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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM074210-04S1
Application #
8003038
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Hamlet, Michelle R
Project Start
2010-01-08
Project End
2010-12-31
Budget Start
2010-01-08
Budget End
2010-12-31
Support Year
4
Fiscal Year
2010
Total Cost
$110,437
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
030811269
City
Boston
State
MA
Country
United States
Zip Code
02115
Zhou, Linli; Tian, Xiaoying; Zhu, Cailei et al. (2014) Polo-like kinase-1 triggers histone phosphorylation by Haspin in mitosis. EMBO Rep 15:273-81
Niedzialkowska, Ewa; Wang, Fangwei; Porebski, Przemyslaw J et al. (2012) Molecular basis for phosphospecific recognition of histone H3 tails by Survivin paralogues at inner centromeres. Mol Biol Cell 23:1457-66
Wang, Fangwei; Ulyanova, Natalia P; Daum, John R et al. (2012) Haspin inhibitors reveal centromeric functions of Aurora B in chromosome segregation. J Cell Biol 199:251-68
Czakai, Kristin; Muller, Katja; Mosesso, Pasquale et al. (2011) Perturbation of mitosis through inhibition of histone acetyltransferases: the key to ochratoxin a toxicity and carcinogenicity? Toxicol Sci 122:317-29
Wang, Fangwei; Ulyanova, Natalia P; van der Waal, Maike S et al. (2011) A positive feedback loop involving Haspin and Aurora B promotes CPC accumulation at centromeres in mitosis. Curr Biol 21:1061-9
Varier, Radhika A; Outchkourov, Nikolay S; de Graaf, Petra et al. (2010) A phospho/methyl switch at histone H3 regulates TFIID association with mitotic chromosomes. EMBO J 29:3967-78
Higgins, Jonathan M G (2010) Haspin: a newly discovered regulator of mitotic chromosome behavior. Chromosoma 119:137-47
Wang, Fangwei; Dai, Jun; Daum, John R et al. (2010) Histone H3 Thr-3 phosphorylation by Haspin positions Aurora B at centromeres in mitosis. Science 330:231-5
Dai, Jun; Kateneva, Anna V; Higgins, Jonathan M G (2009) Studies of haspin-depleted cells reveal that spindle-pole integrity in mitosis requires chromosome cohesion. J Cell Sci 122:4168-76
Eswaran, Jeyanthy; Patnaik, Debasis; Filippakopoulos, Panagis et al. (2009) Structure and functional characterization of the atypical human kinase haspin. Proc Natl Acad Sci U S A 106:20198-203

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