The epigenetic system is essential for specifying cell fates during development because it is used to select and reinforce the portions of the genome that will be expressed or silenced in a given cell type. The highly conserved histone H2A variant, H2A.Z, is a key component of this system that is required for embryonic development in animals and regulates many developmental processes in plants. Research has focused on H2A.Z because it has been suggested to play a causal role in both pancreatic and breast cancer cell proliferation, yet its function remains poorly understood. H2A.Z is selectively deposited into chromatin by the SWR1 remodeling complex at thousands of genes, where it paradoxically promotes the transcription of some genes, while silencing others. Despite the biological importance of this histone variant, key questions regarding its targeting and function persist. For example: how is the SWR1 complex targeted to specific genomic sites for H2A.Z deposition? What are the mechanisms by which H2A.Z represses transcription? Given that plants have historically been a rich model system to inform animal epigenetics and that, unlike animals, H2A.Z-deficient plants are viable, our goal is to use the powerful experimental tools available in the model plant Arabidopsis thaliana to answer both of these questions. We recently identified several unexpected SWR1-interacting proteins in Arabidopsis that were not previously associated with H2A.Z deposition, including methyl-CpG- binding domain 9 (MBD9) and several other proteins known to bind nucleosomes. We have now observed that MBD9 is required for H2A.Z incorporation at a subset of H2A.Z-enriched sites that share a common histone modification profile, suggesting that MBD9 and other SWR1-associated proteins provide specific homing functions for SWR1.
In Aim 1, we will define the roles of these proteins in H2A.Z targeting in order to understand the mechanisms that shape the genomic distribution of this variant. Regarding transcription, we have recently found that H2A.Z is required for gene silencing by the conserved polycomb system. Among the genes that require H2A.Z and the polycomb system for silencing are the Phosphate Starvation Response genes, which can be rapidly converted from silent to active and back again by shifting plants between phosphate-rich and phosphate-depleted growth media.
In Aim 2 we will take advantage of this inducible and repressible system to define the order of events that occur during H2A.Z-mediated polycomb silencing in the root hair cell type, where phosphate concentration changes are first detected by the plant. We will follow these studies with inducible genetic ablation of various players during the establishment or maintenance phases of silencing in order to define the role H2A.Z in gene silencing. The powerful genetic tools available in Arabidopsis provide a unique opportunity to understand the mechanisms of H2A.Z targeting and function, and we anticipate that this work will advance the field by answering long-standing questions about histone variant function. Our results may also provide rationale for manipulation of H2A.Z as a target for cancer therapy.
The proposed work aims to understand the operation of an essential gene regulatory mechanism that is required for proper development and has also been associated with cancer cell proliferation. The research is highly relevant to public health in that the results of the proposed studies will provide a deeper understanding of how defects in gene regulation cause disease, and should also yield insight into how the mechanism under study could be manipulated to control cancer cell proliferation.
