Paul Kalitsis

University of Vic, Vic, Catalonia, Spain

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Publications (105)

  • Paul Kalitsis · Tao Zhang · Kathryn M. Marshall · [...] · Damien F. Hudson
    [Show abstract] [Hide abstract] ABSTRACT: A fundamental requirement in nature is for a cell to correctly package and divide its replicated genome. Condensin is a mechanical multisubunit complex critical to this process. Condensin uses ATP to power conformational changes in DNA to enable to correct DNA compaction, organization, and segregation of DNA from the simplest bacteria to humans. The highly conserved nature of the condensin complex and the structural similarities it shares with the related cohesin complex have provided important clues as to how it functions in cells. The fundamental requirement for condensin in mitosis and meiosis is well established, yet the precise mechanism of action is still an open question. Mutation or removal of condensin subunits across a range of species disrupts orderly chromosome condensation leading to errors in chromosome segregation and likely death of the cell. There are divergences in function across species for condensin. Once considered to function solely in mitosis and meiosis, an accumulating body of evidence suggests that condensin has key roles in also regulating the interphase genome. This review will examine how condensin organizes our genomes, explain where and how it binds the genome at a mechanical level, and highlight controversies and future directions as the complex continues to fascinate and baffle biologists.
    Article · Feb 2017 · Chromosome Research
  • Data: S1 Fig
    Damien F. Hudson · David J. Amor · Amber Boys · [...] · Paul Kalitsis
    [Show abstract] [Hide abstract] ABSTRACT: SNP-array results across the deleted region. Each sib, (A) S1 and (B) S2, display a small, 80-kb homozygous interstitial deletion (orange vertical bar) spanning the RMI2 gene on chromosome 16. Note the lack of SNP heterozygosity across the region, exhibited by blue dots. (PDF)
    File available · Data · Dec 2016
  • Data: S3 Fig
    Damien F. Hudson · David J. Amor · Amber Boys · [...] · Paul Kalitsis
    [Show abstract] [Hide abstract] ABSTRACT: Elevated chromatid breaks in RMI2-deleted individuals. Block stained metaphase chromosomes from control lymphocytes show intact chromosomes (A). Siblings 1 (B) and 2 (C) contain chromatid breaks, shown by arrow. (PDF)
    File available · Data · Dec 2016
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    Damien F. Hudson · David J. Amor · Amber Boys · [...] · Paul Kalitsis
    [Show abstract] [Hide abstract] ABSTRACT: Author Summary Cells contain specific protein complexes that are needed to correct errors during the replication and segregation of DNA. Impairment in the activity of these proteins can be detrimental to the viability of the cell and organism development. Bloom syndrome is an example of a genome instability disorder where cells cannot efficiently untangle DNA after replication. The only gene that is known to cause Bloom syndrome is the BLM helicase. In this article, we describe two affected individuals with Bloom-like features with a homozygous deletion of the RMI2 gene. The RMI2 protein has previously been shown to form a complex with BLM, topoisomerase III alpha and RMI1. Deletion of RMI2 in patient and unrelated cell lines show hyper-recombination and chromosome entanglements during cell division. Furthermore, we show that the BLM and FANCD2 proteins are diminished in the binding of DNA bridges that need to be dissolved during the late stages of cell division. Therefore, loss of RMI2 produces a milder Bloom phenotype and impairs the full activity of the BLM complex.
    Full-text available · Article · Dec 2016 · PLoS Genetics
  • Data: S8 Fig
    Damien F. Hudson · David J. Amor · Amber Boys · [...] · Paul Kalitsis
    [Show abstract] [Hide abstract] ABSTRACT: Analysis of BLM fibers in Anaphase A wild-type and RMI2 null cells. Representative anaphase A images of parental heterozygous, P1 and P2, and homozygous siblings S1 and S2, fibroblasts (A) and RMI2 wild-type and null HCT-116 cells (B) stained with anti-BLM (green), anti-α-tubulin (red) and DAPI for DNA (blue). Scale bar 5 μm. Quantification of detection of BLM fibers in anaphase A cells in (C) parent (P1, P2) and sibling (S1, S2), and (D) wild-type HCT-116 control and RIM2 null cells (1–2, 1–3, 4–6). Data taken from three independent experiments, with a minimum of 15 anaphases A cells scored for each fibroblast cell line (P1, P2, S1, S2) per experiment and also for each HCT-116 cell line (wild type, 1–2, 1–3, 4–6) per experiment. Error bars represent standard error of the mean. (PDF)
    File available · Data · Dec 2016
  • Data: S5 Fig
    Damien F. Hudson · David J. Amor · Amber Boys · [...] · Paul Kalitsis
    [Show abstract] [Hide abstract] ABSTRACT: Sister chromatid exchange and colony forming analyses on RMI2 null clones. (A–D) Differential chromatid staining on representative metaphase cells from HCT-116 and RMI2 null clones, 1–2, 1–3 and 4–6. (E, F) Colony forming assays on HCT-116 and RMI2 null cell lines displayed as numbers of colonies and total area occupied in a 6-well tray (arbitrary units). (PDF)
    File available · Data · Dec 2016
  • Data: S2 Fig
    Damien F. Hudson · David J. Amor · Amber Boys · [...] · Paul Kalitsis
    [Show abstract] [Hide abstract] ABSTRACT: Sequence characteristics of the deletion junction. Two Alu elements on the telomere (tel) and centromere (cen) sides of the deleted region show evidence of a non-allelic recombination event. (A) Sequence alignment of the two elements reveals a high level of homology and the position of the recombination event (arrow). Sequence chromatogram across the deletion junction (arrow). (PDF)
    File available · Data · Dec 2016
  • Data: S9 Fig
    Damien F. Hudson · David J. Amor · Amber Boys · [...] · Paul Kalitsis
    [Show abstract] [Hide abstract] ABSTRACT: FANCD2 occasionally localises to UFBs during anaphase. Examples of FANCD2 localisation to sister chromatid foci in parental fibroblast cells, P2. A fine thread of FANCD2 signal can be seen in an anaphase cell of sibling S2. Fibroblast cells stained with anti-FANCD2 (green), anti-α-tubulin (red) and DAPI for DNA (blue). Scale bar 5 μm. (PDF)
    File available · Data · Dec 2016
  • Data: S4 Fig
    Damien F. Hudson · David J. Amor · Amber Boys · [...] · Paul Kalitsis
    [Show abstract] [Hide abstract] ABSTRACT: RMI2 CRISPR-Cas9 mutation sequence sites of null clones. CRISPR-Cas9 induced RMI2 mutation regions were PCR-amplified, cloned and sequenced. Deletion or insertion region is shown against the reference genome for each nickase pair. Guide pairs are shown in blue with the protospacer adjacent motif (PAM) site shown in red. (PDF)
    File available · Data · Dec 2016
  • Data: S6 Fig
    Damien F. Hudson · David J. Amor · Amber Boys · [...] · Paul Kalitsis
    [Show abstract] [Hide abstract] ABSTRACT: Examples of anaphase bridges and chromosome laggards in RMI2 null cells. Representative images of dividing fibroblasts showing bridges and lagging chromosomes. Cells were co-stained with anti-α-tubulin (red) and DAPI to visualise DNA (blue). Scale bar 5 μm. (PDF)
    File available · Data · Dec 2016
  • Data: S7 Fig
    Damien F. Hudson · David J. Amor · Amber Boys · [...] · Paul Kalitsis
    [Show abstract] [Hide abstract] ABSTRACT: DNA content analysis on fibroblasts and knockout cell lines Exponentially growing cells were measured for DNA content using flow cytometry. (PDF)
    File available · Data · Dec 2016
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    Thian T. Beh · Ruth N. MacKinnon · Paul Kalitsis
    [Show abstract] [Hide abstract] ABSTRACT: The centromere plays a crucial role in ensuring the fidelity of chromosome segregation during cell divisions. However, in cancer and constitutional disorders, the presence of more than one active centromere on a chromosome may be a contributing factor to chromosome instability and could also have predictive value in disease progression, making the detection of properly functioning centromeres important. Thus far, antibodies that are widely used for functional centromere detection mainly work on freshly harvested cells whereas most cytogenetic samples are stored long-term in methanol-acetic acid fixative. Hence, we aimed to identify antibodies that would recognise active centromere antigens on methanol-acetic acid fixed cells. A panel of active centromere protein antibodies was tested and we found that a rabbit monoclonal antibody against human CENP-C recognises the active centromeres of cells fixed in methanol-acetic acid. We then tested and compared combinations of established methods namely centromere fluorescence in situ hybridisation (cenFISH), centromere protein immunofluorescence (CENP-IF) and multicolour FISH (mFISH), and showed the usefulness of CENP-IF together with cenFISH followed by mFISH (CENP-IF-cenFISH-mFISH) with the aforementioned anti-CENP-C antibody. We further demonstrated the utility of our method in two cancer cell lines with high proportion of centromere defects namely neocentromere and functional dicentric. We propose the incorporation of the CENP-IF-cenFISH-mFISH method using a commercially available rabbit monoclonal anti-CENP-C into established methods such as dicentric chromosome assay (DCA), prenatal karyotype screening in addition to constitutional and cancer karyotyping. This method will provide a more accurate assessment of centromere abnormality status in chromosome instability disorders.
    Full-text available · Article · Dec 2016 · Molecular Cytogenetics
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    [Show abstract] [Hide abstract] ABSTRACT: Condensin is an integral component of the mitotic chromosome condensation machinery, which ensures orderly segregation of chromosomes during cell division. In metazoans, condensin exists as two complexes, condensin I and II. It is not yet clear what roles these complexes may play outside mitosis, and so we have examined their behaviour both in normal interphase and in premature chromosome condensation (PCC). We find that a small fraction of condensin I is retained in interphase nuclei, and our data suggests that this interphase nuclear condensin I is active in both gene regulation and chromosome condensation. Furthermore, live cell imaging demonstrates condensin II dramatically increases on G1 nuclei following completion of mitosis. Our PCC studies show condensins I and II and topoisomerase II localise to the chromosome axis in G1-PCC and G2/M-PCC, while KIF4 binding is altered. Individually, condensins I and II are dispensable for PCC. However, when both are knocked out, G1-PCC chromatids are less well structured. Our results define new roles for the condensins during interphase and provide new information about the mechanism of PCC.
    Full-text available · Article · Mar 2016 · Chromosome Research
  • [Show abstract] [Hide abstract] ABSTRACT: Over-expression of growth factors can contribute to the development and progression of cancer, and gastrins in particular have been implicated in accelerating the development of gastrointestinal cancers. Previously our group showed that hypoxia, cobalt chloride (a hypoxia mimetic) and zinc chloride could activate the expression of the gastrin gene in vitro. To characterise activation of the gastrin promoter by zinc ions further in vivo, TALEN technology was used to engineer a luciferase reporter construct into the endogenous human gastrin gene promoter in SW480 colon cancer cells. Gastrin promoter activity in the resultant Gast(luc) SW480 colon cancer cells was then measured by bioluminescence in cell culture and in tumour xenografts in SCID mice. Activation of intracellular signalling pathways was assessed by Western blotting. Activation of the gastrin promoter by zinc ions was concentration dependent in vitro and in vivo. Zinc ions significantly stimulated phosphorylation of ERK1/2 (MAPK pathway) but not of Akt (PI3K pathway). We conclude that the endogenous gastrin promoter is responsive to zinc ions, likely via activation of the MAPK pathway.
    Article · Sep 2015 · Metallomics
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    [Show abstract] [Hide abstract] ABSTRACT: Histones package DNA and regulate epigenetic states. For the latter, probably the most important histone is H3. Mammals have three near-identical H3 isoforms: canonical H3.1 and H3.2, and the replication-independent variant H3.3. This variant can accumulate in slowly dividing somatic cells, replacing canonical H3. Some replication-independent histones, through their ability to incorporate outside S-phase, are functionally important in the very slowly dividing mammalian germ line. Much remains to be learned of H3.3 functions in germ cell development. Histone H3.3 presents a unique genetic paradigm in that two conventional intron-containing genes encode the identical protein. Here, we present a comprehensive analysis of the developmental effects of null mutations in each of these genes. H3f3a mutants were viable to adulthood. Females were fertile, while males were subfertile with dysmorphic spermatozoa. H3f3b mutants were growth-deficient, dying at birth. H3f3b heterozygotes were also growth-deficient, with males being sterile because of arrest of round spermatids. This sterility was not accompanied by abnormalities in sex chromosome inactivation in meiosis I. Conditional ablation of H3f3b at the beginning of folliculogenesis resulted in zygote cleavage failure, establishing H3f3b as a maternal-effect gene, and revealing a requirement for H3.3 in the first mitosis. Simultaneous ablation of H3f3a and H3f3b in folliculogenesis resulted in early primary oocyte death, demonstrating a crucial role for H3.3 in oogenesis. These findings reveal a heavy reliance on H3.3 for growth, gametogenesis, and fertilization, identifying developmental processes that are particularly susceptible to H3.3 deficiency. They also reveal partial redundancy in function of H3f3a and H3f3b, with the latter gene being generally the most important.
    Full-text available · Article · Feb 2015 · PLoS Genetics
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    [Show abstract] [Hide abstract] ABSTRACT: The condensin complex plays a key role in organizing mitotic chromosomes. In vertebrates, there are two condensin complexes that have independent and cooperative roles in folding mitotic chromosomes. In this study, we dissect the role of a putative Cdk1 site on the condensin II subunit CAP-D3 in chicken DT40 cells. This conserved site has been shown to activate condensin II during prophase in human cells, and facilitate further phosphorylation by polo-like kinase I. We examined the functional significance of this phosphorylation mark by mutating the orthologous site of CAP-D3 (CAP-D3T1403A) in chicken DT40 cells. We show that this mutation is a gain of function mutant in chicken cells; it disrupts prophase, results in a dramatic shortening of the mitotic chromosome axis, and leads to abnormal INCENP localization. Our results imply phosphorylation of CAP-D3 acts to limit condensin II binding onto mitotic chromosomes. We present the first in vivo example that alters the ratio of condensin I:II on mitotic chromosomes. Our results demonstrate this ratio is a critical determinant in shaping mitotic chromosomes.
    Full-text available · Article · Jan 2015 · Journal of Biological Chemistry
  • Thian Thian Beh · Paul Kalitsis
    [Show abstract] [Hide abstract] ABSTRACT: The centromere is a protein–DNA structure that is essential for the correct segregation of chromosomes to daughter cells. In human cells it is located at a specific region of each chromosome that is devoid of genes and characterized by heterochromatin. The underlying DNA is made up of a 171-bp tandem repeat known as alpha satellite, which is organized in a head-to-tail arrangement and spans up to several megabases in length. The centromere DNA is the foundation onto which over 100 proteins form a structure known as the kinetochore. This structure fully assembles during mitosis and is instrumental in the capture of spindle microtubules and the movement of chromosomes to opposing poles of the cell. In this chapter we describe the main cellular functions of the centromere in a healthy cell and show how errors in its normal role can contribute to human diseases such as developmental disorders, cancer, infertility and premature aging. In addition, we examine the evolution of centromere DNA and proteins in humans and primates and the emergence of new centromeres in non-satellite DNA locations.
    Chapter · Jan 2015
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    Alison N Graham · Paul Kalitsis
    [Show abstract] [Hide abstract] ABSTRACT: The centromere is an essential chromosomal structure that is required for the faithful distribution of replicated chromosomes to daughter cells. Defects in the centromere can compromise the stability of chromosomes resulting in segregation errors. We have characterised the centromeric structure of the spontaneous mutant mouse strain, BALB/cWt, which exhibits a high rate of Y chromosome instability. The Y centromere DNA array shows a de novo interstitial deletion and a reduction in the level of the foundation centromere protein, CENP-A, when compared to the non-deleted centromere array in the progenitor strain. These results suggest there is a lower threshold limit of centromere size that ensures full kinetochore function during cell division.
    Full-text available · Article · Jan 2014 · PLoS ONE
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    [Show abstract] [Hide abstract] ABSTRACT: The condensin complex is essential for correct packaging and segregation of chromosomes during mitosis and meiosis in all eukaryotes. To date, the genome-wide location and the nature of condensin-binding sites have remained elusive in vertebrates. Here we report the genome-wide map of condensin I in chicken DT40 cells. Unexpectedly, we find that condensin I binds predominantly to promoter sequences in mitotic cells. We also find a striking enrichment at both centromeres and telomeres, highlighting the importance of the complex in chromosome segregation. Taken together, the results show that condensin I is largely absent from heterochromatic regions. This map of the condensin I binding sites on the chicken genome reveals that patterns of condensin distribution on chromosomes are conserved from prokaryotes, through yeasts to vertebrates. Thus in three kingdoms of life, condensin is enriched on promoters of actively transcribed genes and at loci important for chromosome segregation.
    Full-text available · Article · Oct 2013 · Nature Communications
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    [Show abstract] [Hide abstract] ABSTRACT: Immunofluorescence for markers of pericentric heterochromatin. (A) Panels: First row; metaphase spreads were probed with an antibody specific for H3K9me3. Second row; CREST antibody is specific for the centromere. Third row; merge of the two images directly above, concomitant with detection of DNA with DAPI, reveals the pericentric localization of H3K9me3. (B) As for A, except that CBX5 (synonym HP1α) is shown to be localized to the pericentric region. (C) As for A, except that H4K20me3 is shown to be localized to the pericentric region. Dicer1c/− panels at left are the EP parental cell line. (PDF)
    File available · Data · Sep 2012