우리 금융 감사 지원 건강의 국가 학회에서 부여 (R01GM112003 YZ), 웰 치 재단 (YZ에 될-1913 년), 미국 암 협회 (YZ에 RSG-16-215-01 TBE), 암 예방 및 연구 연구소의 텍사스 (YH에 RR140053 YZ RP170660), 미국 심장 협회 (YH에 16IRG27250155), 그리고 존 S. 던 재단 공동 연구 보너스 프로그램.

1. 세포 문화, 플라스 미드 Transfection 및 화학 유도
<ol>
	<li>문화는 부착 셀 라인 (예, 인간 미 발달 신장 HEK293T 셀) Dulbecco의 수정된이 글의 중간 (DMEM)에 10% 열 비활성화 태아 둔감 한 혈 청 100 U/mL 페니실린, 5% CO2와 37 ° C에서 스 보완.</li>
	<li>사과 쥬 스 플라스 미드와 셀 transfect
	<ol>
		<li>Transfection 전에 적어도 18 h, 2.5 ml 당 적절 한 DMEM의 씨앗 0.3 x 106 셀 6 잘 플레이트에 고 37 ° C 하룻밤 60-80 %confluency 도달에서 세포를 품 어.</li>
		<li>미리 사용 하기 전에 실내 온도에 transfection 시 따뜻한.</li>
		<li>무 균 튜브에 혈 청 자유로운 매체의 장소 250 µ L. 참고:이 금액은 80 %confluency 6 잘 플레이트에 대 한 맞춤형. 필요한 경우, 비례 또는 셀 숫자의 크기에 따라 중간 금액을 조정 합니다.</li>
		<li>사과 쥬 스18 또는 TET2의 촉매 도메인 인코딩 플라스 미드의 2.5 µ g 추가 (긍정적인 제어 TET2CD)12,13 혈 청 무료 매체와 부드럽게 pipetting으로 혼합.</li>
		<li>7.5 µ L transfection 시 약의 희석된 DNA 혼합물에 추가 하 고 부드럽게 pipetting으로 혼합.</li>
		<li>20-30 분 동안 실내 온도에 혼합물을 품 어.</li>
		<li>미리 데워 진된 미디어 변경 우물에 drop-wise 혼합물을 추가 하 고 부드럽게 흔들어 균일 하 게 배포 transfection 시 약 및 DNA 혼합물 셀 문화 접시.</li>
		<li>하룻밤 사이, 세포와 혼합 incubate 고 transfection 시 약 2 ml 신선한 완전 한 DMEM 미디어를 포함 하를 바꿉니다.</li>
	</ol>
	</li>
	<li>화학 유도
	<ol>
		<li>Rapamycin 재고 (DMSO에 용 해) 적절 한 완전 한 매체에서 20 µ M의 농도의 희석 2 mM.</li>
		<li>20 µ L 희석된 rapamycin의 200의 최종 농도 도달 2 mL 매체에 유지 하는 transfected 세포에 추가 nM. 참고: 48 h에 대 한 보육, 후 셀은 5hmC 세대의 정량화 또는 다른 다운스트림 응용 프로그램에 대 한 준비. 필요한 경우, 유도 효율은 셀 아래 설명 된 대로 추가 5hmC 부 량에 대 한 모든 3 h를 취 함으로써 모니터링 고 그림 1에 표시 된 수 있습니다.</li>
		<li>5hmC 유도 효율성의 더 나은 정량화에 대 한 시드 웰 스 모니터 셀 3 시간 마다 구분 합니다.</li>
	</ol>
	</li>
</ol>
2. Immunostaining에 의해 Rapamycin 유도 5hmC 생산의 정량화
<ol>
	<li>5hmC 면역 형광 염색 참고: 접시의 모든 세포를 충당 하기 위해 3 N HCl와 PBS에서 충분 한 양의 4 %paraformaldehyde, 0.2% 계면 활성 제 (Triton X-100) 등으로 구성 된 고정 솔루션을 추가 합니다. 예를 들어 솔루션의 500 µ L 6-잘 접시에 잘에 대 한 충분 한 것입니다. 실 온에서 모든 단계를 수행 합니다.<ol>
		<li>셀을 수정, 추가 4% paraformaldehyde PBS에 rapamycin 치료 셀 주위 온도에 15 분 동안에. 컨트롤 그룹에 대 한 mCherry-사과주 rapamycin 치료 없이 표현 하는 셀을 사용 합니다. 교 반 하십시오 하지 않습니다. 주의: Paraformaldehyde 독성이 있다. 적절 한 보호를 착용 하십시오.</li>
		<li>통 솔루션을 삭제 합니다. 세포 막 permeabilize를 30 분 PBS에 0.2% 계면 활성 제와 고정된 셀을 품 어.</li>
		<li>Permeabilization 솔루션을 삭제 합니다. Permeabilized 셀에 3 N HCl를 추가 하 고 DNA를 변성을 15 분 동안 품 어.</li>
		<li>HCl 추가 100 mM Tris HCl 버퍼 (pH 8.0) pH의 중성화에 대 일 분에 대 한 셀을 삭제 합니다.</li>
		<li>모든 솔루션을 삭제 합니다. 셀에 대 한 PBS와 철저 하 게 씻어 3-5 시간. PBS를 철저 하 게 제거 합니다. 참고: 모든 세척 단계에 대 한 PBS의 충분 한 양을 추가 합니다. 예를 들어 1 mL의 PBS 6 잘 플레이트에 대 한 충분 하다.</li>
		<li>1 %BSA 및 0.05%의 계면 활성 제 (예: 트윈 20) PBS에 부드러운 동요와 1 시간에 대 한 블로킹 버퍼를 추가 하 여 셀을 차단 합니다.</li>
		<li>삭제 차단 솔루션. 셀에 희석된 5hmC 항 체 (1: 200)을 추가 하 고 실 온에서 2 시간 또는 하룻밤 4 ° c.에 품 어</li>
		<li>3 시간 15 분 대 한 PBS 가진 세포를 씻어.</li>
		<li>추가 희석된 fluorophore (FITC)-셀에 이차 항 체 (1: 200)을 활용 하 고 1 시간에 품 어.</li>
		<li>워시 PBS 가진 셀 세 번 광범위 하 게 잔여 항 체를 제거 하.</li>
		<li>4'의 250 ng/mL을 추가, 6-diamidino-2-phenylindole (DAPI) 5 분에 대 한 핵 얼룩 제거 얼룩 솔루션.</li>
		<li>Immunostained 셀 confocal 영상에 대 한 슬라이드 또는 유리 하단 문화 접시를 탑재 합니다. 대표 결과 그림 1B에 보였다.</li>
	</ol>
	</li>
	<li>Cytometry 참고: 다음 얼룩 절차에 대 한 96-잘 라운드 바닥판을 사용 합니다. 일반적으로, 150 µ L의 시 약은 각 잘 충분 합니다.<ol>
		<li>FACS 버퍼 (1 %BSA, 2 mM EDTA와 PBS)에 rapamycin 유도 transfected 세포 resuspend Rapamycin 치료 없이 사과 표현 셀을 사용 하 여 컨트롤 그룹에 대 한 있습니다.</li>
		<li>수정 하 고 단계 2.1에 설명 된 대로 세포를 permeabilize.</li>
		<li>10 분 동안 DNA를 변성 하 셀 2 N HCl를 추가 합니다.</li>
		<li>HCl 중화를 취소 하 고 단계 2.1에 설명 된 대로 세포를 차단 합니다.</li>
		<li>셀에 희석된 안티-5hmC 항 체 (1: 200)을 추가 하 고 1 시간 실내 온도에 또는 하룻밤 4 ° c.에 대 한 품 어</li>
		<li>FACS 버퍼 셀 세 번 씻어. FACS 버퍼를 철저 하 게 제거 합니다.</li>
		<li>추가 희석된 fluorophore (FITC)-형광 활성화 셀 정렬 (FACS) 분석, 이차 항 체 (1:400)를 활용 하 고 실 온에서 1 h에 품 어.</li>
		<li>FACS 버퍼 셀 세 번 씻어.</li>
		<li>샘플은 FACS 분석에 대 한 준비가 되었습니다. 대표 결과 그림 1 c-D에 표시 했다.</li>
	</ol>
	</li>
</ol>
3. 점 오 점 분석 결과 5hmC 및 5-methylcytosine (5mC) 척도를 도달
<ol>
	<li>상업적인 DNA 추출 키트를 사용 하 여 transfected 및 rapamycin 유도 세포에서 게놈 DNA를 격리 합니다. Rapamycin 치료 없이 사과 쥬 스 페 셀을 사용 하 여 컨트롤 그룹에 대 한 있습니다.</li>
	<li>80 ° C와 사전 담가 니트로 막 이중 증 류 H2O 그리고 염 분 나트륨 시트르산 (SSC) 버퍼 10 분 x 6에서에 진공 오븐이 열.</li>
	<li>96-잘 PCR 접시에 같은 양의 DNA (TE 버퍼에 총 µ 2 g)의 60 µ L을 로드 합니다. 나머지 우물에 테 버퍼 (10 mM Tris HCl, 1 mM EDTA, pH 8.0)의 30 µ L를 추가 합니다.</li>
	<li>확인 두 배 직렬 희석 세로 96 잘 접시에 2 행에 동일한 열 위치를 30 µ L 솔루션의 행 1에 전송 하 여 합니다. 다시 혼합 하 고 경계를 도달까지 후속 행에서 웰 스 솔루션의 30 µ L를 전송.</li></ol>마지막으로 30 µ L을 제거 합니다.
	<li>각 음을 1m NaOH와 10 mM EDTA의 20 µ L을 추가 하 고 접시를 봉인 완전히 DNA 이중 변성에 95 ° C에서 10 분을 위한 혼합물을가 열.</li>
	<li>즉시 얼음에 샘플 진정 차가운 2 M 염화 아세테이트 (pH 7.0)의 50 µ L를 추가 하 고 10 분 동안 얼음에 품 어. 참고: 단계 3.7-3.10 진공의 사용을 포함.</li>
	<li>미리 젖된 막으로 점 오 점 장치를 조립 하 고 잔여 버퍼 풀 진공을 사용 하 여 제거 합니다.</li>
	<li>점 오 점 기구의 각 잘 하 테 버퍼의 200 µ L을 추가 하 여 막 씻으십시오. TE 버퍼를 제거 하는 진공을 켭니다.</li>
	<li>점 오 점 기구의 각 우물에 변성된 DNA (이전 단계 3.1-3.6에서에서 준비)을 로드 합니다. 솔루션을 제거 하는 진공을 켭니다.</li>
	<li>SSC 및 제거 잔여 솔루션 완전히 진공을 사용 하 여 x 2의 200 µ L을 추가 하 여 막 씻으십시오.</li>
	<li>점 오 점 장치를 분해, 막 밖으로가 고 2의 20 mL와 함께 전체 막 씻어 x SSC.</li>
	<li>20 분 동안 막 건조</li>
	<li>2 시간에 대 한 진공에서 80 ° C에서 막 구워.</li>
	<li>막에 차단 솔루션 (TBST에 5% 탈지 우유)를 추가 하 고 실 온에서 1 h에 품 어.</li>
	<li>삭제 차단 솔루션. 희석된 5hmC (1:5, 000) 또는 (1:1, 000) 5mC 추가 막에 항 체 및 4 ° c.에서 밤새 품 어</li>
	<li>잔여 항 체를 제거 하려면 3 시간 10 분 동안 TBST 가진 막 씻으십시오. TBST를 철저 하 게 제거 합니다.</li>
	<li>HRP 활용 된 이차 항 체를 추가 (1:10, 000 안티 토끼 HRP(5hmC) 또는 반대로 마우스 HRP(5mC)) 막에 대 한 1:3,000 및 실 온에서 1 h에 품 어.</li>
	<li>3 시간 10 분 동안 TBST 가진 막 씻으십시오.</li>
	<li>ECL 기판 화학 검출기를 사용 하 여 5hmC 신호의 시각화에 대 한 막에 더럽혀 서 부를 추가 합니다. 대표 결과 그림 1E에 표시 했다.</li>

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H., 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=High+cleavage+efficiency+of+a+2A+peptide+derived+from+porcine+teschovirus-1+in+human+cell+lines,+zebrafish+and+mice.">High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.</a> PLoS One. 6 (4), e18556 (2011).</li><li>Lee, 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=Engineered+Split-TET2+Enzyme+for+Inducible+Epigenetic+Remodeling.">Engineered Split-TET2 Enzyme for Inducible Epigenetic Remodeling.</a> J Am Chem Soc. 139 (13), 4659-4662 (2017).</li><li>Huang, Y., Pastor, W. A., Zepeda-Mart&#237;nez, J. A., Rao, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anti-CMS+technique+for+genome-wide+mapping+of+5-hydroxymethylcytosine.">The anti-CMS technique for genome-wide mapping of 5-hydroxymethylcytosine.</a> Nat Protoc. 7 (10), 1897-1908 (2012).</li><li>Buenrostro, J. D., Wu, B., Chang, H. Y., Greenleaf, 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=ATAC-seq:+A+Method+for+Assaying+Chromatin+Accessibility+Genome-Wide.">ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide.</a> Curr Protoc Mol Biol. 109 (21.29), 1-9 (2015).</li></ol>

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여기 우리 DNA hydroxymethylation의 일시적인 제어를 달성 하기 위해 설계 된 분할-TET2 효소를 사용 하 여를 일러스트 있다. TET 가족 5-methycytosine dioxygenase의의 발견, 많은 연구 해독 TET 단백질과 그들의 주요 촉매 제품 5hmC,12,3,의 생물 학적 기능 수행 되었습니다. 4,5,<sup clas...

DNA 메 틸 화, 주로 5-methylcytosine (5mC) 형성 시 토 신의 탄소 5 위치에 메 틸 그룹의 추가를 나타냅니다, 그리고 DNA 메 틸-transferases (DNMTs)에 의해 촉매. 5mC transcriptional 억제, x 염색체 비활성화와 transposon 입을1에 대 한 신호를 종종 포유류 게놈에 있는 주요 epigenetic 마크 역할을 합니다. DNA 메 틸 화의 반전 10-엘 프 전 (TET) 단백질 가족에 의해 중재 됩니다. TET 효소 철 (II)에 속하는 5mC 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5 fC) 및 5-carboxycytosine (5caC)의 연속 산화 촉매는 종속 dioxygenases 2 oxoglutarate. TET 중재 5mC 산화 epigenetic 제어할 포유류 게놈의 추가 계층을 부과 한다. TET의 발견 TET 단백질의 생물 학적 기능 및 그들의 주요 촉매 제품 5hmC 공개 후 필드에 강렬한 관심을 촉발 했다. 5hmC 하지만 중간 TET 중재 활성 DNA demethylation2,,34일 동안 이지만 또한 epigenetic 안정 역할 표시5,6,7,8 . DNA hydroxymethylation에서 변경 일부 인간의 장애9,,1011, 인과 관계와 연결 된 DNA hydroxymethylation은 높은 유전자 발현과 관련 된 탈 선 후 성적인 수정 DNA에는 고기 사이 종종 남아 설립 도전, 시간적 및 공간적 정의 신뢰할 수 있는 도구를 정확 하 게 추가 또는에서 게놈에 있는 DNA 수정 제거의 부족에 부분적으로 관찰 될 수 있습니다. 해상도입니다.
여기 우리는 화학을 유도할 수 있는 epigenome 도구 (사과주) 인과 관계의 연구를 직면 하는 장애물을 극복 하기 위해 DNA hydroxymethylation와 유전자 전사 사이 개장의 사용을 보고 합니다. 가정에는 디자인을 기반으로 하는 TET2의 촉매 도메인 (TET2CD) 2 개의 비활성 파편 포유류 세포에서 표현 하는 때로 분할할 수 있습니다 및 화학적으로 유도할 수 있는 이합체 화 접근법 ( 여 효소 기능을 복원할 수 있습니다 그림 1A). 분할-TET2CD 시스템을 설정 하기 위해 우리는 TET2CD, Cys 풍부한 지역 및 더블-좌초 β 나선 (DSBH) 배, TET2 DNA 복잡 한 낮은 복잡성 지역12부족의 보고 된 결정 구조 기초의 구성에서 6 사이트를 선택. 합성 유전자 인코딩 rapamycin 유도할 수 있는 이합체 화 모듈 FK506 바인딩 단백질 12 (FKBP12)와 FKBP rapamycin 바인딩 도메인 (FRB) rapamycin14,15, 자체 cleaving 펩타이드 T2A 함께 포유류 대상의 polypeptide 시퀀스16,17, TET2CD 내의 선택한 분할 사이트에 개별적으로 삽입 되었다. 우리의 엔지니어링 대상으로 TET2의 선택 다음과 같은 고려 사항을 기반으로 합니다. 수 반하는 감소 된 DNA hydroxymethylation TET2에서 첫 번째, 체세포 돌연변이 골수성 질환 등 암10, 대 피할 수 민감한 사이트에 유용한 정보를 제공 하는 인간의 장애에서 자주 관찰 된다 삽입입니다. 둘째, TET2 촉매 도메인, 특히 낮은 복잡성 지역의 큰 분수는 효소 기능12, 유도할 수 있는 후 성적인 수정에 대 한 최소화 된 분할 TET2 공예를 활성화를 위해 불가결 이다. 15 구조, 심사 후 포유류 시스템에서 그것의 효소 활동의 가장 높은 rapamycin 유도할 수 있는 복원 전시 하는 구문 선택 되었고 사과 쥬 스18로 지정. 우리는 여기 유도할 수 있는 DNA hydroxymethylation 및 rapamycin와 리 모델링 후 사과 mCherry 태그를 사용 하 여 설명 하 고 사과 중재 5hmC 생산 모델 셀룰러 시스템 HEK293T에서에서 유효성 검사에 세 가지 방법 제시.

<table><tbody><tr><td>Lipofectamine 2000 Transfection Reagent</td><td>Thermo Fisher Scientific</td><td>11668027</td><td></td></tr><tr><td>Rapamycin</td><td>Sigma-Aldrich</td><td>37094</td><td></td></tr><tr><td>Paraformaldehyde, 16% Solution</td><td>VWR</td><td>100503-916</td><td></td></tr><tr><td>Triton X-100</td><td>Amresco</td><td>0694-1L</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Amresco</td><td>0369-500ML</td><td></td></tr><tr><td>Albumin, Bovine</td><td>Amresco</td><td>0332-100G</td><td></td></tr><tr><td>Tween 20</td><td>Amresco</td><td>0777-1L</td><td></td></tr><tr><td>5-Hydroxymethylcytosine (5-hmC) antibody (pAb)</td><td>Active Motif</td><td>39769</td><td></td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td><td>Thermo Fisher Scientific</td><td>A-11008</td><td></td></tr><tr><td>4,6-Diamidino-2-phenylindolde (DAPI)</td><td>Biotium</td><td>40043</td><td></td></tr><tr><td>SVAC1E SHEL LAB Economy Vacuum Oven, 0.6 Cu.Ft. (17 L)</td><td>SHEL LAB</td><td>SVAC1E</td><td></td></tr><tr><td>UltraPure SSC, 20X</td><td>Thermo Fisher Scientific</td><td>15557036</td><td></td></tr><tr><td>Bio-Dot Apparatus</td><td>BIO-RAD</td><td>1706545</td><td></td></tr><tr><td>Anti-5-methylcytosine (5mC) Antibody, clone 33D3</td><td>Millipore</td><td>MABE146</td><td></td></tr><tr><td>Anti-mouse IgG, HRP-linked Antibody</td><td>Cell Signaling Technology</td><td>7076S</td><td></td></tr><tr><td>Anti-rabbit IgG, HRP-linked Antibody</td><td>Cell Signaling Technology</td><td>7074S</td><td></td></tr><tr><td>West-Q Pico Dura ECL Solution</td><td>Gendepot</td><td>W3653-100</td><td></td></tr></tbody></table>

an engineered split-tet2 enzyme for chemical-inducible dna hydroxymethylation and epigenetic remodeling

DNA 메 틸 화 포유류 게놈에 안정적이 고 상속 후 성적인 수정 이며, 세포 기능 제어 유전자 발현 조절에 관여. DNA 메 틸 화, 또는 DNA demethylation의 반전은 dioxygenases의 10-11 전 (TET) 단백질 가족에 의해 중재 됩니다. 그것은 널리 알려졌다 탈 DNA 메 틸 화와 demethylation 발달 결함과 암와 관련 된, 있지만 어떻게 이러한 후 성적인 변경을 직접 기여할 진 식 또는 질병 진행에 후속 변경 신뢰할 수 있는 도구를 정확 하 게 추가 또는 정의 된 시간적, 공간적 해상도에서 게놈에 있는 DNA 수정 제거의 부족 때문에 크게 불분명 남아 있습니다. 이 장애물을 극복 하기 위해 우리는 분할 TET2 효소를 5-methylcytosine (5mC) 산화의 일시적인 제어 및 후속 개장 하는 포유류 세포에 있는 후 성적인 국가의 단순히 화학 물질을 추가 하 여 사용할 수 있도록 설계 되었습니다. 여기, 우리 화학을 유도할 수 있는 epigenome 개장 도구 (사이 다), 포유류 세포와 함께 5-hydroxymethylcytosine (5hmC)의 화학 유도할 수 있는 생산 측정 조작된 분할-TET2 효소에 따라 도입 방법 설명 immunostaining, cytometry 또는 점 오 점 분석 결과. 이 화학을 유도할 수 있는 epigenome 개장 도구 유전자 코드를 변경 하지 않고 셀룰러 시스템 질문 뿐만 아니라 다양 한 생물 학적 시스템에서 epigenotype−phenotype 관계를 프로 빙에 광범위 한 사용을 찾을 것입니다.

유도할 수 있는 화학 5mC-5hmC 변환 단일 셀 수준, 교류 cytometry 분석, transfected (mCherry 양성) 세포 인구에 또는 더 양적 점 오 점 분석 결과 그림 1에서 볼 수 있듯이 immunostaining에 의해 유효성 수 있습니다. TET2, 또는 TET2CD의 촉매 도메인 긍정적인 통제로 연구에 걸쳐 사용 되었다. 5hmC 및 chromatin 접근성에 rapamycin 유도 변경의 게놈 넓은 매핑에 대 한 자세한 ...

Watch this Scientific Journal Video about An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling at JoVE.com

An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling

화학을 유도할 수 있는 DNA Hydroxymethylation 및 후 리 모델링 설계 분할-TET2 효소

자세한 프로토콜 설계 분할-TET2 효소 (사이 다) 화학 유도할 수 있는 DNA hydroxymethylation 및 후 리 모델링 포유류 세포에 도입 되 게 됩니다.

DNA methylation is a stable and heritable epigenetic modification in the mammalian genome and is involved in regulating gene expression to control ...

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Research

JoVE Journal

Chemistry

6.5K 조회수.  Texas A&M University. 자세한 프로토콜 설계 분할-TET2 효소 (사이 다) 화학 유도할 수 있는 DNA hydroxymethylation 및 후 리 모델링 포유류 세포에 도입 되 게 됩니다.

비디오: An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling

High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research. Mammalian DNA is enzymatically modified at the 5th carbon position of cytosine (C) residues to 5-mC, predominately in the context of CpG dinucleotides. 5-mC is amenable to enzymatic oxidation to 5-hmC by the Tet family of enzymes, which are believed to be involved in development and disease. Currently, the biological role of 5-hmC is not fully understood, but is generating a lot of interest due to its potential as a biomarker. This is due to several groundbreaking studies identifying 5-hydroxymethylcytosine in mouse embryonic stem (ES) and neuronal cells. Research techniques, including bisulfite sequencing methods, are unable to easily distinguish between 5-mC and 5-hmC . A few protocols exist that can measure global amounts of 5-hydroxymethylcytosine in the genome, including liquid chromatography coupled with mass spectrometry analysis or thin layer chromatography of single nucleosides digested from genomic DNA. Antibodies that target 5-hydroxymethylcytosine also exist, which can be used for dot blot analysis, immunofluorescence, or precipitation of hydroxymethylated DNA, but these antibodies do not have single base resolution.In addition, resolution depends on the size of the immunoprecipitated DNA and for microarray experiments, depends on probe design. Since it is unknown exactly where 5-hydroxymethylcytosine exists in the genome or its role in epigenetic regulation, new techniques are required that can identify locus specific hydroxymethylation. The EpiMark 5-hmC and 5-mC Analysis Kit provides a solution for distinguishing between these two modifications at specific loci. The EpiMark 5-hmC and 5-mC Analysis Kit is a simple and robust method for the identification and quantitation of 5-methylcytosine and 5-hydroxymethylcytosine within a specific DNA locus. This enzymatic approach utilizes the differential methylation sensitivity of the isoschizomers MspI and HpaII in a simple 3-step protocol. Genomic DNA of interest is treated with T4-BGT, adding a glucose moeity to 5-hydroxymethylcytosine. This reaction is sequence-independent, therefore all 5-hmC will be glucosylated; unmodified or 5-mC containing DNA will not be affected. This glucosylation is then followed by restriction endonuclease digestion. MspI and HpaII recognize the same sequence (CCGG) but are sensitive to different methylation states. HpaII cleaves only a completely unmodified site: any modification (5-mC, 5-hmC or 5-ghmC) at either cytosine blocks cleavage. MspI recognizes and cleaves 5-mC and 5-hmC, but not 5-ghmC. The third part of the protocol is interrogation of the locus by PCR. As little as 20 ng of input DNA can be used. Amplification of the experimental (glucosylated and digested) and control (mock glucosylated and digested) target DNA with primers flanking a CCGG site of interest (100-200 bp) is performed. If the CpG site contains 5-hydroxymethylcytosine, a band is detected after glucosylation and digestion, but not in the non-glucosylated control reaction. Real time PCR will give an approximation of how much hydroxymethylcytosine is in this particular site. In this experiment, we will analyze the 5-hydroxymethylcytosine amount in a mouse Babl/C brain sample by end point PCR.

The EpiMark 5-hmC and 5-mC Analysis Kit can be used to analyze and quantitate 5-methylcytosine and 5-hydroxymethylcytosine within a spe cific locus. The kit distinguishes 5-mC from 5-hmC by the addition of glucose to the hydroxyl group of 5-hmC via an enzymatic reaction utilizing &beta;-glucosyltransferase (T4-BGT). When 5-hmC occurs In the context of CCGG, this modification converts a cleavable MspI site to a non-cleavable site.

Balb / C 뇌 조직에 높은 민감도는 5 hydroxymethylcytosine 감지

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research.   Mammalian DNA is enzymatically ...

연구

신경과학

Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these epigenetic marks is accomplished by sequential oxidation by ten-eleven translocation dioxygenases (TET1-3), followed by the thymine-DNA glycosylase-dependent base excision repair. Inactivation of the TET2 gene due to genetic mutations or by other epigenetic mechanisms is associated with a poor prognosis in patients with diverse cancers, especially hematopoietic malignancies. Here, we describe an efficient single step purification of enzymatically active untagged human TET2 dioxygenase using cation exchange chromatography. We further provide a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach that can separate and quantify the four normal DNA bases (A, T, G, and C), as well as the four modified cytosine bases (5-methyl, 5-hydroxymethyl, 5-formyl, and 5-carboxyl). This assay can be used to evaluate the activity of wild type and mutant TET2 dioxygenases.

Here, we present a protocol for an efficient single step purification of the active untagged human ten-eleven translocation-2 (TET2) 5-methylcytosine dioxygenase using ion-exchange chromatography and its assay using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach.

효율적인 정화와 10-11 전-2 5-Methylcytosine Dioxygenase에 대 한 LC-MS/MS 기반 분석 결과 개발

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these ...

생화학

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

인간으로 Epigenome 편집 도구의 효율적인 배달 lentiviral 벡터 플랫폼 유도 만능 줄기 세포 유래 질병 모델

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

유전학

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

Restriction endonuclease (REase) specificity engineering is extremely difficult. Here we describe a multistep protocol that helps to produce REase variants that have more stringent specificity than the parental enzyme. The protocol requires the creation of a library of expression selection cassettes (ESCs) for variants of the REase, ideally with variability in positions likely to affect DNA binding. The ESC is flanked on one side by a sequence for the restriction site activity desired and a biotin tag and on the other side by a restriction site for the undesired activity and a primer annealing site. The ESCs are transcribed and translated in a water-in-oil emulsion, in conditions that make the presence of more than one DNA molecule per droplet unlikely. Therefore, the DNA in each cassette molecule is subjected only to the activity of the translated, encoded enzyme. REase variants of the desired specificity remove the biotin tag but not the primer annealing site. After breaking the emulsion, the DNA molecules are subjected to a biotin pulldown, and only those in the supernatant are retained. This step assures that only ESCs for variants that have not lost the desired activity are retained. These DNA molecules are then subjected to a first PCR reaction. Cleavage in the undesired sequence cuts off the primer binding site for one of the primers. Therefore, PCR amplifies only ESCs from droplets without the undesired activity. A second PCR reaction is then carried out to reintroduce the restriction site for the desired specificity and the biotin tag, so that the selection step can be reiterated. Selected open reading frames can be overexpressed in bacterial cells that also express the cognate methyltransferase of the parental REase, because the newly evolved REase targets only a subset of the methyltransferase target sites.

Restriction endonucleases with new sequence specificity can be developed from enzymes recognizing a partially degenerate sequence. Here we provide a detailed protocol that we successfully used to alter the sequence specificity of NlaIV enzyme. Key ingredients of the protocol are the in vitro compartmentalization of the transcription/translation reaction and selection of variants with new sequence specificities.

더 엄격한 특이성을 가진 제한 endonuclease의 시험관 진화

Restriction endonuclease (REase) specificity engineering is extremely difficult. Here we describe a multistep protocol that helps to produce REase ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

세포질 기계 화학 Epigenetic 한정자를 리디렉션하여 억압 유전자 전사

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

생체공학

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

의 DNA Methylation : Bisulphite의 변경 및 분석

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

생물학

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work demonstrates that concepts from DNA nanotechnology can be used to program the enzymatic activity of the phage-derived T7 RNA polymerase (RNAP) and build scalable synthetic gene regulatory networks. First, an oligonucleotide-tethered T7 RNAP is engineered via expression of an N-terminally SNAP-tagged RNAP and subsequent chemical coupling of the SNAP-tag with a benzylguanine (BG)-modified oligonucleotide. Next, nucleic-acid strand displacement is used to program polymerase transcription on-demand. In addition, auxiliary nucleic acid assemblies can be used as &#34;artificial transcription factors&#34; to regulate the interactions between the DNA-programmed T7 RNAP with its DNA templates. This in vitro transcription regulatory mechanism can implement a variety of circuit behaviors such as digital logic, feedback, cascading, and multiplexing. The composability of this gene regulatory architecture facilitates design abstraction, standardization, and scaling. These features will enable the rapid prototyping of in vitro&#160;genetic devices for applications such as bio-sensing, disease detection, and data storage.

We describe the engineering of a novel DNA-tethered T7 RNA polymerase to regulate in vitro transcription reactions. We discuss the steps for protein synthesis and characterization, validate proof-of-concept transcriptional regulation, and discuss its applications in molecular computing, diagnostics, and molecular information processing.

시험관 내 전사 및 분자 계산을 위한 DNA 테더드 RNA 폴리머라제

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

게놈 DNA에서 도보로 5 hydroxymethylcytosine의 선택적 캡처

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks.
ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs&#39; position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome.
Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final&#160;evaluation in the therapeutically relevant setting of interest.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

In Vitro (시험관 ) 표적 후성유전학적 침묵을 위한 엔지니어링된 전사 억제제 선택

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

Methyltransferases과 공동 인자 유사체와 핵산과 단백질의 순서 특정 레이블

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...