Impact of a new histone H2A variant on chromatin structure and dynamics

DNA is wrapped around nucleosomes made of eight core histones H2A, H2B, H3 and H4. We have identified a new core histone variant H2A.W that occupies the major portion of the condensed heterochromatin in plants. Heterochromatin is a key feature that is essential for genome integrity through repression of transposons and for transmission through cell division because of the link between heterochromatin and the mitotic kinetochores. We showed that H2A.W is largely responsible for organization of heterochromatin into higher order dense chromocenters but do not know the mechanisms involved. Our major objectives are to unravel the function of H2A.W, to assess its impact on chromatin and to identify mechanisms that target H2A.W to heterochromatin. Our preliminary work showed that complete removal of H2A.W causes lethality and partial chromic depletion of H2A.W is counteracted by DNA methylation, thus obscuring the primary function of H2A.W. In order to address the function of H2A.W we propose a series of strategies to remove H2A.W in a transient manner either by reducing the amount of transcripts or by using a novel approach to remove the functional domain of H2A.W after its incorporation into the chromatin. The impact of the transient loss of function of H2A.W will be assessed using genome wide profiling to record changes in a variety of chromatin and epigenetic marks. In addition, the impact on chromatin structure will be monitored in vivo using state-of-the art microscopy. To identify how H2A.W is targeted to heterochromatin we will combine genetic and biochemical strategies. In parallel we will use affinity purification/mass spectrometry approach to identify proteins binding to H2A.W in the cytoplasm and in the heterochromatin. Two genetic screens will identify mutants in genes involved in H2A.W dynamics and its function in organizing larger functional heterochromatin domains. Altogether the project will enhance our understanding of the mechanisms that participate to heterochromatin function and to its organization into higher order functional domains.

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