자료유형  

 학위논문

Control Number  

0016932309

International Standard Book Number  

9798379612801

Dewey Decimal Classification Number  

574

Main Entry-Personal Name  

Lue, Nicholas Zuo-Bing.

Publication, Distribution, etc. (Imprint  

[S.l.] : Harvard University., 2023

Publication, Distribution, etc. (Imprint  

Ann Arbor : ProQuest Dissertations & Theses, 2023

Physical Description  

1 online resource(180 p.)

General Note  

Source: Dissertations Abstracts International, Volume: 84-12, Section: B.

General Note  

Advisor: Liau, Brian.

Dissertation Note  

Thesis (Ph.D.)--Harvard University, 2023.

Restrictions on Access Note  

This item must not be sold to any third party vendors.

Summary, Etc.  

요약Chromatin-based regulatory processes are indispensable for proper gene expression throughout development. Modifications of DNA and histones interact with a host of chromatin regulatory factors to carry out gene expression programs and ensure genomic stability. DNA methylation is an archetypal chromatin modification that is well understood to help mediate transcriptional silencing of protein-coding genes and repetitive DNA. Dynamics of DNA methylation are mediated by several proteins including DNMT3A, one of two enzymes in mammals responsible for installing DNA methylation de novo. DNMT3A is essential for development and over recent years has emerged as a critical regulator of hematopoiesis. DNMT3A is frequently mutated in hematologic cancers such as acute myeloid leukemia as well as in clonal hematopoiesis, a disorder that broadly affects aging populations. Understanding how mutations in DNMT3A affect its function is therefore an important goal that promises to shed light on disease mechanisms and inspire novel therapeutic approaches.The development and rapid adoption of CRISPR technologies over the past decade has revolutionized our ability to interrogate protein mutations at scale and directly within the native cellular context. Notably, functional genomics screens employing CRISPR-Cas9 mutagenesis have enabled both genome-wide knockout screens to probe for gene-level effect, as well as higher resolution screens tiling across individual genes. More recently, next-generation tools such as base editors have been developed that enable precise introduction of amino acid substitutions into endogenous proteins. Base editors have been rapidly adopted to conduct in situ mutational scanning, yet early screens have predominately utilized viability-based selection strategies, limiting their applicability to proteins like DNMT3A that are dispensable in cultured cells.Here, I discuss our application of base editor screening coupled with an innovative reporter strategy to enable high-throughput in situ evaluation of mutations in DNMT3A for discovery of novel biochemical insights. In Chapter 2, I discuss the development and implementation of a fluorescence-based reporter for endogenous DNMT3A activity. DNMT3A base editor scanning using this reporter reveals a wide variety of mutations across the DNMT3A coding sequence perturbing function. We biochemically characterize several mutations, which critically highlight the importance of an interdomain interface in DNMT3A allosteric regulation. Through rigorous sequencing-based validation, we also gain insights into key challenges facing base editor scanning, which I discuss in detail.In Chapter 3, I focus on our investigation of several mutations in the PWWP histone reader domain arising from our base editor screen. We show that PWWP domain mutations have variable effects on DNMT3A stability, explaining why a subset of our screening hits lead to DNMT3A loss-of-function. Nevertheless, we also identify mutations that appear to alter DNMT3A function without perturbing stability. Through deep biochemical investigation, we establish that these mutations affect the ability of DNMT3A's PWWP domain to bind DNA, and therefore affect the ability of DNMT3A to associate with chromatin in cells. We demonstrate that loss of PWWP DNA binding impairs DNMT3A catalytic activity in vitro and in cells, revealing a noncanonical role for this histone reader domain that suggests possible allosteric crosstalk with the catalytic domain.Finally, in Chapter 4 I discuss future directions for research into DNMT3A as well as for base editor scanning more broadly. Further biochemical investigation into potential PWWP-MTase allosteric crosstalk is warranted and may reveal novel mechanisms by which DNMT3A activity is regulated. Adapting the reporter strategy demonstrated here can also open the door to gaining new insights into DNTM3A activation as well as more broadly studying a variety of nonessential chromatin regulators. This includes the demethylase protein TET2, which is also highly mutated in hematologic disorders. Such studies have the potential to transform our understanding of DNMT3A and chromatin regulation more broadly, revealing novel ways to treat human disease.

Subject Added Entry-Topical Term  

Biochemistry.

Subject Added Entry-Topical Term  

Molecular biology.

Subject Added Entry-Topical Term  

Genetics.

Index Term-Uncontrolled  

Base editing

Index Term-Uncontrolled  

Biochemistry

Index Term-Uncontrolled  

CRISPR

Index Term-Uncontrolled  

DNA methylation

Index Term-Uncontrolled  

DNMT3A

Index Term-Uncontrolled  

Functional genomics

Added Entry-Corporate Name  

Harvard University Chemical Biology

Host Item Entry  

Dissertations Abstracts International. 84-12B.

Host Item Entry  

Dissertation Abstract International

Electronic Location and Access  

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Control Number  

joongbu:641823