Our research goal is to understand the role of aneuploidy (chromosome loss or gain) in childhood cancer development. We are currently investigating the mechanism of CIN (chromosome instability) in pediatric tumors.
The molecular mechanisms that ensure accurate chromosome segregation in mitosis and meiosis are of fundamental importance to the conservation of euploidy in eukaryotes. Errors in this process (e.g., chromosome nondisjunction and chromosome loss) result in aneuploidy—the phenotypic consequences of which are usually profound, including cancer, birth defects, and developmental disorders such as Down syndrome. In humans, errors in chromosome segregation may trigger the onset of neoplasia by uncovering the expression of recessive oncogenic phenotypes, or by contributing to the development of specific aneuploidies. The centromere, a single locus per chromosome, is essential to ensure high fidelity of chromosome transmission. The kinetochore (the protein complex at the centromere) mediates attachment of chromosomes to spindle microtubules and directs chromosome movement during mitosis. Cells have a surveillance system, the spindle checkpoint, which can delay mitotic progression by transiently inhibiting the anaphase-promoting complex in response to defective kinetochore-microtubule attachment. Defects in kinetochore function and the spindle checkpoint result in aneuploidy. Considerable evidence indicates a role of a dysfunctional spindle checkpoint in tumorigenesis.
In most eukaryotes, the centromere is associated with large arrays of repetitive DNA, but has no defined DNA sequence. Consequently, heritability of the centromere is thought to involve epigenetic modifications. CENP-A, the centromeric histone H3 variant, is thought to be a strong candidate for the epigenetic mark. After DNA replication, centromeric nucleosomes, including existing CENP-A, are distributed to the replicated chromatids, and newly synthesized CENP-A deposition occurs at the centromere in G1 in humans. This regulation is crucial for proper centromere inheritance and function. However, the molecular mechanism that determines the precise CENP-A deposition epigenetically remains obscure. One of our aims is to determine the function of post-translational modifications (PTMs) of CENP-A in the regulation of CENP-A deposition at the centromere and the assembly of kinetochore complexes. The contribution of our work will be significant because understanding the role of PTMs of CENP-A in regulating centromere formation should advance the understanding of development of diseases associated with chromosome instability (CIN) such as tumors.
Neocentromeres originate from non-centromeric regions of chromosomes, (i.e., not alpha-satellite DNA). The formation of complex rearranged chromosomes, each containing a neocentromere, has been observed in cancer cells, particularly hematological malignancies. Addition of a neocentromere to a chromosome with an endogenous centromere creates a dicentric state, which results in extensive genomic instability displaying hallmarks of cellular transformation. In colon cancer, CENP-A is overexpressed, and this overexpression is associated with mistargeting of CENP-A to non-centromeric chromatin. These findings suggest that overexpression of CENP-A might cause aneuploidy by creating neocentromeres. Genomic amplification of the CENP-A locus occurred in neuroendocrine prostate cancer (15% of cases) and breast cancer (10% of cases). We have found that CENP-A is highly expressed in several pediatric tumors.
Thus, elucidating the mechanism of neocentromere formation will contribute to understanding the mechanism of “cancer evolution” that results in resistance to cancer therapy.
We will investigate the role of aneuploidy in the development of pediatric cancers.
The centromere is the chromosomal site where microtubules bind. The kinetochore means the protein complexes that bind to centromeric DNA. However, they are occasionally used equally. The centromere is required for proper chromosome segregation, and if this mitotic mechanism fails, chromosome gain or loss occurs.
The histone H3 variant CENP-A is the prime candidate to carry the epigenetic information that specifies the chromosomal location of the centromere. As you see here, the kinetochore complexes are built on the centromere specific nucleosomes containing CENP-A.
This slide is the summary of our findings about this pathway from Sgt1 to CENP-A ubiquitylation. Sgt1 is a co-chaperone protein that works together with Hsp90. We found that Sgt1-Hsp90 complex is required for the recognition of CENP-A by the Cul4A E3 ligase, and this enzyme ubiquitylates CENP-A. Ubiquitylated CENP-A binds efficiently to HJURP the histone chaperone protein, and CENP-A goes to the centromere.
This picture shows mitotic chromosomes. CENP-B binds to alpha-satellite DNA directly and the red signals serve as a centromere control in this experiment. When we express Flag-tagged CENP-A in cells, it goes to the centromere. But, non-ubiquitylated mutant K124R does not. Then, we put mono-ubiquitin at the C-terminal end, the mono-ubiquitin fusion protein goes to the centromere. This experiment shows that mono-ubiquitylation is important for CENP-A deposition at the centromere, and we used this mono-ubiquitin fusion mutant for several experiments that I will talk about from now.
Before S phase, both CENP-A are ubiquitylated, and after DNA replication, ubiquitylated CENP-A is distributed, and then, HJURP localizes at the centromere by binding to ubiquitylated CENP-A, and new CENP-A is recruited to the centromere. Then, now this CENP-A heterodimer is recognized by the Cul4 E3 ligase and this new CENP-A is ubiquitylated. There are advantages of this model compared to the previous ones. The position of the centromere can be “memorized” by CENP-A ubiquitylation. This mechanism can prevent formation of centromeres at other places on chromosomes.
EwS cells exhibit chromosome instability. A. Asynchronously growing indicated cells (ES-2, ES-4 and hTERT-RPE1) were fixed with 4% formaldehyde on a cover glass, and stained with DAPI, anti-tubulin and anti-CENP-B antibodies. Mitotic cells were analyzed for chromosome segregational defects, such as misaligned chromosomes during metaphase, lagging chromosomes during anaphase and chromosome bridges during anaphase and telophase. Representative images for each defect are shown. B. Abnormal mitotic cells were counted for each cell line. ES-2 (n=46), ES-4 (n=30) and hTERT-RPE1 (n=83). C. A representative image of micronuclei is shown. D. The average number (±SD) of cells with micronuclei (ES-2: n=621, ES-4: n=164 and hTERT-RPE: n=217) was obtained. **p < 0.01 compared with hTERT-PRE1 (Student’s t test). Scale bar 5 𝜇
- 1993 - Toyobo, short-term fellowship for attending a meeting abroad
- 1995 - Japan Society for the Promotion of Science (JSPS) Fellowship for Research at Centers of Excellence Abroad
- 1996 - Human Frontier Science Program Organization, long-term research fellowships
- 2000 - Uehara Memorial Foundation, long-term research fellowship
- 2006 - Wendy Will Case Cancer Award
- 2006 - The Susan G. Komen Breast Cancer Foundation Research Grant
- 2007 - American Cancer Society Research Scholar Grant
- Niikura Y, Kitagawa R and Kitagawa K. CENP-A Ubiquitylation is the epigenetic mark for centromere identify. Cell Reports 2016, 15: 61-76.
- Niikura Y and Kitagawa K. Immunofluorescence analysis of endogenous and exogenous centromere-kinetochore proteins. J Vis Exp. 2016 Mar 3;(109). doi: 10.3791/53732.
- Ogi H, Sakuraba Y, Kitagawa R, Xiao L, Shen C, Cynthia M, Ohta S, Arnold MA, Ramirez N, Houghton PJ, and Kitagawa K. The Oncogenic Role of the Cochaperone Sgt1. Oncogenesis 2015 2015 May 18;4:e149.
- Niikura Y, Kitagawa R, Ogi H, Abdulle R, Pagala V, and Kitagawa K. CENP-A K124 Ubiquitylation Is Required for CENP-A Deposition at the Centromere. Developmental Cell 2015 Mar 9;32(5):589-603.
- Ohkuni K, Abdulle R, and Kitagawa K. Degradation of centromeric histone H3 variant Cse4 requires the Fpr3 peptidyl-prolyl cis-trans isomerase. Genetics 2014; 196(4):1041-5.
- Bian Y, Kitagawa , Bansal PK, Fujii Yo, Stepanov A, and Kitagawa K. Synthetic genetic array screen identifies PP2A as a therapeutic target in Mad2-overexpressing tumors. Proc Natl Acad Sci U S A. 2014; 111(4):1628-33.
- Kikuchi K, Narita T, Pham VT, Iijima J, Hirota K, Keka IS, Mohiuddin M, Okawa K, Hori T, Fukagawa T, Essers J, Kanaar R, Whitby MC, Sugasawa K, Taniguchi Y, Kitagawa K, Takeda S. Structure-specific endonucleases Xpf and Mus81 play overlapping but essential roles in DNA repair by homologous recombination. Cancer Res. 2013; 73(14):4362-71.
- Ohkuni K and Kitagawa K, Role of transcription at centromeres in budding yeast. Transcription 2012; 3(4).
- Ohkuni K and Kitagawa K, Endogenous Transcription at the Centromere Facilitates Centromere Activity in Budding Yeast. Current Biology 2011; 21(20):1695-703.
- Yang C, Tang X, Guo X, Niikura Y, Kitagawa K, Cui K, Wong STC, Fu L, and Xu B, Aurora-B Mediated ATM Serine 1403 Phosphorylation Is Required For Mitotic ATM Activation and the Spindle Checkpoint. Molecular Cell 2011; 44(4):597-608.
- Kitagawa K, Too early to say, "no targeting of mitosis!" Nat Rev Clin Oncol. 2011; 8(7):444.
- Goto GH, Mishra A, Abdulle R, Slaughter CA, and Kitagawa K, Bub1-mediated Adaptation of the Spindle Checkpoint. PLoS Genetics 2011; 7(1):e1001282.
- Kikuchi K, Niikura Y, Kitagawa K, and Kikuchi A, Dishevelled, a Wnt signaling component, is involved in mitotic progression in cooperation with Plk1. EMBO J. 2010; 29(20):3470-83.
- Niikura Y, Ogi H, Kikuchi K,Kitagawa K, BUB3 that dissociates from BUB1 activates caspase-independent mitotic death (CIMD). Cell Death Differ. 2010; 17(6):1011-24.
- Bansal PK, Mishra A, High AA, Abdulle R, Kitagawa K, Sgt1 dimerization is negatively regulated by protein kinase CK2-mediated phosphorylation at S361; J Biol Chem. 2009; 284(28):18692-8.
- Bansal PK, Nourse A, Abdulle R, Kitagawa K. Sgt1 dimerization is required for yeast kinetochore assembly. J Biol Chem. 2009; 284(6):3586-92.
- Kitagawa K* and Niikura Y. Caspase-Independent Mitotic Death (CIMD). Cell Cycle. 2008; 7(8):1001-5.
- Ohkuni K, Abdulle R, Tong AH, Boone C, and Kitagawa K. Ybp2 Associates with the Central Kinetochore of Saccharomyces cerevisiae and Mediates Proper Mitotic Progression. PLoS ONE. 2008;3(2):e1617.
- Niikura Y, Dixit A, Scott R, Perkins G and Kitagawa K. BUB1 mediation of caspase-independent mitotic death determines cell fate. J. Cell Biol. 2007;178(2):283-96. (Faculty of 1000 Biology: F1000 Factor 6.0: Must Read http://www.f1000biology.com/article/id/1088744/evaluation).
- Scaglione KM, Bansal PK, Deffenbaugh AE, Kiss A, Moore JM, Korolev S, Cocklin R, Goebl M, Kitagawa K, and Skowyra D. SCF E3 -mediated autoubiquitination negatively regulates activity of the Cdc34 E2 but plays a nonessential role in the catalytic cycle in vitro and in vivo. Mol. Cell. Biol. 2007;27(16):5860-70.
- Niikura Y, Ohta S, Vandenbeldt KJ, Abdulle R, McEwen BF and Kitagawa K. 17-AAG, an Hsp90 inhibitor, causes kinetochore defects: a novel mechanism by which 17-AAG inhibits cell proliferation. Oncogene 2006; 25:4133-46 (Faculty of 1000 Biology: F1000 Factor 3.0: Recommended http://www.f1000biology.com/article/id/1033229/evaluation).
- Kondo-Okamoto N, Ohkuni K, Kitagawa K, McCaffery JM, Shaw JM, Okamoto K. The novel F-box protein Mfb1p regulates mitochondrial connectivity and exhibits asymmetric localization in yeast. Mol. Biol. Cell 2006; 17:3756-67.
- Bansal PK, Abdulle R, and Kitagawa K. Sgt1 associates with molecular chaperones: an initial step of assembly of the core kinetochore complex. Mol. Cell. Biol. 2004 Sep;24(18):8069-79.
- Steensgaard P, Garre M, Muradore I, Transidico P, Nigg EA, Kitagawa K, Earnshaw WC, Faretta M, Musacchio A. Sgt1 is required for human kinetochore assembly. EMBO Rep. 2004 Jun;5(6):626-31. Epub 2004 May 07.
- Niikura Y and Kitagawa K. Identification of a Novel Splice Variant: Human SGT1B (SUGT1B). DNA sequence. 2003 Dec;14(6):436-41.
- Kitagawa K, Abdulle R, Bansal PK, Cagney G, Fields S, and Hieter P. Requirement of Skp1-Bub1 interaction for kinetochore-mediated activation of the spindle checkpoint. Mol. Cell. 2003 May;11(5):1201-13.
- Nowotny M, Spiechowicz M, Jastrzebska B, Filipek A, Kitagawa K, Kuznicki J. Calcium-regulated interaction of Sgt1 with S100A6 (calcyclin) and other S100 proteins. J. Biol. Chem. 2003 Jul 18;278(29):26923-8. Epub 2003 May 13.
- Kitagawa K and Abdulle R. In vivo site-directed mutagenesis of yeast plasmids by using a three-fragment homologous recombination system. Biotechniques. 2002 Aug;33(2):288, 290, 292 passim.
- Schadick KH, Fourcade M, Boumenot P, Seitz JJ, Morrell JL, Chang L, Gould KL, Partridge JF, Allshire RC, Kitagawa K, Hieter P, and Hoffman CS.* Schizosaccharomyces pombe Git7p, a member of the Saccharomyces cerevisiae Sgt1p family, is required for Pglucose/cAMP signaling, cell wall integrity, and septation. Eukaryot Cell. 2002 Aug;1 (4):558-67.
- Dubacq C, Guerois R, Courbeyrette R, Kitagawa K, and Mann C. Sgt1p contributes to cAMP pathway activity and physically interacts with the adenylyl cyclase Cyr1p/Cdc35p in budding yeast. Eukaryot Cell. 2002 Aug;1 (4):568-82.
- Azevedo C, Sadanandom A, Kitagawa K., Freialdenhoven A., Shirasu K, and Schulze-Lefert P. The RAR1 interactor SGT1, an essential component of R gene–triggered disease resistance. Science. 2002 Mar 15;295(5562):2073-76.
- Kitagawa K and Hieter P. Evolutionary conservation between budding yeast and human kinetochores. Nature Reviews Mol. Cel. Biol. 2001 Sep;2 (9):678-87. Review.
Funding Agency National Cancer Institute
Title Formation of a Neocentromere at a DSB Site
Status Active Active
Grant Detail To determine the role of CENP-A (centromeric histone H3) in DNA damage response.