Abstract
The tumour suppressor Adenomatous Polyposis Coli (APC) is required for proper mitosis; however, the exact role of APC in mitosis is not understood. Using demembranated sperm chromatin exposed to meiotic Xenopus egg extract and HeLa cells expressing fluorescently labelled histones, we established that APC contributes to chromatin compaction. Sperm chromatin in APC-depleted Xenopus egg extract frequently formed tight round or elongated structures. Such abnormally compacted chromatin predominantly formed spindles with low microtubule content. Furthermore, in mitotic HeLa cells expressing GFP- and mCherry-labelled H2B histones, depletion of APC caused a decrease in the donor fluorescence lifetime of neighbouring fluorophores, indicative of excessive chromatin compaction. Profiling the chromatin-associated proteome of sperm chromatin incubated with Xenopus egg extracts revealed temporal APC-dependent changes in the abundance of histones, closely mirrored by chromatin-associated Topoisomerase IIa, condensin I complex and Kif4. In the absence of APC these factors initially accumulated on chromatin, but then decreased faster than in controls. We also found and validated significant APC-dependent changes in chromatin modifiers Set-a and Rbbp7. Both were decreased on chromatin in APC-depleted extract; in addition, the kinetics of association of Set-a with chromatin was altered in the absence of APC.
Highlights
The tumor suppressor Adenomatous Polyposis Coli (APC) is a large multifunctional multi-domain protein that regulates betacatenin turnover and organizes the microtubule cytoskeleton [1]
We previously established a requirement for APC in spindle formation in Xenopus egg extracts arrested at metaphase of meiosis II (CSF extracts)
APC depletion, or overexpression of a truncated APC in various models induces a plethora of mitotic defects ranging from centrosomal dysfunction, spindle destabilization, abnormally oriented kinetochores, cytokinetic failure to compromised spindle assembly checkpoint resulting in chromosome segregation errors [19] [9,10] [8] [7] [34,35]
Summary
The tumor suppressor Adenomatous Polyposis Coli (APC) is a large multifunctional multi-domain protein that regulates betacatenin turnover and organizes the microtubule cytoskeleton [1]. Intestinal crypt cells lacking functional APC are hyperproliferative and display differentiation and migration defects [2,3]. Loss of APC correlates with chromosomal instability (CIN) in cancers and induces CIN in cultured cells and mouse models [4]. While some of these phenotypes are mediated by the enhanced beta-catenin signaling that results from the absence of wild type APC [5,6], loss of beta-catenin–independent function(s) of APC contributes to defective mitoses responsible for CIN [7,8,9]. The ability of APC to crosslink and stabilise microtubules during spindle formation [10] explains some mitotic defects that occur in the absence of APC
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