Active centromere and chromosome identification in fixed cell lines

Abstract

BackgroundThe 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.ResultsA 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.ConclusionsWe 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.

The bromodomain inhibitor JQ1 is a molecular glue targeting centromeres

“…Metaphase spreads were prepared using a standard method for karyotype analysis using Carnoy’s fixative and dropping the sample onto a microscope slide 56 . This process produces high resolution metaphase chromosome features, but under these fixation conditions centromeric antibodies do not detect their epitope 57 , which prevents fluorescence microscopy. Briefly, 1 well of a 6-well plate per sample was seeded two days prior to metaphase chromosome preparation.…”

The Hidden Genomic and Transcriptomic Plasticity of Giant Marker Chromosomes in Cancer

“…Immuno-FISH on elongated chromosomes was performed to validate the putative structure of neocentromeres, as previously described (Earnshaw et al 1989). As recommended by Beh et al (2016), we used a rabbit anti-CENP-C polyclonal antibody and a goat anti-rabbit FITC-conjugate antibody to label functional centromeres (Beh et al 2016). CENP-A and CENP-C were reported to localize exclusively to active centromeres, as part of the constitutive centromere-associated network (Klare et al 2015;Shono et al 2015;Beh et al 2016).…”

“…As recommended by Beh et al (2016), we used a rabbit anti-CENP-C polyclonal antibody and a goat anti-rabbit FITC-conjugate antibody to label functional centromeres (Beh et al 2016). CENP-A and CENP-C were reported to localize exclusively to active centromeres, as part of the constitutive centromere-associated network (Klare et al 2015;Shono et al 2015;Beh et al 2016). Alphoid and BAC probes spanning candidate neocentromeric regions were selected and labeled as reported (Trombetta et al 2012;Macchia et al 2015) (Table S1).…”