Author ORCiD
Date of Award
2026
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Hironori Funabiki
Keywords
DNA methylation, H2A.Z, ZHX, cryo-EM, xenopus egg extract
Abstract
DNA methylation represents a widespread and heritable modification that exerts substantial influence on chromatin organization and function in many eukaryotic genomes. The presence of DNA methylation has been strongly linked to transcriptional regulation, in both activating as well as repressive ways, though exactly how a small chemical mark can enact such drastic and seemingly opposing effects on gene expression remains an open question. In my thesis work, I describe the influence DNA methylation has on two classes of chromatin proteins, the histone variant H2A.Z and the Zinc Finger and Homeoboxes (ZHX) family of transcription factors. DNA methylation and H2A.Z preferentially occupy exclusive areas of the genome, biasing against their colocalization in the same genomic space. However, exactly how this mutual exclusion occurs has largely been left a mystery. We find that DNA methylation impacts H2A.Z nucleosome dynamics via both physical destabilization effects and influences on H2A.Z-specific nucleosome remodeler activity. Using single particle cryo-EM, we solved structures of human H2A.Z nucleosomes on methylated and unmethylated DNA, suggesting that the presence of methylated DNA can destabilize histone-DNA contacts and increase linker DNA opening/flexibility. This conclusion was corroborated by that observation that DNA methylation on H2A.Z nucleosomes increases its accessibility to a restriction enzyme, while the influence of DNA methylation on H2A nucleosomes is minimal. However, this change occurred to a much lesser degree than that induced generally by H2A.Z nucleosomes compared to canonical H2A nucleosomes. Through sequencing studies and the use of crude physiological cytoplasmic extract from Xenopus laevis eggs coupled with sperm nuclei, we confirm that the H2A.Z and DNA methylation antagonism is conserved in Xenopus laevis. We further find that the degree of this H2A.Z and DNA methylation antagonism increases, and that the amount of overlap between them decreases, in nuclei from a Xenopus fibroblast cell line compared to egg extract. Using the egg extract system, we show that DNA methylation inhibits the binding of the SRCAP complex, the primary nucleosome remodeler responsible for H2A.Z deposition, and that the loss of SRCAP abolishes H2A.Z’s preference for unmethylated DNA. From these results, we conclude that the SRCAP complex is the primary mediator driving the deposition of H2A.Z on unmethylated regions of the genome. During the course of characterizing proteins whose chromatin binding is affected by DNA methylation and H2A.Z, we identified members of the ZHX transcription factor family, ZHX2 and ZHX3, as preferential methylated DNA binders in Xenopus egg extract. The ZHX proteins are an enigmatic group of homeodomain (HD) proteins characterized by the presence of two N-terminal C2H2 zinc fingers followed by four to five tandem HDs. This family has largely been viewed as repressive factors, preventing gene transcription through inhibition of the pioneer factor, NF-YA. We find that the ZHX family members robustly and specifically bind to methylated DNA substrates over unmethylated. Many HD proteins have come to be recognized as DNA methylation sensors; however, conventional HD proteins typically only contain one HD and mediate methylated DNA binding through conserved residues. We asked whether the unique repetitive HD structure of the ZHX proteins play a role in DNA methylation recognition that differs from classic HDs. Using truncation mutants, we identify the first and second homeodomains as necessary for DNA binding in all ZHX family members (ZHX1, ZHX2, ZHX3). Structural predictions and sequence alignment analysis indicate that homeodomain 2 serves as the primary DNA binding domain and may recognize DNA methylation via a mechanism distinct from currently known methyl-binding homeodomain proteins. Additionally, we find that ZHX members bind chromatin in a cell cycle-dependent manner and that mitotic eviction of ZHX proteins is driven by a highly conserved N-terminal stretch of 13 amino acids which contains several predicted phosphorylation sites. Finally, we show that the ZHX2-ZHX3 heterodimer is the preferential dimer formed between ZHX members and that the ZHX members do not associate with NF-YA in either Xenopus egg extract or fibroblast cell lines. Altogether, our findings on how DNA methylation is able to repel chromatin elements like H2A.Z, which has a well-known regulatory role at promotors of active genes, as well as recruit putative repressive factors like the ZHX protein family help to shed light on the ways DNA methylation shapes the genomic regulatory landscape.
License and Reuse Information
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.
Recommended Citation
Shih, Rochelle M., "The Influence of DNA Methylation on Both H2A.Z Nucleosome Dynamics and the Zinc Finger and Homeoboxes Transcription Factor Family" (2026). Student Theses and Dissertations. 843.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/843
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy