Effect of APC depletion on association of Set-a and Rbbp7 with chromatin.

<p>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.</p

APC depletion increases chromatin compaction in HeLa cells.

<p>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. <b><i>A</i></b><i>.</i> 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><i>B</i></b><i>.</i> 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. <b><i>C</i></b><i>.</i> 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).</p

APC depletion changes chromatin appearance in <i>Xenopus</i> egg extract.

<p>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. <b><i>A</i></b><i>.</i> Two representative examples of “strong” spindles formed in such extract, with a dense microtubule network and well-resolved, mitotically condensed chromatin. <b><i>B</i></b><i>.</i> Two representative examples of “weak” spindles formed in CSF extract with low microtubule content and abnormally compacted chromatin. <b><i>C</i></b><i>.</i> 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. <b><i>D</i></b><i>.</i> Proportion of phenotypically “weak” 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. <b><i>E</i></b><i>.</i> Quantification of chromatin morphology by categories depicted in C, after incubation in untreated (control), mock- or APC-depleted extracts for 1 h. 80–165 chromatin figures were scored for each condition. <b><i>F</i></b><i>.</i> 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 +12°C. 70–90 chromatin figures were scored for each condition. <b><i>G</i></b><i>.</i> 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. <b><i>H</i></b><i>.</i> Representative images of demembranated sperm chromatin after incubation in ULSS extract at 23.5°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 µm. <b><i>I</i></b><i>.</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. 74–133 chromatin figures were scored for each condition.</p

Temporal chromatin association profiles of proteins identified in proteomic screen in APC-depleted and control extracts.

<p>Data from 231 protein ID’s that display similar dynamics in all eight analyses (in three independent extracts, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038102#pone-0038102-g003" target="_blank">Fig. 3</a> 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.</p

Proteins reduced on chromatin in the absence of APC, but with unchanged dynamics of chromatin association.

<p>Data were processed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038102#pone-0038102-t002" target="_blank">table 2</a>. The fold change decrease (“fc down”) was calculated as a ratio between control and APC-depleted total values calculated as above; the fold change down for individual sets, calculated from raw data are also given. Only data with a reproducible decrease in the total signal in the absence of APC are presented. The positive Pearson coefficient (P) (P>0.3) given in the last row confirms similarity in dynamics of these proteins between APC-deficient and control samples.</p

Proteins elevated on chromatin in the absence of APC, but with unchanged dynamics of chromatin association.

<p>The total amount of the signal for each protein ID in control or APC-depleted sample was calculated as a sum of the values in all time points from the smoothened profile, and averaged between the two sets. These values were further normalized to the total signal from all protein IDs in either control or APC-depleted samples in this dataset. The fold change increase (“fc up”) was calculated as a ratio between APC-depleted and control total values calculated as above. For individual sets, the fold change increase (“fc up set 1” and “fc up set 2”) was calculated from the raw data (not smoothened and not normalized to the total signal in the whole sample). Only data with a reproducible increase in the total signal in the absence of APC are presented. The positive Pearson coefficient (P) (P>0.3) given in the last row confirms similarity in dynamics of these proteins between APC-deficient and control samples. The maximum number of peptides detected in mock-depleted extract (set I), APC-depleted extract (set I)/mock-depleted extract (set II), APC-depleted extract (set II) are provided.</p

Effect of APC depletion on known mediators of chromatin compaction.

<p><b><i>A.</i></b> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038102#pone-0038102-g004" target="_blank">fig. 4</a>. <b><i>B</i></b><i>.</i> 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. <b><i>C</i></b><i>.</i> Immunoblotting of the chromatin samples prepared as for the proteomic screen, with antibodies recognizing <i>Xenopus</i> TopoII (top panel) and Smc2 (bottom panel). Each lane contains the equivalent of 200 ng chromatin.</p

The adenomatous polyposis Coli protein contributes to normal compaction of mitotic chromatin

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