Friday, May 9, 2025
5/9/2025
Visualizing transcription factor binding and chromatin assembly on newly synthesized DNA - PROJECT SUMMARY Newly synthesized DNA represents an ideal system to understand the molecule mechanisms governing chromatin structure and gene expression. Chromatin structure controls transcription by modulating DNA accessibility. During DNA replication, chromatin structure must be disassembled to allow for passage of the replication fork, which generates an intermediate chromatin structure distinct from mature chromatin. Failure to reestablish proper chromatin structure leads to spurious gene expression and is associated with a variety of human diseases. Transcription factors (TFs) are a critical component of chromatin structure that regulate transcription by binding to targeted sequences on DNA and altering chromatin structure. Following replication fork passage, TF binding sites become occluded by nucleosomes. As nucleosomes are refractory to TF binding, it is unclear when or how TFs rebind to targeted DNA sequences to reestablish mature chromatin structure. Furthermore, in-spite of the established role for TFs in regulating gene expression, it is unclear what effect TF binding has on chromatin structure. To address these gaps in knowledge, I will use novel techniques termed Nascent CUT&Tag and Nascent Fiber-seq to profile TF binding on nascent DNA and during the subsequent steps of chromatin maturation. In Aim 1, I will use Nascent CUT&Tag to determine the kinetics and mechanisms of TF binding to nascent chromatin. This aim will test whether TFs can associate with nucleosomal DNA during the process of rebinding, or whether accessible DNA is necessary for TFs to bind following replication fork passage. In Aim 2, I will visualize TF binding and nascent chromatin maturation on the single molecule level. By simultaneously visualizing TF binding and surrounding chromatin structure, I will be able to assess the direct impacts of TF binding on critical chromatin features such as nucleosome positioning and RNA polymerase II occupancy. In Aim 3, I will characterize the impacts of nucleosome turnover on TF binding and chromatin maturation. Using the anti-cancer drug aclarubicin, I will drive elevated nucleosome turnover and observe the effects on TF binding and nascent chromatin maturation. These studies will provide critical insights into the features regulating TF binding and chromatin assembly, which will inform our understanding of the underlying mechanisms regulating gene expression and cell fate specification. The training outlined in this proposal will provide a strong foundation to develop as an independent investigator studying the critical relationship between chromatin structure and gene expression.
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