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In Vivo Surveillance of Transcription by RNA Polymerase II in Budding Yeast
In Vivo Surveillance of Transcription by RNA Polymerase II in Budding Yeast
상세정보
- 자료유형
- 학위논문 서양
- 최종처리일시
- 20250211153022
- ISBN
- 9798346858645
- DDC
- 574
- 저자명
- VanBelzen, Jake.
- 서명/저자
- In Vivo Surveillance of Transcription by RNA Polymerase II in Budding Yeast
- 발행사항
- [Sl] : Northwestern University, 2024
- 발행사항
- Ann Arbor : ProQuest Dissertations & Theses, 2024
- 형태사항
- 147 p
- 주기사항
- Source: Dissertations Abstracts International, Volume: 86-06, Section: B.
- 주기사항
- Includes supplementary digital materials.
- 주기사항
- Advisor: Brickner, Jason H.
- 학위논문주기
- Thesis (Ph.D.)--Northwestern University, 2024.
- 초록/해제
- 요약Chromatin immunoprecipitation and sequencing (ChIP-seq) is the most common approach to observe global binding of proteins to DNA in vivo. Recently, an alternative method, ChEC-seq, was shown to identify transcription factor motifs. ChEC-seq, in which an endogenous TF is fused to micrococcal nuclease, requires neither fixation nor antibodies. However, the specificity of DNA cleavage during ChEC-seq has been suggested to be lower than the specificity of the peaks identified by ChIP-seq or ChIP-exo, perhaps reflecting non-specific binding of transcription factors to DNA. I simplified the ChEC-seq protocol to minimize nuclease digestion while increasing the yield of cleaved DNA, which improved sensitivity, precision, and throughput. ChEC-seq2 readily and reliably identified transcription factor motifs and binding sites. Interestingly, when I applied ChEC-seq2 to RNA Polymerase II, I identified a pattern of enrichment that disagreed with ChIP-seq.ChIP-seq and ChEC-seq2 reveal strikingly different patterns of enrichment of yeast RNA polymerase II. I hypothesized that this reflects distinct populations of RNAPII, some of which are captured by ChIP-seq and some of which are captured by ChEC-seq2. RNAPII association with enhancers and promoters - predicted from biochemical studies - is detected well by ChEC-seq2 but not by ChIP-seq. Enhancer/promoter bound RNAPII correlates with transcription levels and matches predicted occupancy based on published rates of enhancer recruitment, preinitiation assembly, initiation, elongation and termination. The occupancy from ChEC-seq2 allowed us to develop a stochastic model for global kinetics of RNAPII transcription which captured both the ChEC-seq2 data and changes upon chemical-genetic perturbations to transcription. Finally, RNAPII ChEC-seq2 and kinetic modeling suggests that a mutation in the Gcn4 transcription factor that blocks interaction with the NPC destabilizes promoter-associated RNAPII without altering its recruitment to the enhancer.
- 일반주제명
- Molecular biology
- 일반주제명
- Genetics
- 일반주제명
- Cellular biology
- 키워드
- Mutation
- 기타저자
- Northwestern University Interdepartmental Biological Sciences (IBiS) Graduate Program
- 기본자료저록
- Dissertations Abstracts International. 86-06B.
- 전자적 위치 및 접속
- 로그인 후 원문을 볼 수 있습니다.
MARC
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■00520250211153022
■006m o d
■007cr#unu||||||||
■020 ▼a9798346858645
■035 ▼a(MiAaPQ)AAI31632240
■040 ▼aMiAaPQ▼cMiAaPQ
■0820 ▼a574
■1001 ▼aVanBelzen, Jake.
■24510▼aIn Vivo Surveillance of Transcription by RNA Polymerase II in Budding Yeast
■260 ▼a[Sl]▼bNorthwestern University▼c2024
■260 1▼aAnn Arbor▼bProQuest Dissertations & Theses▼c2024
■300 ▼a147 p
■500 ▼aSource: Dissertations Abstracts International, Volume: 86-06, Section: B.
■500 ▼aIncludes supplementary digital materials.
■500 ▼aAdvisor: Brickner, Jason H.
■5021 ▼aThesis (Ph.D.)--Northwestern University, 2024.
■520 ▼aChromatin immunoprecipitation and sequencing (ChIP-seq) is the most common approach to observe global binding of proteins to DNA in vivo. Recently, an alternative method, ChEC-seq, was shown to identify transcription factor motifs. ChEC-seq, in which an endogenous TF is fused to micrococcal nuclease, requires neither fixation nor antibodies. However, the specificity of DNA cleavage during ChEC-seq has been suggested to be lower than the specificity of the peaks identified by ChIP-seq or ChIP-exo, perhaps reflecting non-specific binding of transcription factors to DNA. I simplified the ChEC-seq protocol to minimize nuclease digestion while increasing the yield of cleaved DNA, which improved sensitivity, precision, and throughput. ChEC-seq2 readily and reliably identified transcription factor motifs and binding sites. Interestingly, when I applied ChEC-seq2 to RNA Polymerase II, I identified a pattern of enrichment that disagreed with ChIP-seq.ChIP-seq and ChEC-seq2 reveal strikingly different patterns of enrichment of yeast RNA polymerase II. I hypothesized that this reflects distinct populations of RNAPII, some of which are captured by ChIP-seq and some of which are captured by ChEC-seq2. RNAPII association with enhancers and promoters - predicted from biochemical studies - is detected well by ChEC-seq2 but not by ChIP-seq. Enhancer/promoter bound RNAPII correlates with transcription levels and matches predicted occupancy based on published rates of enhancer recruitment, preinitiation assembly, initiation, elongation and termination. The occupancy from ChEC-seq2 allowed us to develop a stochastic model for global kinetics of RNAPII transcription which captured both the ChEC-seq2 data and changes upon chemical-genetic perturbations to transcription. Finally, RNAPII ChEC-seq2 and kinetic modeling suggests that a mutation in the Gcn4 transcription factor that blocks interaction with the NPC destabilizes promoter-associated RNAPII without altering its recruitment to the enhancer.
■590 ▼aSchool code: 0163.
■650 4▼aMolecular biology
■650 4▼aGenetics
■650 4▼aCellular biology
■653 ▼aChromatin immunoprecipitation
■653 ▼aMicrococcal nuclease
■653 ▼aMutation
■653 ▼aChemical-genetic perturbations
■690 ▼a0307
■690 ▼a0369
■690 ▼a0379
■71020▼aNorthwestern University▼bInterdepartmental Biological Sciences (IBiS) Graduate Program.
■7730 ▼tDissertations Abstracts International▼g86-06B.
■790 ▼a0163
■791 ▼aPh.D.
■792 ▼a2024
■793 ▼aEnglish
■85640▼uhttp://www.riss.kr/pdu/ddodLink.do?id=T17164606▼nKERIS▼z이 자료의 원문은 한국교육학술정보원에서 제공합니다.
