Abstract
The kinetochore (centromeric DNA and associated proteins) is a key determinant for high fidelity chromosome transmission. Evolutionarily conserved Scm3p is an essential component of centromeric chromatin and is required for assembly and function of kinetochores in humans, fission yeast, and budding yeast. Overexpression of HJURP, the mammalian homolog of budding yeast Scm3p, has been observed in lung and breast cancers and is associated with poor prognosis; however, the physiological relevance of these observations is not well understood. We overexpressed SCM3 and HJURP in Saccharomyces cerevisiae and HJURP in human cells and defined domains within Scm3p that mediate its chromosome loss phenotype. Our results showed that the overexpression of SCM3 (GALSCM3) or HJURP (GALHJURP) caused chromosome loss in a wild-type yeast strain, and overexpression of HJURP led to mitotic defects in human cells. GALSCM3 resulted in reduced viability in kinetochore mutants, premature separation of sister chromatids, and reduction in Cse4p and histone H4 at centromeres. Overexpression of CSE4 or histone H4 suppressed chromosome loss and restored levels of Cse4p at centromeres in GALSCM3 strains. Using mutant alleles of scm3, we identified a domain in the N-terminus of Scm3p that mediates its interaction with CEN DNA and determined that the chromosome loss phenotype of GALSCM3 is due to centromeric association of Scm3p devoid of Cse4p/H4. Furthermore, we determined that similar to other systems the centromeric association of Scm3p is cell cycle regulated. Our results show that altered stoichiometry of Scm3p/HJURP, Cse4p, and histone H4 lead to defects in chromosome segregation. We conclude that stringent regulation of HJURP and SCM3 expression are critical for genome stability.
Highlights
Proper chromosome segregation is essential for normal cell proliferation
We determined that altered dosage of HJURP and its budding yeast homolog SCM3 leads to defects in chromosome segregation in yeast and human cells
We identified the centromeric DNA–interacting domain of Scm3p and determined that association of Scm3p devoid of Cse4p leads to chromosome segregation defects
Summary
Proper chromosome segregation is essential for normal cell proliferation. Errors in this process lead to birth defects, developmental disorders, aneuploidy and possibly cancer [1]. Budding yeast contains ‘‘point’’ centromeres, whereas other eukaryotes, for example fission yeast, fruit fly, plants and mammals have ‘‘regional’’ centromeres. Despite CEN DNA sequence variation, replacement of histone H3 in centromeric chromatin by the centromere-specific histone H3 variant CenH3 is universally conserved [6]. CenH3 homologs (Cse4p in budding yeast, Cnp in fission yeast, CID in fruit fly, HTR12 in Arabidopsis and CENP-A in humans) function as an epigenetic mark in all organisms and is essential for determining centromere identity and proper kinetochore function [8,9,10,11,12,13,14]. Kinetochore sub-complexes Ctf3p (Ctf3p, Mcm16p and Mcm22p) and COMA (Ame1p, Ctf19p, Mcm21p and Okp1p) exhibit genetic and physical interactions with Cse4p [15,16]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
