Dikovskaya D et al. (2012), The adenomatous polyposis coli protein contribu...

XB-ART-45454

PLoS One 2012 Jan 01;76:e38102. doi: 10.1371/journal.pone.0038102.

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The adenomatous polyposis coli protein contributes to normal compaction of mitotic chromatin.

Dikovskaya D , Khoudoli G , Newton IP , Chadha GS , Klotz D , Visvanathan A , Lamond A , Swedlow JR , Näthke IS .

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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.

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Species referenced: Xenopus
Genes referenced: h2ac21 h2bc21 rbbp7 set smc2

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	Figure 1. APC depletion changes chromatin appearance in Xenopus egg extract.Demembranated sperm chromatin was incubated with CSF extract in the presence of a small amount of Rhodamine-labelled tubulin for 1 h, overlayed with DAPI-containing fixative and imaged. A. Two representative examples of âstrongâ spindles formed in such extract, with a dense microtubule network and well-resolved, mitotically condensed chromatin. B. Two representative examples of âweakâ spindles formed in CSF extract with low microtubule content and abnormally compacted chromatin. C. Typical morphology of chromatin classified here as normal (left panel), rod-like (middle panel) and compacted (right panel) used for the quantification shown in D-F. D. Proportion of phenotypically âweakâ spindles formed around chromatin with normal, rod-like and compacted morphology was determined in three different preparations from the same extract. Between 62 and 85 spindles per slide were counted. E. Quantification of chromatin morphology by categories depicted in C, after incubation in untreated (control), mock- or APC-depleted extracts for 1 h. 80â165 chromatin figures were scored for each condition. F. Quantification of different chromatin morphologies according to categories depicted in C, after incubation in mock- or APC-depleted extracts for 1 h, followed by exposure to +12Â°C. 70â90 chromatin figures were scored for each condition. G. Level of APC depletion in CSF extract visualized by immunoblotting mock- and APC-depleted extracts with anti-APC antibody. Tubulin was used as a loading control. H. Representative images of demembranated sperm chromatin after incubation in ULSS extract at 23.5Â°C for 50 min, classified as normal, rod-like and compacted for quantification in I. Sum intensity projections of deconvolved z-stack images capturing the total chromatin fluorescence are shown. Size bar 15 Âµm. I. Percent of normal, rod-like and compacted chromatin (as depicted in H) detected after incubation in mock- or APC-depleted ULSS extract after 40 (ULSS 1) or 50 (ULSS 2) min. Data from two independent experiments are shown. 74â133 chromatin figures were scored for each condition.
	Figure 2. APC depletion increases chromatin compaction in HeLa cells.Asynchronously growing HeLa cells stably expressing GFP-H2B and mCherry-H2B were depleted of APC using siRNA (siAPC) or transfected with non-targeting siRNA (siContr), and donor fluorescence lifetime was measured using time-correlated single photon counting technique. A. Normalised lifetime values for APC- (grey bars) and mock- (white bars) depleted interphase cells (left) and mitotic cells (right). P-values are given underneath the plots. Please note that APC-depleted mitotic cells display significantly shorter FRET lifetime, indicating a higher degree of chromatin compaction. At least ten cells were measured for each condition. B. Level of APC depletion of cells used in A is visualized by immunobloting the corresponding lysates with anti-APC antibodies. Tubulin is used as a loading control. C. Representative chromatin images from control (left) and APC-depleted (right) mitotic cells. FRET efficiency is shown in false colors, with blue corresponding to low FRET efficiency (i.e. less compaction), and red corresponding to high FRET efficiency (i.e. more compaction).
	Figure 3. Experimental layout for proteomic analysis of chromatin-associated proteins in APC-depleted and mock-depleted Xenopus extract.A. Schematic representation of extract preparation. CSF (crude) extract obtained from Xenopus eggs was spun as in Methods, and a clear golden fraction was collected (ULSS extract). It was then incubated for 1 h with Dynabeads loaded either with affinity purified APC-specific antibody or non-specific rabbit IgG in the same concentration. Antibody-bound Dynabeads were magnetically retrieved, and the depleted extracts were used in the experiment. B. Schematic representation of chromatin manipulation in the experiment. Demembranated chromatin was added to the extract at concentration 20 ng per 1 Âµl of extract, mixed and incubated for indicated length of time (as in D.) before isolation by centrifugation through 30% sucrose cushion. After several washes chromatin was trypsinised and processed for mass spectrometry as described in Materials and Methods. C. Level of APC depletion in three extract preparations used in proteomics screen visualized by immunoblotting with anti-APC antibodies (APCII) and anti-tubulin antibodies as a loading control. 1 Âµl of corresponding extract were loaded into each lane and separated on PAGE prior immunoblotting. D. Experimental layout. In the 1st set (left), mock- and APC-depleted 1st ULSS extract were each separated into two halves and analysed as two technical replicas (exp. 1 and exp 2). Chromatin incubated for 5, 20, 40 or 60 min in these extracts was isolated as in B., and analysed by mass-spectrometry twice for each point (r.1 and r.2). In the 2nd set, two independently derived and immunodepleted extracts (2nd and 3rd ULSS) were used in two identically performed experiments. Within each experiment, both APC and mock-depleted extract were separated into two halves (repl.1 and repl.2) and each was incubated with chromatin for 10, 20, 30, 40, 50 or 60 min. Chromatin associated peptides were measured with two different settings for each point, producing one extended measurement. (r.1, 2).
	Figure 4. Temporal chromatin association profiles of proteins identified in proteomic screen in APC-depleted and control extracts.Data from 231 protein IDâs that display similar dynamics in all eight analyses (in three independent extracts, see Fig. 3 D) were clustered using TreeView, and displayed as a heat map. Green, black and red represent low, intermediate and high signal, respectively. An enlarged insert depicts a cluster of proteins with dynamics that vary significantly after APC depletion.
	Figure 5. Effect of APC depletion on known mediators of chromatin compaction.A. Dynamics of chromatin association of indicated components of condensin I and II complexes, Topoisomerase IIa, Kif4 and two linker histones in mock-and APC-depleted extract, as determined in the proteomic screen is represented in a heat map, color-coded as in fig. 4. B. Average values from all histones identified in the screen (H2B, H2A, H2A.ZI2, H1C, hist1H4I and B4) normalized to corresponding total signal in mock-depleted samples. C. Immunoblotting of the chromatin samples prepared as for the proteomic screen, with antibodies recognizing Xenopus TopoII (top panel) and Smc2 (bottom panel). Each lane contains the equivalent of 200 ng chromatin.
	Figure 6. Effect of APC depletion on association of Set-a and Rbbp7 with chromatin.Immunoblotting of chromatin samples prepared as for proteomic screen, with antibodies against Set-a (A) and Rbbp7 (B), as well as the Coomassie staining of the bottom portions of the gel used as a loading control. Each lane contains the equivalent of 200 ng chromatin.

References [+] :

Adachi, Chromosome assembly in vitro: topoisomerase II is required for condensation. 1991, Pubmed, Xenbase