Katsumi Kitagawa, Pharm.D., PhD
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.UTHSA Faculty Profile: Katsumi Kitagawa, Pharm.D., PhD
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 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 the defective kinetochore-microtubule attachment. Defects in kinetochore function and the spindle checkpoint resulting in aneuploidy. Considerable evidence indicates a role of a dysfunctional spindle checkpoint in tumorigenesis.
In most eukaryotes, the centromere is associated with vast arrays of repetitive DNA but has no defined DNA sequence. Consequently, the heritability of the centromere is thought to involve epigenetic modifications. CENP-A, the centromeric histone H3 variant, is believed to be a strong candidate for the epigenetic mark. After DNA replication, centromeric nucleosome (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 the 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, have been observed in cancer cells, particularly hematological malignancies. The 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, the CENP-A is overexpressed, and this overexpression is associated with the 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 of this pathway from Sgt1 to CENP-A ubiquitylation. Sgt1 is a co-chaperone protein that works together with Hsp90. We found that the 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 the 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 essential 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 the S phase, both CENP-A is ubiquitylated. 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 to this model compared to the previous ones. The position of the centromere can be “memorized” by CENP-A ubiquitylation. This mechanism can prevent the 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 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) obtained. **p < 0.01 compared with hTERT-PRE1 (Student’s t-test). Scale bar 5 𝜇
Risa Kitagawa, PhD
Research Scientist Senior
Yuankai Dong, PhD
Mahmoud Ibrahim, PhD
Graduate Research Scientist
- Jingsi Zhou
- Please welcome the Greehey CCRI summer 2021 interns June 7, 2021
- Frontiers in Molecular Biosciences: The Role of Human Centromeric RNA in Chromosome StabilityThe Role of Human Centromeric RNA in Chromosome Stability (Kitagawa Lab) April 20, 2021
- APPLICATIONS NOW BEING ACCEPTED: Greehey CCRI Donald G McEwen, Memorial Summer 2021 Undergraduate Research Program April 6, 2021
Kitagawa Lab, “In the News”