We have recently discovered a novel mitotic histone kinase, haspin, that has homologs in diverse eukaryotes. Haspin phosphorylates Thr-3 in the N-terminal tail of histone H3. In human cells, H3 Thr-3 phosphorylation is first detected on chromosome arms in late G2/early prophase, becomes focused at centromeres by prometaphase, and declines during anaphase. In vitro, haspin specifically phosphorylates histone H3 at Thr-3, and depletion of haspin by RNA interference (RNAi) reveals that it is required for H3 Thr-3 phosphorylation in mitotic cells. Haspin associates with condensed chromosomes, particularly at centromeres, and is also found at the centrosomes during mitosis. Importantly, haspin RNAi causes misalignment of metaphase chromosomes and spindle defects, and overexpression delays progression through early mitosis. Our more recent data suggest that haspin is required for the maintenance of sister chromatid cohesion and centromeric aurora B localization prior to anaphase. We have also isolated candidate haspin-binding proteins that are consistent with haspin function at the centrosome and spindle. This work reveals a new enzyme involved in composing the histone code and adds haspin to the select group of kinases that regulate chromosome dynamics and spindle activity during mitosis. We wish to determine the mechanistic basis for haspin action during mitosis.
In Aim 1 we will use immunofluorescence and live cell imaging to examine the defects underlying chromosome misalignment caused by haspin depletion. We will define in detail defects in cohesion, chromosome-spindle attachment, spindle checkpoint activation, aurora B activity and spindle/centrosome function following haspin RNAi.
In Aim 2 we will determine how H3 Thr-3 phosphorylation regulates centromeric chromatin. First, we will delineate the location of Thr-3 phosphorylation with respect to cohesin and other molecules, allowing refinement of current centromere structure models. Then we will use RNAi and overexpression to determine the influence of haspin on cohesin, chromosome passenger and heterochromatin protein binding at centromeres, and on patterns of histone modification. Expression of H3 molecules mutated at Thr-3 and in vitro binding studies will reveal the role of Thr-3 phosphorylation in these effects.
In Aim 3 we explore functional interactions of haspin to understand its role in centrosome and spindle activity. Regulation of chromosome behavior during cell division is critical to allow accurate passage of the genome to daughter cells. Cancer cells have atypical numbers of abnormal chromosomes, suggesting that disruption of these mechanisms contributes to the generation of malignancy. A greater knowledge of the role of human haspin in this process will help us understand the defects that underlie transformation and may lead to new approaches to block the division of cancer cells. ? ? ? ?
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