Undergraduate Thesis Or Project
 

A role for CENP-S,T,W,X at Neurospora crassa centromeres_Mu Feng.pptx

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  • During mitosis, sister chromatids are separated by the action of the mitotic spindle. The mitotic spindle attaches to chromosomes at centromeres through a protein complex called the kinetochore. While ~100 proteins have been identified at kinetochores and centromeres, the function of most is poorly understood. The centromere protein, CENP-T (Centromere Protein T), is conserved throughout eukaryotes (1) where it plays a key role in linking centromeric DNA to the kinetochore (2, 3). This likely occurs through the concerted action of an N-terminal alpha-helix and a C-terminal histone fold domain, which are linked by a central region devoid of predicted structure . The N-terminal helix interacts with the NDC80 complex in a phospho-dependant manner (3), while the C-terminal histone fold associates with CENP-W, a small protein consisting almost entirely of a histone fold domain. Furthermore, the CENP-T/CENP-W dimer forms heterotetramers with two additional histone fold-containing proteins, CENP-S and CENP-X, in vitro (3). These heterotetramers are reminiscent of nucleosomes and bind DNA in vitro. This has led to a compelling model in which CENP-T plays a key role in bridging centromeric DNA, the kinetochore, and ultimately the mitotic spindle. We use Neurospora crassa as a platform for the investigation of these proteins. The N. crassa orthologs, CEN-T (NCU02161), -W (NCU03400), -S (NCU03629) and -X (NCU09478) maintain the domain structure of their human counterparts . GFP-tagged CEN-T co-localizes with mCherry-tagged CenH3 at N. crassa centromeres and ChIP-sequencing confirms its centromeric localization. We were unable to isolate strains in which cen-t or cen-w were deleted, suggesting that these proteins are essential for life. In contrast, strains lacking cen-s or cen-x appear normal, calling into question the importance of a nucleosome-like tetramer in N. crassa. Our immediate goals are: (i) to determine if the CEN-T-W-S-X tetramer exists in N. crassa and what aspects of it are essential for chromosome segregation; (ii) to understand how CEN-T and the CEN-T-W-S-X tetramer interact with centromeric DNA; and (iii) to understand the chromatin context of CEN-T and the CEN-T-W-S-X tetramer with a focus on what types of nucleosomes are immediately adjacent.
  • Keywords: Neurospora, centromere
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