Volume 49, Issue 4, Pages 759-771 (February 2013) A Network Model of the Molecular Organization of Chromatin in Drosophila  Joke G. van Bemmel, Guillaume J. Filion, Arantxa Rosado, Wendy Talhout, Marcel de Haas, Tibor van Welsem, Fred van Leeuwen, Bas van Steensel  Molecular Cell  Volume 49, Issue 4, Pages 759-771 (February 2013) DOI: 10.1016/j.molcel.2013.01.040 Copyright © 2013 Elsevier Inc. Terms and Conditions

Molecular Cell 2013 49, 759-771DOI: (10.1016/j.molcel.2013.01.040) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 Identification of 42 Previously Unknown (Set N) Chromatin Proteins by Generation of DamID Binding Profiles (A) Overview of the testing of 112 candidate proteins by DamID. Pie charts show the representation of the different selection criteria for the initial selected candidate proteins, for proteins which exhibit detectable DNA methylation (DamID products) when fused to Dam and for proteins which yielded a specific and reproducible binding profile. (B) DamID binding profiles of the 42 Set N chromatin proteins along a 2 Mb region on chromosome 2L. y axis depicts log2 enrichment of Dam-fusion over Dam-only control; positive values are plotted in black and negative values in gray for contrast. Rows were arranged by genome-wide hierarchical clustering. Below the profiles, genes on both strands are depicted as lines with blocks indicating exons. Colored domains represent the previously identified five principal chromatin types (Filion et al., 2010). (See also Figure S1.) Molecular Cell 2013 49, 759-771DOI: (10.1016/j.molcel.2013.01.040) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 Bayesian Network Model of the Interactions among 112 Chromatin Components (A) Nodes represent chromatin components. Dark gray, newly identified chromatin proteins; light gray, known chromatin proteins; gray border, histone marks. Solid lines represent predicted interactions with a confidence score of at least 70%. Proteins for which all confidence scores are below 70% are tentatively connected to the network via the edge that has the highest score (dotted lines). Confidence scores are listed in Table S3. (B–D) Agreement of the Bayesian network model with published data. Analysis was restricted to the 65 previously known chromatin proteins. Bar graphs show the proportion of protein pairs that are directly linked (black) or not directly linked (gray) in BN70 for (B) protein pairs co-occurring in at least two publications in the FlyBase literature list; (C) protein pairs with physical interactions according to the DroID database, and (D) protein pairs with genetic interactions of the corresponding genes according to BioGRID. White bars show the degree of overlap after random scrambling of edges or nodes in the network. P values are according to Fisher’s exact test (gray versus black bars) or estimated from the number of random permutations that yielded at least the same degree of overlap as the unscrambled set (white versus black bars; 100,000 permutations). (See also Figure S2.) Molecular Cell 2013 49, 759-771DOI: (10.1016/j.molcel.2013.01.040) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 TRIP1 Is Essential for S Phase Entry (A) BN70 connectivity of replication machinery components MUS209 and ASF1, and the Set N components TRIP1 and CC3. (B) Knockdown efficiency determined by quantitative RT-PCR analysis of TRIP1 and CC3 mRNA expression levels, after treatment with dsRNA fragments against the control gene white (gray), Trip1 (green), and CC3 (blue) with two independent dsRNA fragments each (#1 and #2). y axis depicts average mRNA level of three replicates relative to the control knockdown, with error bars depicting the standard deviation. (C) Cell-cycle profile after control knockdown (gray histogram), TRIP1 knockdown (green lines), and CC3 knockdown (blue lines). For each sample ∼50,000 cells were counted. x axis depicts DNA content determined by propidium iodide (PI) labeling and the y axis the averaged relative frequency of two replicate experiments. (D) EdU incorporation after TRIP1 knockdown (Fischer’s Exact test: ∗ indicates p < 0.05). Colored bars represent knockdowns as in (A). y axis depicts average percentage of nucleotide incorporating cells of two replicates, with error bars depicting the standard deviation. For each treatment five fields (about 200–1000 cells) were scored. (E and F) Percentage of TRIP1, MUS209, and ASF1 binding sites (gray) that overlap with ORC2 peaks (E) or with early origin regions (F) as mapped by (Eaton et al., 2011). White bars depict overlap expected by random chance (mean ± SD, based on 1000 circular permutations; ∗ indicates p < 0.001). Molecular Cell 2013 49, 759-771DOI: (10.1016/j.molcel.2013.01.040) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 Set N Proteins in the Principal Chromatin Types (A) Principal component analysis of the newly generated binding data of the 42 proteins in Set N and the 12 known chromatin proteins. Figure shows a three-dimensional projection of the first three principal components. Each dot represents a probed locus and is colored according to the previously identified chromatin types (Filion et al., 2010). Separation of the colors in five distinct groups confirms the previous classification into five known chromatin types. (B–F) Occupancy of all mapped chromatin components within each of the five principal chromatin types, projected onto BN70 as color scales. A value of 1 on the color scale means that 100% of the genomic loci in this chromatin type are bound by the protein represented by the node. (See also Figure S3.) Molecular Cell 2013 49, 759-771DOI: (10.1016/j.molcel.2013.01.040) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 5 HP1 Is Required for Recruitment of CC26 to Pericentric Heterochromatin (A) Cluster of GREEN chromatin including five Set N proteins (gray border). Green color scale represents the occupancy of each protein within GREEN chromatin as in Figure 4B. (B) Microscopy images of Kc167 cells transfected with GFP tagged CC26 and stained with anti-HP1 antibody and DAPI, after knockdown of white (negative control) (top panel) or of HP1 (bottom panel). White lines indicate the nuclear rim (based on DAPI signal). (C) Percentage of cells with chromocenter enrichment of GFP-CC26; ∗ indicates p < 0.05 according to Fischer’s Exact test; n, numbers of cells scored. Qualitative analysis of an independent replicate experiment showed the same altered localization pattern of CC26 after HP1 knockdown (data not shown). (D) No detectable loss of chromocenter localization of GFP-CC4 upon HP1 knockdown. (E and F) Changes in CC4 (E) and CC26 (F) DamID patterns after HP1 knockdown relative to white control knockdown. Distribution of changes is shown for loci in GREEN chromatin (green) and all other genomic regions (black). In (F) GREEN chromatin regions are further divided in pericentric (dashed) and nonpericentric regions (dotted). (See also Figure S4.) Molecular Cell 2013 49, 759-771DOI: (10.1016/j.molcel.2013.01.040) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 6 Protein-Protein Interactions in GREEN and BLACK Chromatin (A–F) In (A) and (D), BN70 connectivity of GREEN and BLACK chromatin clusters is shown. In (B), (C), (E), and (F), Y2H assays for CC26 and its candidate partners in GREEN chromatin (B and C) and SUUR and partners in BLACK chromatin (E and F) are shown. Ten-fold dilution series of yeast strain PJ69-4a expressing indicated chromatin components fused to the GAL4 DNA-binding domain (DBD) or activating domain (AD) were grown under selective conditions (-his), where interaction of the tested proteins is required for growth. In addition, the color of the colonies reflects the expression of an independent reporter gene (ADE2): the weaker the red staining, the stronger the interaction between the tested proteins. In (C) and (F), semiquantitative analysis of colony outgrowth, under -his selection (gray bars) or under nonselective conditions, where interaction of the tested proteins is not required for growth (white bars), is shown. Error bars depict range of growth scores (n = 3); when no error bars are shown n = 1. (See also Figure S5.) Molecular Cell 2013 49, 759-771DOI: (10.1016/j.molcel.2013.01.040) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 7 Functional Compartmentalization of BN70 (A) Average mRNA expression levels of the target genes of each protein in BN70 plotted as a color scale. (B) BN70 highlighting predicted activators (red), putative repressors (green), nonsignificant proteins (dark gray), and histone marks (light gray) (p < 0.0001). (See also Figure S6). (D–H) Six examples illustrating the enrichment of GO categories among the target genes of proteins in BN70. Proteins with their target genes significantly enriched for the indicated GO category are depicted in red. A complete overview of all significant GO categories is shown in Figure S7. Molecular Cell 2013 49, 759-771DOI: (10.1016/j.molcel.2013.01.040) Copyright © 2013 Elsevier Inc. Terms and Conditions