Investigation of MBD9’s Role in H2A.Z Deposition in Arabidopsis thaliana 公开

Farrugia, Stefan (Spring 2019)

Permanent URL: https://etd.library.emory.edu/concern/etds/pr76f4501?locale=zh
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Abstract

Gene expression regulation is vital to the proper functioning of an organism. This is accomplished in a variety of ways, one of which is with nucleosomes. Nucleosomes consist of DNA wrapped around proteins called histones, which can conceal or expose DNA to transcription factors. Besides the four canonical histones H2A, H2B, H3, and H4, there are histone variants like H2A.Z which seem to provide more specialized roles. The plant Arabidopsis thaliana is a great model system to study H2A.Z as it is viable without any of its three copies of the H2A.Z gene, it has easy methods to introduce new genetic material, and it has several visible phenotypes for H2A.Z mutants. The protein complex that is responsible for depositing H2A.Z in A. thaliana is called SWR1, and only recently has work been done to establish the identity of the SWR1’s subunits, one of which is MBD9. Not much is understood about MBD9, including its status as a stable subunit of SWR1 and its role in the deposition of H2A.Z. A proposed hypothesis is that MBD9 recognizes histone tail modifications and also interacts with SWR1, thereby guiding the complex to specific genomic regions for H2A.Z deposition. In order to test this, molecular tools for assays like histone peptide microarrays need to be developed. Here, a MBD9-MYC tag fusion protein was constructed and successfully translated. Additionally, a system to create MBD9 joined with a variety of tags was initiated for the purpose of protein interaction assays. Finally, phenotypic analysis of A. thaliana H2A.Z CRISPR mutant lines was accomplished. These novel H2A.Z mutants will be utilized to test protein interactions of MBD9-tag fusion proteins in vivo. Together, these results provide the groundwork for further experiments in understanding MBD9 and H2A.Z in A. thaliana.

Table of Contents

Table of Contents

Introduction                                                                                                    Pages 1-5

           Histones as regulators of gene expression                                                    1

           H2A.Z’s significance and deposition                                                             1-2

           Arabidopsis thaliana as a model organism for studying H2A.Z           2-3

           MBD9 is required for H2A.Z deposition                                                        3-5

Objectives                                                                                                        Page 5

Methods                                                                                                          Pages 6-10

           Plasmid and Primer Design                                                                              6

           SPRI Bead DNA Purification                                                                          7

           Gibson Assembly                                                                                             7

           Transformation                                                                                    7-8

           Inoculation                                                                                                      8

           Miniprep Plasmid Preparation                                                             8

           In Vitro Transcription and Translation                                                 8-9

           Gateway Cloning Recombination                                                                   9

           Assessment of A. thaliana Phenotypes                                                  9-10

Results and Discussion                                                                         Pages 10-13

           Expression of cMBD9-MYC Protein                                                               10-12

           Establishment of a MBD9-Tag Construct System                                       12

           Analysis of H2A.Z Mutant Phenotypes                                                 13

Conclusion                                                                                                      Pages 14

Figures                                                                                                            Pages 15-24

References                                                                                                      Pages 25-28

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