Function of the chromosomal kinase haspin in mitosis

描述(申请人提供)：我们最近发现了一种新的有丝分裂组蛋白激酶，haspin，它在不同的真核生物中具有同源物。Haspin使组蛋白H3的N端的Thr-3磷酸化。在人类细胞中，H3Thr-3的磷酸化在G2末期/早期在染色体臂上首次检测到，在前中期集中在着丝粒，在后期下降。在体外，haspin特异性地在Thr-3处磷酸化组蛋白H3，通过RNA干扰(RNAi)去除haspin表明它是有丝分裂细胞中H3Thr-3磷酸化所必需的。Haspin与浓缩的染色体结合，特别是在着丝粒上，在有丝分裂过程中也在着丝粒上发现。重要的是，haspin RNAi导致中期染色体错位和纺锤体缺陷，过度表达通过早期有丝分裂延迟进展。我们最近的数据表明，haspin是维持姐妹染色单体凝聚力和着丝粒极光B后期定位所必需的。我们还分离了与中心体和纺锤体上的haspin功能一致的候选haspin结合蛋白。这项工作揭示了一种参与构成组蛋白密码的新酶，并将haspin添加到选定的一组激酶中，这些激酶在有丝分裂过程中调节染色体动态和纺锤体活动。我们希望确定haspin在有丝分裂过程中作用的机制基础。在目标1中，我们将使用免疫荧光和活细胞成像来检查haspin耗尽引起的染色体错位的潜在缺陷。我们将详细定义haspin RNAi后在凝聚力、染色体纺锤体附着、纺锤体检查点激活、极光B活性和纺锤体/中心体功能方面的缺陷。在目标2中，我们将确定H3Thr-3磷酸化如何调节着丝粒染色质。首先，我们将描述Thr-3磷酸化相对于粘附素和其他分子的位置，从而改进当前的着丝粒结构模型。然后，我们将使用RNAi和过度表达来确定haspin对着丝粒上的粘附素、染色体乘客和异染色质蛋白结合的影响，以及对组蛋白修饰模式的影响。Thr-3突变的H3分子的表达和体外结合研究将揭示Thr-3磷酸化在这些效应中的作用。在目标3中，我们探讨了haspin的功能相互作用，以了解它在中心体和纺锤体活动中的作用。对细胞分裂过程中染色体行为的调节是使基因组准确传递到子代细胞的关键。癌细胞具有非典型数量的异常染色体，这表明这些机制的破坏导致了恶性肿瘤的产生。更多地了解人类haspin在这一过程中的作用将有助于我们理解转化背后的缺陷，并可能导致阻止癌细胞分裂的新方法。

DESCRIPTION (provided by applicant): 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.