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Biochemical and Genetic Approaches Identify Novel Factors Involved in Somatic Hypermutation Targeting
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| 자료유형 | 학위논문 |
|---|---|
| 서명/저자사항 | Biochemical and Genetic Approaches Identify Novel Factors Involved in Somatic Hypermutation Targeting. |
| 개인저자 | Dinesh, Ravi Kumar. |
| 단체저자명 | Yale University. Immunobiology. |
| 발행사항 | [S.l.]: Yale University., 2019. |
| 발행사항 | Ann Arbor: ProQuest Dissertations & Theses, 2019. |
| 형태사항 | 127 p. |
| 기본자료 저록 | Dissertations Abstracts International 81-04B. Dissertation Abstract International |
| ISBN | 9781088319666 |
| 학위논문주기 | Thesis (Ph.D.)--Yale University, 2019. |
| 일반주기 |
Source: Dissertations Abstracts International, Volume: 81-04, Section: B.
Advisor: Schatz, David G. |
| 이용제한사항 | This item must not be sold to any third party vendors.This item must not be added to any third party search indexes. |
| 요약 | Secondary diversification of the antibody repertoire in humans and mice occurs through the processes of somatic hypermutation (SHM) and class switch recombination (CSR), and is catalyzed by the enzymatic activity of activation induced cytidine deaminase (AID). The specific targeting of SHM to immunoglobulin loci in germinal center B cells is poorly understood. Previous work in our lab has shown that cis- regulatory sequences called DIVACs (for Diversification Activator) derived from immunoglobulin loci are responsible for targeting SHM to proximal transcribed genes. Leveraging recent advances in reporter assays for SHM in our lab, I devised two strategies to uncover potential trans-acting factors that target SHM.In the first strategy, I created a cell line where SHM is rapid, robust, and inducible, and used this cell line to perform a genome-wide CRISPR-Cas9 screen for factors involved in SHM. A number of negative regulators of NF-觀B signaling were revealed to be potentially necessary for proper SHM. Using genome editing in Ramos cells, I performed studies to validate two factors-I觀B慣 and TRAF2-and show that their ablation causes a reduction in SHM that is linked to a reduction in the expression of AID.In the second strategy, I identified potential trans-factors that bind to DIVACs using Immobilized Template Analysis (ITA) followed by mass spectrometry. Using ChIP-qPCR coupled with SHM reporter assays, I show that for two factors, E2A and MEF2B, recruitment to the IgH intronic enhancer DIVAC element correlates with SHM. My studies also uncovered a tight link between transcription and SHM in our reporter assays performed in Ramos cells-a finding warranting further investigation. |
| 일반주제명 | Immunology. Molecular biology. |
| 언어 | 영어 |
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