Name: biochemical assays for analyzing activities of atp-dependent chromatin remodeling enzymes
Uploaded: 2014-10-25T14:00:00.000+00:00
Duration: 10 min 14 s
Description: Watch this Scientific Journal Video about Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes at JoVE.com

Work in the authors' laboratory is supported by a grant from the National Institute of General Medical Sciences (GM41628) and by a grant to the Stowers Institute for Medical Research from the Helen Nelson Medical Research Fund at the Greater Kansas City Community Foundation.

1 ATP에 의존하는 뉴 클레오 리모델링 분석 실험 뉴 클레오 리모델링 활동 ATP-의존성을 측정하기 위해, INO80를 immunopurified 또는 INO80의 서브 콤플렉스는 ATP와 216 bp의, 32 P-표지 된 DNA 단편의 일 단부에 위치하는 단일 뉴 클레오를 포함 mononucleosomal 기판과 함께 배양된다. 반응 생성물은 다음 네이티브 폴리 아크릴 아마이드 겔에서 전기 영동한다. <ol><li> 32 P-표지 된 &#39;, 601&#39;DNA 단편을 생성하기에서 증폭을 pGEM-3Z-601 4 올리고 뉴클레오티드로서 5&#39;-ACAGGATGTATATATCTGACACGTGCCTGG 및 5&#39;-AATACTCAAGCTTGGATGCCTGCAG을 사용하여 최종 위치 (601) 뉴 클레오 솜 위치 서열을 포함하는 216 염기쌍의 DNA 단편, 순방향 및 역방향 프라이머. <ol><li> 표 1에 기재된 바와 같이 100 ㎕의 PCR 반응을 설정한다. </li><li> 다음 프로그램을 사용하여 서열 증폭기에서 PCR 반응을 수행하여 1 분을 96 ° C, followe에서94 ° C, 57 ° C, 30주기 동안 72 ° C에서 60 초에서 30 초에서 45 초만큼 D; 72 ° C에서 7 분으로 끝나는. </li><li> PCR 반응이 완료되면, 클레아없는 스핀 컬럼을 통해 회 반응 생성물을 통과하여 통합되지 않은 뉴클레오타이드를 제거한다. </li><li> PCR 반응은 원하는 ~ 216 염기쌍의 DNA 단편을 생성 된 것을 확인하는 1.2 % 아가 로즈 겔에서 정제 된 PCR 산물의 5 μL를 실행. </li><li> 정제 된 PCR 생성물 20 배량의 5 μL 희석하고 UV 분광 광도계를 이용하여 DNA 농도를 측정한다. 평균 수율은 ~ 40 NG / μL이다. </li><li> 섬광 계수기에서 정제 된 PCR 생성물의 1 μL의 방사능을 측정한다. 노출 당 비용 (CPM) / NG를 계산하여 라벨 효율을 추정한다. 성공적인 라벨링 반응물을 601 DNA 단편 ~ 15,000 CPM / NG을 수득 할 것으로 예상된다. </li></ol></li><li> 용액을 희석 방법 5를 사용하여 표시 601 DNA에 헬라 세포 뉴 클레오을 전송합니다. <ol><li> 2 pmol의 혼합32 P-표지 된 601 DNA 단편 헬라 뉴 클레오 6 μg 1.0 M의 NaCl, 10 mM 트리스 - 염산, pH가 8.0, 1 mM의 EDTA, 0.1 mM의 PMSF 및 1 ㎜를 포함하는 완충액 50 μL에 (같이 제조 5 참조) DTT와 30 분 동안 30 ° C에서 배양한다. </li><li> 순차적 0.8 M, 0.6 M로 혼합물을 희석하고, 10 mM Tris-HCl (pH8.0)을, 1 mM의 EDTA, 0.1 mM의 PMSF의 각각 12.5 μL, 20.8 μL, 41.6 μL, 희석 0.4 M의 NaCl, 및 1 mM의 DTT, 각 희석 한 후 30 ° C에서 30 분 배양과 함께. </li><li> 또한 30 분 동안 배양하여 다음 0.1 % Nonidet P-40, 20 % 글리세롤, 및 200 μg / ㎖ BSA를 함유하는 동일한 완충액 250 μL을 125 μL 첨가함으로써, 0.1 M의 NaCl로 한 다음 0.2 M 및로 혼합물을 희석 마지막이 희석 사이에 30 ° C에서. 최대 3 개월 동안 4 ° C에서 mononucleosome 기판을 저장합니다. </li></ol></li><li> 10 μL의 총 부피에 ATP에 의존하는 뉴 클레오 슬라이딩 반응을 수행합니다. 정말이에요INO80 뉴 클레오 리모델링 활성을위한 최적의 NaCl 농도이기 때문에 ction의 구성 요소. 표 2에 나와있는 것은 ~ 50 mM의 염화나트륨 (미발표 결과), 고려에 함유 된 염화나트륨의 양을 가지고, 50 mm의 각 반응에서의 NaCl의 총 농도를 조정 뉴 클레오 효소의 준비. <ol><li> 분석을 설정하기 시작하기 전에 기본 폴리 아크릴 아마이드 젤 (18 X 16cm)를 캐스팅. 5 %의 아크릴 아미드를 함유 한 겔을 제조 (아크릴 아미드 : 37.5 비스 아크릴 아미드 : 1), 0.5의 TBE (45 mM 트리스 보레이트, 1 mM의 EDTA), 0.01 %과 황산 암모늄 (APS), 및 0.001 % N, N, N&#39;, N&#39;의 -tetramethylethylenediamine (TEMED는). 표 3에 나와있는 재료를 혼합 겔을 실온에서 적어도 2 시간 동안 중합하도록 허용합니다. </li><li> 각각의 반응을 수행하는 한편, 프리 실리콘이 냉장 된 1.5 ml의 마이크로 원심 튜브 ~ 20 nM의 INO80 또는 INO80의 subcomplex에 결합 (EB100 버퍼의 양 (같이 제조 3 참조) <str옹&gt; 충분한 표 4) 4.75 μL의 부피를 수득 하였다. 가루 즉시 드라이 아이스 또는 액체 질소에 남아 INO80 - 함유 분획을 다시 동결. </li><li> &#39;X&#39;의 요소에 의해 확장, 나머지 재료와 함께 마스터 칵테일을 설정합니다 (여기서 반응의 X = 총 세). 단일 10 μL 반응에 필요한 각 성분의 양은 표 5에 열거한다; 레시피가 수행되는 반응의 수에 따라 확장 할 것이다. Pipetman와 튜브를 눌러 또는 피펫 팅 의해 아래로 잘 섞어 벤치 탑의 microcentrifuge에 몇 초 동안 튜브를 회전. </li><li> 단계 1.3.2에서 설정 반응 관 각각에 마스터 칵테일 5.25 μl를 분배. 로 pipetting 아래로 잘 섞는다. 30 ° C 열 블록 또는 물을 욕조에 반응 튜브를 전송하여 반응을 시작하고 2 시간 동안 배양한다. </li><li> 한편, 경쟁 DNA를 포함하는 칵테일 &#39;믹스를 제거하는&#39;준비와 뉴 클레오는 X (반응 + 4 X = 총)의 요인에 의해 확장. 단일 반응 혼합물을 1.5 ㎕의 제거를 준비하기 위해 필요한 각 성분의 양을 표 6에 열거한다; 레시피가 수행되는 분석의 수에 따라 확장 할 것이다. </li><li> 제거 믹스의 1.5 μl를 첨가하여 반응을 종료합니다. 잘 믹스 스핀 다운, 그리고 추가로 30 분 동안 30 ° C에서 배양. </li><li> 한편, 일정한 버퍼 순환을 유지하기 위해 하부 챔버 내부의 자기 교반 막대 버퍼를 실행으로 0.5 배의 TBE를 사용하여, 4 ° C에서 30 분 동안 100 V에서 수직 전기 영동 장치의 기본 폴리 아크릴 아마이드 겔을 미리 실행합니다. </li><li> 샘플을로드 배 TBE, 30 % 글리세롤, 0.25 % 브로 모 페놀 블루, 0.25 % 크실렌 Cyanol FF를 포함 로딩 염료의 2.5 μl를 추가합니다. 간단히 샘플을 회전, 잘 혼합, 및로드 정보를 사용하여 겔에로드합니다. </li><li> 버퍼 순환 4 ° C에서 4.5 시간 동안 200 V에서 젤을 실행합니다.</li><li> 신호를 검출하기 위해, 여과지의 두 시트의 스택 겔 옮긴다. 투명한 플라스틱 포장을 사용하여 상단에있는 겔 필터 종이 랩, 다음 원하는 시간 동안 4 ° C에서 저장 형광체 화면에 노출. </li><li> 동위 원소 촬상 스캐너 시스템의 화면을 검색하고 적합한 소프트웨어를 사용하여 데이터를 분석한다. </li></ol></li></ol> 2 Mononucleosome 결합 분석 mononucleosomes위한 소정 INO80 복합체의 결합 친화도 분석법을 위해, 단계 120에서 생성 mononucleosomal 기판을 이용한 전기 영동 이동성 시프트 분석법 (EMSA)을 수행. <ol><li> 반응은 뉴 클레오 리모델링 분석에 대해 설명하지만 반응에서 ATP 및 제거 믹스를 생략으로 분​​석을 바인딩 믹스 설정; 30 분 동안 30 ° C에서 배양한다. </li><li> 각 반응 혼합물에 로딩 염료의 2.5 μl를 추가하고, 3.5 %의 아크릴 아마이드 (아크릴 아미드가 포함 된 기본 폴리 아크릴 아마이드 겔에 적용 : 세 비스7.5 : 1), 1 % 글리세롤, 0.5 배의 TBE, 0.01 %의 APS 및 0.001 % TEMED. </li><li> 버퍼를 실행으로 0.5 배의 TBE를 사용하여 버퍼 순환 4 ° C에서 2.5 시간 동안 200 V에서 젤을 실행하고 저장 형광체 화면에 노출. </li></ol> 3 DNA- 및 뉴 클레오 의존 ATPase의 분석 실험 20 mM 트리스 - 염산 (pH를 7.5), 60 mM의 염화나트륨, 6.6 mM의 MgCl2를, 0.8 mM의 EDTA, 0.015 % Nonidet P-40, 2.5 % 글리세롤, 0.1 ㎎ / ㎖ BSA, 1을 포함하는 5 ㎕의 반응 혼합물에 ATPase를 세이를 수행 mM의 DTT, 0.1 mM의 PMSF, 2 mM의 ATP [α- 32 P] ATP의 2 μCi (3000 CI / mmol)을 첨가 하였다. INO80 단지의 각 INO80의 복잡하거나 시간 동안 원형 플라스미드 DNA 측정 (5,000 bp의, ~ 30 nm의) 폐쇄 함유 DNA- 또는 뉴 클레오 독립적 ATPase의 하나를 측정하는 세 병렬 반응, 일 포함 EB100 버퍼를 설정 정량 할 DNA- 의존적 ATPase를, 및 뉴 클레오 의존적 ATPase를 측정하는 한 헬러 oligonucleosomes (~ 185 나노 미터)을 포함. 얼음에 모두 반응을 설정합니다. <oL&gt; <li> 각 반응의 경우, 사전 냉장 윤활 1.5 ML의 microcentrifuge 튜브에 2.2 μL의 볼륨을 제공하기에 충분 EB100 버퍼의 양 immunopurified INO80 또는 INO80 서브 콤플렉스의 10 ~ 50 nm의 결합. 가루 즉시 드라이 아이스 또는 액체 질소에있는 INO80 - 함유 분획을 다시 동결. </li><li> 마스터 칵테일을 설정합니다. 단일 반응에 필요한 각 성분의 양을 표 7에 표시됩니다. X는 = INO80 준비의 수를 정량 할 3 (X + 2) 하나의 요인에 의해 확장하여 조리법을 확장 할 수 있습니다. </li><li> &#39;서브 칵테일&#39;포함 버퍼를 준비하려면 만, DNA, 또는 뉴 클레오는 3 개의 별도의 튜브에 마스터 칵테일의 2.5 (X + 2) μl를 분배. 0.3 어느 EB100의 (X + 2) μL 폐쇄 원형 플라스미드 DNA (1.5 μg / μL), 또는 헬라 oligonucleosomes (1.5 μg / μL)을 넣고 잘 섞는다. </li><li> 성에서 설정 한 효소 함유 반응 튜브에 적절한 서브 칵테일의 2.8 μl를 분배EP 3.1. 부드럽게 섞어 아래로 피펫; 거품을 도입하지 마십시오. </li><li> , 반응을 시작하는 30 ° C 열 블록에 반응 튜브를 전송합니다. </li><li> 적어도 1.5 cm 떨어진 하단 가장자리로부터 직선 5, 15, 30, 및 인큐베이션 60 분 후, 셀룰로스 폴리에틸렌 박층 크로마토에 각각 반응 혼합물의 0.5 ㎕를 스팟 (TLC) 플레이트 (20 × 10cm) . 바로 그래서 여러 시점이 하나의 튜브에서 취할 수 있습니다 30 ° C 열 블록에 반응 튜브를 반환합니다. 스포팅 후, 타격 건조기를 사용하여 TLC 판을 건조. </li><li> TLC 플레이트의 하단은 0.5 cm 용액에 침지 될 수 있도록 충분히 0.375 M 인산 칼륨 (pH를 3.5)를 함유하는 유리 챔버 TLC 플레이트를 옮긴다. </li><li> 챔버를 포함하고, 액상의 전면 TLC 플레이트의 상단에 도달 할 때까지 개발한다. 즉시 철저하게 타격 건조기를 사용하여 판을 건조. </li><li> RT에서 저장 형광면에 건조 TLC 판을 노출.동위 원소 촬상 스캐너 시스템의 화면을 스캔 방사성 ATP와 ADP 기판 생성물의 양을 결정한다. </li><li> 가수 분해 ATP의 양을 계산하기 위하여, 다음과 같은 수식을 사용하여 개시, 반응 혼합물 중의 ATP 존재하는 양에 의해 가수 분해 %의 ATP를 곱 pmol의의 ATP 가수 분해 = 10 pmol의의 ATP를 반응 X 개시까지 [ADP / (ATP + ADP)] </li></ol>

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D., Thastrom, A., Widom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+access+of+proteins+to+buried+nucleosomal+DNA+target+sites+occurs+via+a+mechanism+that+is+distinct+from+nucleosome+translocation,+Mol.Cell+Biol.">Spontaneous access of proteins to buried nucleosomal DNA target sites occurs via a mechanism that is distinct from nucleosome translocation, Mol.Cell Biol.</a> 22 (20), 7147-7157 (2002).</li><li>Saha, A., Wittmeyer, J., Cairns, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+remodeling+through+directional+DNA+translocation+from+an+internal+nucleosomal+site.">Chromatin remodeling through directional DNA translocation from an internal nucleosomal site.</a> Nature Structural and Molecular Biology. 12 (9), 747-755 (2005).</li><li>Gottschalk, A. 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=Poly(ADP-ribosyl)ation+directs+recruitment+and+activation+of+an+ATP-dependent+chromatin+remodeler,+Proc.Natl.Acad.Sci.U.S.A.">Poly(ADP-ribosyl)ation directs recruitment and activation of an ATP-dependent chromatin remodeler, Proc.Natl.Acad.Sci.U.S.A.</a> 106 (33), 13770-13774 (2009).</li><li>Clapier, C. R., Cairns, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+ISWI+involves+inhibitory+modules+antagonized+by+nucleosomal+epitopes.">Regulation of ISWI involves inhibitory modules antagonized by nucleosomal epitopes.</a> Nature. 492 (7428), 280-284 (2012).</li><li>Brune, M., Hunter, J. L., Corrie, J. E. T., Direct Webb, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-Time+Measurement+of+Rapid+Inorganic+Phosphate+Release+Using+a+Novel+Fluorescent+Probe+and+Its+Application+to+Actomyosin+Subfragment+1+ATPase,+Biochemistry.">Real-Time Measurement of Rapid Inorganic Phosphate Release Using a Novel Fluorescent Probe and Its Application to Actomyosin Subfragment 1 ATPase, Biochemistry.</a> 33 (27), 8262-8271 (1994).</li><li>Luk, E., 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=Stepwise+histone+replacement+by+SWR1+requires+dual+activation+with+histone+H2A.Z+and+canonical+nucleosome.">Stepwise histone replacement by SWR1 requires dual activation with histone H2A.Z and canonical nucleosome.</a> Cell. 143 (5), 725-736 (2010).</li></ol>

The authors declare that they have no competing financial interests.

, 그리고 리모델링 및 / 또는 ATPase의 효소 오염에 우리가 분석에서 관찰하는 뉴 클레오 리모델링 및 ATPase의 활동 INO80 복합체의 촉매 활성에 따라 보장하기 위해, 우리는 정기적으로 분석 뉴 클레오 리모델링 및 INO80 단지의 촉매 비활성 버전의 ATPase의 활동은 정제 야생형 INO80 동일한 절차를 사용하여 평행. INO80 착체 또는 서브 콤플렉스의 다른 제제의 액티비티를 비교하는 실험의 해석을 복잡하게...

SNF2 가족 염색질 리모델링 단지는 중앙 SNF2 같은 ATPase의 서브 유닛 1, 2를 포함한다. 단일 서브 유니트 효소와 같은 일부 SNF2 형상 ATPases 기능, 다른 큰 멀티 소단위 복합체 촉매 서브 유닛으로서 기능하는 동안. 리모델링 복합체가 그들의 활동에 기여 염색질의 서브 유니트 각각은 리모델링 과정을 해부 생화학 분석을 수행 할 수있는 능력을 필요로하는 분자 메커니즘을 해명. 인간 INO80 착물 및 기타 염색질 개조 효소에 의해 ATP 의존적 뉴 클레오 리모델링은 그 DNA- 및 / 또는 뉴 클레오 의존적 ATPase를 활성화 한 후, 뉴 클레오로 리모델링 효소의 결합을 시작하는 다단계 과정으로 구상 될 수있다 뉴 클레오 솜 DNA, 그리고 뉴 클레오 1,2의 최종 위치 조정에 리모델링 효소의 전위. ATP 의존적 크로 마틴 리모델링 프로세스 (R)의 분자 세부 사항을 이해반응의 각 단계에 크로 마틴 리모델링 복합체의 개별 서브 유닛의 기여의 리모델링의 각 단계에 대한 반응 및 정의의 절개를 equires. 이런 분석은 in vitro에서 정의 된 분자의 기판을 사용하여 뉴 클레오 리모델링 및 기타 활동을 분석 할 수있는 능력을 필요로한다. 이전 조브 프로토콜에서, 우리는 정의 서브 유닛 조성 3 INO80 크로 마틴 리모델링 복합체 및 서브 콤플렉스를 생성하는 데 사용되는 절차를 설명했다. 여기서 우리는, DNA- 및 뉴 클레오-ATPase의 활성화, 이러한 복합체와 관련된 뉴 클레오 리모델링 활동을 결합 뉴 클레오의 정량 분석​​을 가능하게 세 가지 생화학 적 분석을 제시한다.

<table><tbody><tr><td>Protease Inhibitor Cocktail</td><td>Sigma</td><td>P8340</td><td></td></tr><tr><td>10x PCR reaction buffer&amp;nbsp;</td><td>Roche Applied Science&amp;nbsp;</td><td>11435094001</td><td></td></tr><tr><td>Roche Taq DNA Polymerase</td><td>Roche Applied Science&amp;nbsp;</td><td>11435094001</td><td></td></tr><tr><td>NucAway Nuclease-free Spin Columns&amp;nbsp;</td><td>Ambion</td><td>Cat. # AM10070</td><td></td></tr><tr><td>ultrapure ATP&amp;nbsp;</td><td>USB/Affymetrix</td><td>77241 25 UM</td><td></td></tr><tr><td>bovine serum albumin&amp;nbsp;</td><td>Sigma</td><td>A9418&amp;nbsp;</td><td></td></tr><tr><td>40% Acrylamide/Bis 37.5:1</td><td>Amresco</td><td>0254-500ML</td><td></td></tr><tr><td>Sonicated salmon sperm DNAs&amp;nbsp;</td><td>GE Healthcare</td><td>27-4565-01</td><td></td></tr><tr><td>10% ammonium persulfate (APS)</td><td>Thermo Scientific</td><td>17874</td><td></td></tr><tr><td>benzonase&amp;nbsp;</td><td>Novagen</td><td>Cat. No. 70664</td><td></td></tr><tr><td>dCTP, [&amp;alpha;-&lt;sup&gt;32&lt;/sup&gt;P]- 6,000 Ci/mmol</td><td>PerkinElmer</td><td>BLU013Z250UC</td><td></td></tr><tr><td>PCR thermal cycler PTC 200</td><td>MJ Research</td><td>PTC 200</td><td></td></tr><tr><td>Hoefer vertical electrophoresis unit</td><td>Hoefer</td><td>SE600X-15-1.5</td><td></td></tr><tr><td>lubricated 1.5 ml microcentrifuge tubes&amp;nbsp;</td><td>Costar</td><td>3207</td><td></td></tr><tr><td>Storage Phosphor Screen&amp;nbsp;</td><td>Molecular Dynamics</td><td>63-0034-79</td><td></td></tr><tr><td>3MM filter paper</td><td>Whatman&amp;nbsp;</td><td>28458-005</td><td>VWR</td></tr><tr><td>Typhoon PhosphorImager&amp;nbsp;</td><td>GE Healthcare</td><td>8600</td><td></td></tr><tr><td>ImageQuant software</td><td>GE Healthcare</td><td>ver2003.02</td><td></td></tr><tr><td>TLC Glass Plates, PEI-Cellulose F</td><td>Millipore</td><td>5725-7</td><td></td></tr><tr><td>Immobilon-FL Transfer Membrane 7 x 8.4</td><td>Millipore</td><td>IPFL07810</td><td></td></tr><tr><td>General purpose survey meter with end-window or pancake GM (Geiger-Mueller) probe</td><td>Biodex</td><td>Model 14C</td><td></td></tr></tbody></table>

biochemical assays for analyzing activities of atp-dependent chromatin remodeling enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.

수치는 뉴 클레오 슬라이딩 (그림 1) 및 바인딩 (그림 2) 분석 및 DNA- 또는 뉴 클레오 의존 ATPase의 분석 (그림 3)을 포함 INO80 활동을 특성화하는 데 사용되는 생화학 적 분석의 대표적인 결과를 보여줍니다. 그림 1의 실험은 그대로 INO80 단지의 능력 FLAG-Ies2 또는 FLAG-INO80E을 통해 정제 INO80은 216 BP, 방사성 동위 원소 표지 된 DNA 단...

Watch this Scientific Journal Video about Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes at JoVE.com

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

ATP에 의존 염색질 리모델링 효소의 활동을 분석하는 생화학 분석 실험

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and ...

biochemical-assays-for-analyzing-activities-atp-dependent-chromatin

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JoVE Journal

Biology

14.7K 조회수.  Stowers Institute for Medical Research. Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

비디오: Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous nature of most mammalian chromatin-modifying complexes. In order to overcome these hurdles, we have adapted a sequential salt extraction (SSE) assay for evaluating the relative binding affinities of chromatin-bound complexes. This easy and straightforward assay can be used by non-experts to evaluate the relative difference in binding affinity of two related complexes, the changes in affinity of a complex when a subunit is lost or an individual domain is inactivated, and the change in binding affinity after alterations to the chromatin landscape. By sequentially re-suspending bulk chromatin in increasing amounts of salt, we are able to profile the elution of a particular protein from chromatin. Using these profiles, we are able to determine how alterations in a chromatin-modifying complex or alterations to the chromatin environment affect binding interactions. Coupling SSE with other in vitro and in vivo assays, we can determine the roles of individual domains and proteins on the functionality of a complex in a variety of chromatin environments.

Sequential salt extraction of chromatin bound proteins is a useful tool for determining the binding properties of large protein complexes. This method can be employed to evaluate the role of individual subunits or domains in the overall affinity of a protein complex to bulk chromatin.

바인딩 속성 수정 복합물 Chromatin의 대량 Chromatin의 분석에 대 한 순차적 소금 기사

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous ...

연구

유전학

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.

바인딩, 전사, 번역, 및 회전율 세포 활성화 중 글로벌 이벤트를 표시 하는 녹음 방송 요인의 실시간 분석

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

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.&#160; Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

체외 유비퀴틴화 및 뉴클레오소말 히스톤의 이중화 율법의 비퀴틴화 어설션

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

생화학

Assays for Validating Histone Acetyltransferase Inhibitors

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene expression. KATs, such as CBP/p300, are under intense investigation as therapeutic targets due to their critical role in tumorigenesis of diverse cancers. The development of novel small molecule inhibitors targeting the histone acetyltransferase (HAT) function of KATs is challenging and requires robust assays that can validate the specificity and potency of potential inhibitors.
This article outlines a pipeline of three methods that provide rigorous in vitro validation for novel HAT inhibitors (HATi). These methods include a test tube HAT assay, Chromatin Hyperacetylation Inhibition (ChHAI) assay, and Chromatin Immunoprecipitation-quantitative PCR (ChIP-qPCR). In the HAT assay, recombinant HATs are incubated with histones in a test tube reaction, allowing for acetylation of specific lysine residues on the histone tails. This reaction can be blocked by a HATi and the relative levels of site-specific histone acetylation can be measured via immunoblotting. Inhibitors identified in the HAT assay need to be confirmed in the cellular environment.
The ChHAI assay uses immunoblotting to screen for novel HATi that attenuate the robust hyperacetylation of histones induced by a histone deacetylase inhibitor (HDACi). The addition of an HDACi is helpful because basal levels of histone acetylation can be difficult to detect via immunoblotting.
The HAT and ChHAI assays measure global changes in histone acetylation, but do not provide information regarding acetylation at specific genomic regions. Therefore, ChIP-qPCR is used to investigate the effects of HATi on histone acetylation levels at gene regulatory elements. This is accomplished through selective immunoprecipitation of histone-DNA complexes and analysis of the purified DNA through qPCR. Together, these three assays allow for the careful validation of the specificity, potency, and mechanism of action of novel HATi.

Inhibitors of histone acetyltransferases (HATs, also known as lysine acetyltransferases), such as CBP/p300, are potential therapeutics for treating cancer. However, rigorous methods for validating these inhibitors are needed. Three in vitro methods for validation include HAT assays with recombinant acetyltransferases, immunoblotting for histone acetylation in cell culture, and ChIP-qPCR.

히스톤 아세틸트랜스퍼효소 억제제 검증을 위한 아세서

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene ...

암 연구학

CD Spectroscopy to Study DNA-Protein Interactions

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent advancements in CD spectroscopy have enabled the study of DNA-protein interactions and conformational dynamics of DNA in different microenvironments in detail for a better understanding of transcriptional regulation in vivo. The area around a potential transcription zone needs to be unwound for transcription to occur. This is a complex process requiring the coordination of histone modifications, binding of the transcription factor to DNA, and other chromatin remodeling activities. Using CD spectroscopy, it is possible to study conformational changes in the promoter region caused by regulatory proteins, such as ATP-dependent chromatin remodelers, to promote transcription. The conformational changes occurring in the protein can also be monitored. In addition, queries regarding the affinity of the protein towards its target DNA and sequence specificity can be addressed by incorporating mutations in the target DNA. In short, the unique understanding of this sensitive and inexpensive method can predict changes in chromatin dynamics, thereby improving the understanding of transcriptional regulation.

The interaction of an ATP-dependent chromatin remodeler with a DNA ligand is described using CD spectroscopy. The induced conformational changes on a gene promoter analyzed by the peaks generated can be used to understand the mechanism of transcriptional regulation.

DNA 단백질 상호 작용을 연구하는 CD 분광법

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent ...

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting of two copies of the core histones H2A, H2B, H3, and H4, wrapped around by the DNA. The octamer is composed of two copies of an H2A/H2B dimer and a single copy of an H3/H4 tetramer. The highly charged core histones are prone to non-specific interactions with several proteins in the cellular cytoplasm and the nucleus. Histone chaperones form a diverse class of proteins that shuttle histones from the cytoplasm into the nucleus and aid their deposition onto the DNA, thus assisting the nucleosome assembly process. Some histone chaperones are specific for either H2A/H2B or H3/H4, and some function as chaperones for both. This protocol describes how in vitro laboratory techniques such as pull-down assays, analytical size-exclusion chromatography, analytical ultra-centrifugation, and histone chaperoning assay could be used in tandem to confirm whether a given protein is functional as a histone chaperone.

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

This protocol describes a battery of methods that includes analytical size-exclusion chromatography to study histone chaperone oligomerization and stability, pull-down assay to unravel histone chaperone-histone interactions, AUC to analyze the stoichiometry of the protein complexes, and histone chaperoning assay to functionally characterize a putative histone chaperone in vitro.

체외 분석, 풀다운 및 샤프롱 분석을 사용한 히스톤 샤페론의 특성 분석

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting ...

Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in response to environmental stimuli. These changes are essential for genomic integrity, epigenetic regulation, and DNA metabolic reactions such as replication, transcription, and repair. Chromatin assembly is crucial for chromatin dynamics and is catalyzed by histone chaperones. Despite extensive studies, the mechanisms by which histone chaperones enable chromatin assembly remains elusive. Moreover, the global features of nucleosomes organized by histone chaperones are poorly understood. To address these problems, this work describes a unique single-molecule imaging technique named DNA curtain, which facilitates the investigation of the molecular details of nucleosome assembly by histone chaperones. DNA curtain is a hybrid technique that combines lipid fluidity, microfluidics, and total internal reflection fluorescence microscopy (TIRFM) to provide a universal platform for real-time imaging of diverse protein-DNA interactions.Using DNA curtain, the histone chaperone function of Abo1, the Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase, is investigated, and the molecular mechanism underlying histone assembly of Abo1 is revealed. DNA curtain provides a unique approach for studying chromatin dynamics.

DNA curtain, a high-throughput single-molecule imaging technique, provides a platform for real-time visualization of diverse protein-DNA interactions. The present protocol utilizes the DNA curtain technique to investigate the biological role and molecular mechanism of Abo1, a Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase.

DNA 커튼 기법에 의한 히스톤 조립의 분자 메커니즘 해독

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

인간의 세포에서 활성 유전자 발현의 어세 동적 히스톤 수정을 염색질 Immunoprecipitation (칩)

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

생물학

HAT1 효소 활성을 스크리닝하기 위한 클릭 기반 분석 개발

An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome assembly. It is required for tumor growth in various systems, making it a potential target for cancer treatment. To facilitate the identification of compounds that can inhibit HAT1 enzymatic activity, we have devised an acetyl-click assay for rapid screening. In this simple assay, we employ recombinant HAT1/Rbap46, which is purified from activated human cells. The method utilizes the acetyl-CoA analog 4-pentynoyl-CoA (4P) in a click-chemistry approach. This involves the enzymatic transfer of an alkyne handle through a HAT1-dependent acylation reaction to a biotinylated H4 N-terminal peptide. The captured peptide is then immobilized on neutravidin plates, followed by click-chemistry functionalization with biotin-azide. Subsequently, streptavidin-peroxidase recruitment is employed to oxidize amplex red, resulting in a quantitative fluorescent output. By introducing chemical inhibitors during the acylation reaction, we can quantify enzymatic inhibition based on a reduction of the fluorescence signal. Importantly, this reaction is scalable, allowing for high throughput screening of potential inhibitors for HAT1 enzymatic activity.

저자 스포트라이트: HAT1 Inhibitor Screening을 위한 Acetyl-Click Assay 개발

Quick and accurate chemical assays to screen for specific inhibitors are an important tool in the drug development arsenal. Here, we present a scalable acetyl-click chemistry assay to measure the inhibition of HAT1 acetylation activity.

히스톤 아세틸전이효소 1 아세틸화를 측정하기 위한 아세틸 클릭 화학 분석

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome ...

ATP에 의존 염색질 리모델링 효소의 활동을 분석하는 생화학 분석 실험

요약

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이 비디오의 챕터

바인딩 속성 수정 복합물 Chromatin의 대량 Chromatin의 분석에 대 한 순차적 소금 기사

바인딩, 전사, 번역, 및 회전율 세포 활성화 중 글로벌 이벤트를 표시 하는 녹음 방송 요인의 실시간 분석

체외 유비퀴틴화 및 뉴클레오소말 히스톤의 이중화 율법의 비퀴틴화 어설션

히스톤 아세틸트랜스퍼효소 억제제 검증을 위한 아세서

DNA 단백질 상호 작용을 연구하는 CD 분광법

체외 분석, 풀다운 및 샤프롱 분석을 사용한 히스톤 샤페론의 특성 분석

DNA 커튼 기법에 의한 히스톤 조립의 분자 메커니즘 해독

인간의 세포에서 활성 유전자 발현의 어세 동적 히스톤 수정을 염색질 Immunoprecipitation (칩)

히스톤 아세틸전이효소 1 아세틸화를 측정하기 위한 아세틸 클릭 화학 분석

히스톤 아세틸전이효소 1 아세틸화를 측정하기 위한 아세틸 클릭 화학 분석