かく砕いて超音波処理した。我々は超遠心分離が架橋筋肉組織を用いて筋原線維から核を分離することができないことを発見したので、重要な筋線維汚染のない高品質の核を得るために逐次濾過手順を考案した。その後、超音波装置を用いてクロマチンを調製し、抗BMAL1抗体を用いたChIPアッセイにより、遺伝子プロモーターを標的とするBMAL1のパターン。この濾過プロトコールは、架橋骨格筋組織から高品質核を単離するための簡単に適用可能な方法を構成し、概日および他の時間に敏感な研究のための一貫した試料処理を可能にする。次世代シークエンシング（NGS）と組み合わせて、本発明者らの方法は、骨格筋機能に焦点を当てた様々な機構およびゲノム研究のために配置することができる。

ここでは、リアルタイムDNA-タンパク質相互作用を保存するために、組織回収直後にホルムアルデヒド架橋を行った。しかし、我々は、核分離23,24に一般的に使用されるスクロースまたはコロイド勾配は、筋原線維から核を分離するのに有効でないことを見出した（データ示さず）。その理由は、架橋が核および筋原繊維に同様の重力を与えたためで?...

Watch this Scientific Journal Video about A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation at JoVE.com

A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation

我々は、超遠心分離の必要性を排除した、架橋マウス骨格筋から高品質の核を単離するための濾過ベースのプロトコールを提示し、容易に適用することができる。本発明者らは、核から調製されたクロマチンが、クロマチン免疫沈降およびクロマチン免疫沈降配列決定研究に適していることを示す。

クロマチン免疫沈降のための架橋された骨格筋からの高品質核の調製のためのろ過に基づく方法

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a ...

a-filtration-based-method-preparing-high-quality-nuclei-from-cross

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

8.8K ビュー.  University of Texas Health Science Center at Houston. 我々は、超遠心分離の必要性を排除した、架橋マウス骨格筋から高品質の核を単離するための濾過ベースのプロトコールを提示し、容易に適用することができる。本発明者らは、核から調製されたクロマチンが、クロマチン免疫沈降およびクロマチン免疫沈降配列決定研究に適していることを示す。 

ビデオ: A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation

Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a high content of structural proteins. Dissected limb muscles from adult mice are physically disrupted by mechanical homogenisation, or a combination of mincing and douncing, in a hypotonic buffer before formaldehyde fixation of the cell lysate. The fixed nuclei are purified by further cycles of mechanical homogenisation or douncing and sequential filtrations to remove cell debris. The purified nuclei can be sonicated immediately or at a later stage after freezing. The chromatin can be efficiently sonicated and is suitable for chromatin immunoprecipitation experiments, as illustrated by the profiles obtained for transcription factors, RNA polymerase II, and covalent histone modifications. The binding events detected using chromatin prepared by this protocol are predominantly those taking place in the muscle fiber nuclei despite the presence of chromatin from other fiber-associated satellite and endothelial cells. This protocol is therefore adapted to study gene regulation in the adult mouse skeletal muscle.

A novel protocol for the preparation of chromatin from adult mouse skeletal muscle adapted to the study of gene regulation in muscle fibers by chromatin immunoprecipitation is presented.

クロマチン免疫沈降マウス骨格筋からの改善されたプロトコル

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a ...

生化学

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are 
reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and 
often challenging.
The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a 
bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, 
and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve 
fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from 
those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, 
histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). 
Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.

We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.

軸索損傷後の脊髄後根神経節の組織からのクロマチン免疫沈降

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

神経科学

Quantitative Analysis of Chromatin Proteomes in Disease

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,1 the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.2 Transcriptional regulation during development and disease have been well studied in this organ,3-5 but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.6 In the developed world, heart disease is the number one cause of mortality for both men and women.7 Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options.
Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,8-13 until recently14 there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.15 In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.16 Additionally, cardiomyocytes are 40% mitochondria by volume17 which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

病におけるクロマチンプロテオームの定量的解析

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due ...

医学

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

Chromatin immunoprecipitation (ChIP) is a widely-used method for determining the interactions of different proteins with DNA in chromatin of living cells. Examples include sequence-specific DNA binding transcription factors, histones and their different modification states, enzymes such as RNA polymerases and ancillary factors, and DNA repair components. Despite its ubiquity, there is a lack of up-to-date, detailed methodologies for both bench preparation of material and for accurate analysis allowing quantitative metrics of interaction. Due to this lack of information, and also because, like any immunoprecipitation, conditions must be re-optimized for new sets of experimental conditions, the ChIP assay is susceptible to inaccurate or poorly quantitative results.
Our protocol is ultimately derived from seminal work on transcription factor:DNA interactions1,2 , but incorporates a number of improvements to sensitivity and reproducibility for difficult-to-obtain cell types. The protocol has been used successfully3,4 , both using qPCR to quantify DNA enrichment, or using a semi-quantitative variant of the below protocol.
This quantitative analysis of PCR-amplified material is performed computationally, and represents a limiting factor in the assay. Important controls and other considerations include the use of an isotype-matched antibody, as well as evaluation of a control region of genomic DNA, such as an intergenic region predicted not to be bound by the protein under study (or anticipated not to show changes under the experimental conditions). In addition, a standard curve of input material for every ChIP sample is used to derive absolute levels of enrichment in the experimental material. Use of standard curves helps to take into account differences between primer sets, regardless of how carefully they are designed, and also efficiency differences throughout the range of template concentrations for a single primer set. Our protocol is different from others that are available5-8 in that we extensively cover the later, analysis phase.

We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.

バイオマスの制限額を使用して効率的なクロマチン免疫沈降

Chromatin immunoprecipitation (ChIP) is a  widely-used method for determining the interactions of different proteins with  DNA in chromatin of living ...

生物学

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

ChroPアプローチは、クロマチンの遺伝子座特異的プロテオームの風景を分析するために、チップと質量分析を組み合わせ

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

発生と人間INO80クロマチンリモデリング複合体とSubcomplexesの精製

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

タンデムアフィニティー精製を使用してのMyoDインタラクトームの同定は、質量分析に結合します

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...

The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision

Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA interactions. ChIP coupled to high throughput sequencing (ChIP-seq) is commonly used to determine the genomic location of proteins that interact with chromatin. However, ChIP-seq is hampered by relatively low mapping resolution of several hundred base pairs and high background signal. The ChIP-exo method is a refined version of ChIP-seq that substantially improves upon both resolution and noise. The key distinction of the ChIP-exo methodology is the incorporation of lambda exonuclease digestion in the library preparation workflow to effectively footprint the left and right 5&#39; DNA borders of the protein-DNA crosslink site. The ChIP-exo libraries are then subjected to high throughput sequencing. The resulting data can be leveraged to provide unique and ultra-high resolution insights into the functional organization of the genome. Here, we describe the ChIP-exo method that we have optimized and streamlined for mammalian systems and next-generation sequencing-by-synthesis platform.

Here, we present a protocol to achieve near base pair resolution of protein-DNA interactions. This is obtained by exonuclease treatment of DNA fragments selectively enriched by chromatin immunoprecipitation (ChIP-exo) followed by high throughput sequencing.

ChIP-エキソ方法：近く塩基対の精度でタンパク質 -  DNA相互作用を同定します

Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA ...

Chromatin Extraction from Frozen Chimeric Liver Tissue for Chromatin Immunoprecipitation Analysis

Crosslinking Chromatin Immunoprecipitation (X-ChIP) is a widely used technique to assess levels of histone marks and occupancy of transcription factors on host and/or pathogen chromatin. Chromatin preparation from tissues creates additional challenges that need to be overcome to obtain reproducible and reliable protocols comparable to those used for cell culture. Tissue disruption and fixation are critical steps to achieve efficient shearing of chromatin. Coexistence of different cell types and clusters may also require different shearing times to reach optimal fragment size and hinders shearing reproducibility. The purpose of this method is to achieve reliable and reproducible host chromatin preparations from frozen tissue (liver) suitable for both ChIP-qPCR and next generation sequencing (NGS) applications. We observed that the combination of liquid nitrogen tissue pulverization followed by homogenization leads to increased reproducibility compared to homogenization only, since it provides a suspension consisting mostly of dissociated single cells that can be efficiently sheared. Moreover, the fixation step should be performed under mild rotation to provide homogeneous crosslinking. The fixed material is then suitable for buffer-based nuclei isolation, to reduce contamination of cytoplasmic protein and pathogen DNAs and RNAs (when applicable), avoiding time-consuming centrifugation gradients. Subsequent sonication will complete nuclear lysis and shear the chromatin, producing a specific size range according to the chosen shearing conditions. The size range should fall between 100 and 300 nt for NGS applications, while it could be higher (300-700 nt) for ChIP-qPCR analysis. Such protocol adaptations can greatly improve chromatin analyses from frozen tissue specimens.

This protocol focuses on chromatin preparation from snap frozen tissues and it is suitable for Crosslinking Chromatin Immunoprecipitation (X-ChIP) followed by either quantitative PCR analysis (X-ChIP-qPCR) or next generation sequencing approaches (X-ChIP-seq).

クロマチン免疫沈降分析のための凍結キメラ肝臓組織からのクロマチン抽出

Crosslinking Chromatin Immunoprecipitation (X-ChIP) is a widely used technique to assess levels of histone marks and occupancy of transcription factors on ...

Immunoprecipitation of Target Protein-DNA Complexes from Motor Neuron Lysate

Source: Montanera, K. N. et al., Rhee, H. S. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/61124">High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo).</a> J. Vis. Exp. (2020)
This video demonstrates the immunoprecipitation of DNA fragments crosslinked with target protein from stem cell-derived motor neuron lysates using target protein-specific antibody-coated magnetic beads.

運動ニューロンライセートからの標的タンパク質-DNA複合体の免疫沈降

NOTE: Autoclaved distilled and deionized water (ddH2O) is recommended for making buffers and reaction master mixes.
1. Harvesting and crosslinking cells

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