ISWI Remodeling Complexes inXenopusEgg Extracts: Identification as Major Chromosomal Components that Are Regulated by INCENP-aurora B

Abstract

We previously characterized major components of mitotic chromosomes assembled in Xenopus laevis egg extracts and collectively referred to them as Xenopus chromosome-associated polypeptides (XCAPs). They included five subunits of the condensin complex essential for chromosome condensation. In an effort to identify novel proteins involved in this process, we have isolated XCAP-F and found it to be the Xenopus ortholog of ISWI, a chromatin remodeling ATPase. ISWI exists in two major complexes in Xenopus egg extracts. The first complex contains ACF1 and two low-molecular-weight subunits, most likely corresponding to Xenopus CHRAC. The second complex is a novel one that contains the Xenopus ortholog of the human Williams syndrome transcription factor (WSTF). In the absence of the ISWI complexes, the deposition of histones onto DNA is apparently normal, but the spacing of nucleosomes is greatly disturbed. Despite the poor spacing of nucleosomes, ISWI depletion has little effect on DNA replication, chromosome condensation or sister chromatid cohesion in the cell-free extracts. The association of ISWI with chromatin is cell cycle regulated and is under the control of the INCENP-aurora B kinase complex that phosphorylates histone H3 during mitosis. Apparently contradictory to the generally accepted model, we find that neither chromosome condensation nor chromosomal targeting of condensin is compromised when H3 phosphorylation is drastically reduced by depletion of INCENP-aurora B.

Molecular analysis of mitotic chromosome condensation using a quantitative time-resolved fluorescence microscopy assay

“…During mitotic entry, the mitotic kinase Aurora B phosphorylates histone H3 (30) and promotes removal of cohesin and remodeling complexes from chromatin (31,32). However, the role of Aurora B in chromosome condensation is less clear.…”

Shugoshin regulates cohesion by driving relocalization of PP2A in Xenopus extracts

“…Rabbit polyclonal sera against Xenopus CENP-A were obtained using a synthetic peptide as immunogen (MRPGSTPPSRRKSRPPRRVC). Other antibodies used in this study were as follows: anti-SMC1, anti-SMC3, and anti-Scc1 (Losada et al 1998); anti-SA1 (Losada et al 2000); anti-XCAP-G (Hirano et al 1997); anti-XINCENP and anti-XAurora B (MacCallum et al 2002); and anti-Plx1 . A mouse monoclonal antibody against the catalytic subunit of PP2A (clone 1D6) was purchased from Upstate.…”

Micromanipulation Studies of Chromatin Fibers in Xenopus Egg Extracts Reveal ATP-dependent Chromatin Assembly Dynamics

“…Other ATPases that might be responsible for the ⌳ and V events include minichromosome maintenance complexes, which are loaded onto chromatin in Xenopus egg extracts when ATP is present, and chromatin-remodeling factors, which are again known to participate in assembly of chromatin in Xenopus egg extracts (MacCallum et al, 2002). The role played by these proteins in the ⌳ and V events could be probed by their immunodepletion from the extracts, by using methods such as those recently used to study the role of linker histone (Maresca et al, 2005).…”

“…We have carried out experiments using ϩATP assembly reactions in the presence of the DNA polymerase inhibitor aphidicolin as well as function-blocking antibodies to condensin subunits, and in both cases we observed ⌳ and V events, indicating that this activity is not due to either DNA polymerase or condensin function. In addition, we have included the topo II inhibitor ICRF-193 in ϩATP reactions, and we have observed the ⌳ and V events, indicating that topo II is not the enzyme responsible for them.Other ATPases that might be responsible for the ⌳ and V events include minichromosome maintenance complexes, which are loaded onto chromatin in Xenopus egg extracts when ATP is present, and chromatin-remodeling factors, which are again known to participate in assembly of chromatin in Xenopus egg extracts (MacCallum et al, 2002). The role played by these proteins in the ⌳ and V events could be probed by their immunodepletion from the extracts, by using methods such as those recently used to study the role of linker histone (Maresca et al, 2005).…”

“…These extracts contain a mixture of enzymes competent to assemble not only nucleosomes but also active chromatin. Despite the high complexity of extract reactions, drugs and antibodies have been used to identify and study the contribution of specific proteins to chromatin structure and function (Dilworth et al, 1987;Kleinschmidt et al, 1990;Hirano and Mitchison, 1994;MacCallum et al, 2002;Maresca et al, 2005). Extracts diluted from 2.5-to 400-fold have been used to assemble chromatin onto single DNAs against low piconewton forces (Ladoux et al, 2000;Wagner et al, 2005).…”