Specification of kinetochore-forming chromatin by the histone H3 variant CENP-A

Abstract

The mechanisms that specify precisely where mammalian kinetochores form within arrays of centromeric heterochromatin remain largely unknown. Localization of CENP-A exclusively beneath kinetochore plates suggests that this distinctive histone might direct kinetochore formation by altering the structure of heterochromatin within a sub-region of the centromere. To test this hypothesis, we experimentally mistargeted CENP-A to non-centromeric regions of chromatin and determined whether other centromere-kinetochore components were recruited. CENP-A-containing non-centromeric chromatin assembles a subset of centromere-kinetochore components, including CENP-C, hSMC1, and HZwint-1 by a mechanism that requires the unique CENP-A N-terminal tail. The sequence-specific DNA-binding protein CENP-B and the microtubule-associated proteins CENP-E and HZW10 were not recruited, and neocentromeric activity was not detected. Experimental mistargeting of CENP-A to inactive centromeres or to acentric double-minute chromosomes was also not sufficient to assemble complete kinetochore activity. The recruitment of centromere-kinetochore proteins to chromatin appears to be a unique function of CENP-A, as the mistargeting of other components was not sufficient for assembly of the same complex. Our results indicate at least two distinct steps in kinetochore assembly: (1) precise targeting of CENP-A, which is sufficient to assemble components of a centromere-prekinetochore scaffold; and (2) targeting of kinetochore microtubule-associated proteins by an additional mechanism present only at active centromeres.

Flavors of Non-Random Meiotic Segregation of Autosomes and Sex Chromosomes

“…Minor satellite DNA is the prominent site for CENP-A and CENP-B assembly [4,11]. However, if the minor satellite region is not big enough, CENP-A can also be formed de novo elsewhere and its localization is not strictly sequence dependent [12][13][14][15]. CENP-B is a minor satellite DNA binding protein.…”

Recent insights into mechanisms preventing ectopic centromere formation

“…The ectopic CENP-A CID chromatin can self-propagate even without lacO/LacI system [43]. Mislocalized CENP-A in fission yeast and humans also can recruit kinetochore components [34,[44][45][46]. Neocentromeres that form at noncentromeric chromatin also always contain CENP-A.…”

CENP-A Regulation and Cancer

“…Thus, consistently, when HJURP is overexpressed, the increased levels of CENP-A that ensue result in an exclusive accumulation at the centromere, without ectopic localization ( Filipescu et al, 2017 ). Strikingly though, exogenous overexpression or abnormal upregulation of endogenous CENP-A leads to its mislocalization at non-centromeric regions ( Van Hooser et al, 2001 ; Gascoigne et al, 2011 ; Lacoste et al, 2014 ; Athwal et al, 2015 ) ( Figure 3 ). This is observed for overexpression of CENP-A either constitutive ( Lacoste et al, 2014 ; Athwal et al, 2015 ; Shrestha et al, 2017 , 2021 ) or inducible ( Van Hooser et al, 2001 ; Gascoigne et al, 2011 ; Shrestha et al, 2017 , 2021 ; Jeffery et al, 2021 ).…”