Khoudoli GA et al. (2008), Temporal profiling of the chromatin proteome re...

XB-ART-37873

Curr Biol 2008 Jun 03;1811:838-43. doi: 10.1016/j.cub.2008.04.075.

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Temporal profiling of the chromatin proteome reveals system-wide responses to replication inhibition.

Khoudoli GA , Gillespie PJ , Stewart G , Andersen JS , Swedlow JR , Blow JJ .

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Although the replication, expression, and maintenance of DNA are well-studied processes, the way that they are coordinated is poorly understood. Here, we report an analysis of the changing association of proteins with chromatin (the chromatin proteome) during progression through interphase of the cell cycle. Sperm nuclei were incubated in Xenopus egg extracts, and chromatin-associated proteins were analyzed by mass spectrometry at different times. Approximately 75% of the proteins varied in abundance on chromatin by more than 15%, suggesting that the chromatin proteome is highly dynamic. Proteins were then assigned to one of 12 different clusters on the basis of their pattern of chromatin association. Each cluster contained functional groups of proteins involved in different nuclear processes related to progression through interphase. We also blocked DNA replication by inhibiting either replication licensing or S phase CDK activity. This revealed an unexpectedly broad system-wide effect on the chromatin proteome, indicating that the response to replication inhibition extends to many other functional modules in addition to the replication machinery. Several proteins that respond to replication inhibition (including nuclear pore proteins) coprecipitated with the Mcm2-7 licensing complex on chromatin, suggesting that Mcm2-7 play a central role in coordinating nuclear structure with DNA replication.

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Species referenced: Xenopus
Genes referenced: gmnn mcm2 ruvbl1

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	Figure 1. Proteomic Data Acquisition, Manipulation, and Verification(A) Sperm chromatin was incubated in egg extract and the efficiency of DNA replication was monitored by [α32P] dATP incorporation. The mean size of approximately 20 randomly selected nuclei at each time point is also shown.(B) Cartoon of cell-cycle events occurring during incubation of sperm nuclei in Xenopus egg extract, showing the approximate timing of mitotic exit, origin licensing (Mcm2–7 loading), nuclear assembly, and DNA replication.(C) Chromatin was isolated in parallel to the mass-spectrometry samples and immunoblotted for the indicated proteins. Below are corresponding heat maps derived from mass spectrometry (red highest, green lowest, black intermediate). The total number of peptides identified for each protein across the entire time course is also shown.
	Figure 2. FCM Clustering and Functional Annotation Analysis of Temporal Profiles of Polypeptides Associated with Replicating ChromatinFCM clustering results for 458 polypeptides identified on replicating chromatin that demonstrated more than 15% variation in abundance during interphase are presented as the heat map. The color coding is as follows: green, 0.0; black, 0.5; and red, 1.0. Reproducibility level (column Rp) is defined as a percentage of cases where at least two individual profiles we present in a cluster together with corresponding averaged one. Functional annotation analysis of FCM clusters is demonstrated next to corresponding FCM cluster. The number of proteins separated into each FCM cluster is indicated in brackets. “FG” indicates functional groups identified by DAVID; “GES” indicates group enrichment score.
	Figure 3. System-wide Effect of Replication Inhibitors on the Chromatin ProteomeSperm nuclei were incubated in either untreated extracts or extracts supplemented with geminin or roscovitine.(A and B) Chromatin proteins were identified and sorted into the 12 FCM clusters. For each protein, the cluster membership was compared between the untreated sample and in the presence of either geminin (A) or roscovitine (B). Column Rp presents reproducibility level. Proteins falling along the highlighted diagonal did not change cluster membership on addition of the inhibitor. Proteins that were present in the replicating chromatin but were not detected on chromatin after addition of inhibitors are recorded in the “lost” column.(C) Effect of replication inhibitors on nuclear size. Error bars indicate the standard deviation (SD).
	Figure 4. Hierarchical Clustering of the Combined Data Set(A–E) The combined data set was constructed by uniting data from three experimental conditions (inhibitor free, geminin, and roscovitine) for each protein. Selected groups of highly correlated proteins are shown.(F) Chromatin was isolated in parallel to the mass-spectrometry samples and immunoblotted for DUF87 and RuvBL1. The color coding is as follows: green, 0.0; black, 0.5; and red, 1.0.

References [+] :

Andersen, Proteomic characterization of the human centrosome by protein correlation profiling. 2003, Pubmed