The ability of an organism to properly regulate development, maintain homeostasis and appropriately respond to environmental stimuli depends on its ability to dynamically regulate transcription. One form of transcriptional regulation involves changing how accessible DNA is to transcriptional machinery by making changes to the surrounding chromatin structure, using mechanisms such as incorporating histone variants into nucleosomes. The highly conserved histone H2A variant, H2A.Z, acts as either an activator or repressor of transcription in different contexts; however, the mechanisms behind the opposing roles of H2A.Z are presently unclear. In the well-established plant model Arabidopsis thaliana, H2A.Z is required to transcriptionally activate genes such as FLC, a developmental switch gene that represses the transition from vegetative to reproductive development. By identifying and characterizing factors that antagonize H2A.Z in its role in transcriptional activation of FLC, we will better understand the mechanisms of how H2A.Z acts as both a positive and negative regulator of transcription. Mutations in BRM, a chromatin remodeling complex subunit, alleviate the requirement for H2A.Z in transcriptional activation of FLC. We hypothesize that H2A.Z activates genes, such as FLC, by destabilizing nucleosomes at regulatory regions of a locus, while transcriptional repressors, such as BRM, antagonize the function of H2A.Z and inhibit transcription by stabilizing or sliding nucleosomes to replace those that were displaced by H2A.Z.
Specific aim 1 of this project will address whether changes in nucleosome stability or composition can explain the changes in transcription observed in brm mutants or mutants defective in incorporating H2A.Z into nucleosomes. Mutants that cannot incorporate H2A.Z into nucleosomes at the FLC locus flower early because they do not activate FLC transcription.
In specific aim 2, we will use mutants from a forward genetic suppressor screen that suppress the early flowering phenotype and restore FLC transcription to detect additional transcriptional repressors that antagonize H2A.Z function at FLC. Suppressor mutations will be mapped using whole genome sequencing and the causal genes that antagonize H2A.Z function will be identified and characterized. Identifying suppressors will allow us to refine our model about why H2A.Z is needed for transcriptional activation. This project takes advantage of a unique genetic interaction between BRM and H2A.Z, shown in Arabidopsis, in order to further understand H2A.Z function through determining how specific transcriptional repressors antagonize H2A.Z function. Results will address our long-term research goal of understanding how the incorporation of histone variants regulates the vital process of transcriptional regulation. This work will provide insight into transcriptional regulatin mechanisms and, since H2A.Z has been implicated in breast, bladder, and pancreatic cancer, it will provide potential targets for therapies to treat diseases in which transcriptional processes are misregulated, such as these cancers.

Public Health Relevance

The ability of every organism to carry out the synchronous processes of development is dependent on its ability to dynamically regulate gene transcription. The long-term goal of this work is to elucidate the molecular mechanisms behind chromatin-based transcriptional regulation, which can be targets for designing disease treatments and therapies. The findings of this study will uncover characteristics of how structures organizing DNA in the nucleus can regulate the expression of genes to control development.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Predoctoral Individual National Research Service Award (F31)
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Special Emphasis Panel (ZRG1)
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Sledjeski, Darren D
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Emory University
Schools of Arts and Sciences
United States
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