Function of the chromosomal kinase haspin in mitosis

描述（由申请人提供）：我们最近发现了一种新的有丝分裂组蛋白激酶haspin，它在多种真核生物中具有同源物。Haspin使组蛋白H3的N-末端尾部的Thr-3磷酸化。在人类细胞中，H3 Thr-3磷酸化首先在G2晚期/早期前期的染色体臂上检测到，在前中期集中在着丝粒，并在后期下降。在体外，haspin特异性地磷酸化组蛋白H3的Thr-3，并通过RNA干扰（RNAi）的haspin的耗竭揭示了它是需要在有丝分裂细胞中的H3 Thr-3磷酸化。Haspin与浓缩的染色体结合，特别是在着丝粒处，并且在有丝分裂期间也在中心体处发现。重要的是，haspin RNAi导致中期染色体的错位和纺锤体缺陷，并且过表达延迟了早期有丝分裂的进展。我们最近的数据表明，haspin是必需的姐妹染色单体凝聚力和着丝粒极光B定位后期前的维护。我们还分离出了候选haspin结合蛋白，这与haspin在中心体和纺锤体的功能是一致的。这项工作揭示了一种参与组蛋白编码的新酶，并将haspin添加到有丝分裂期间调节染色体动力学和纺锤体活性的选择激酶组中。我们希望确定有丝分裂过程中haspin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由haspin缺失引起的染色体错位的潜在缺陷。我们将详细定义haspin RNAi后内聚、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/中心体功能的缺陷。在目标2中，我们将确定H3 Thr-3磷酸化如何调节着丝粒染色质。首先，我们将描绘的位置Thr-3磷酸化相对于粘蛋白和其他分子，允许目前的着丝粒结构模型的细化。然后，我们将使用RNAi和过表达，以确定haspin的影响，在着丝粒的粘附，染色体乘客和异染色质蛋白结合，并对组蛋白修饰的模式。在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。在目的3中，我们探索haspin的功能相互作用，以了解其在中心体和纺锤体活动中的作用。细胞分裂期间染色体行为的调节对于允许基因组准确传递到子细胞至关重要。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有助于恶性肿瘤的产生。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.