Analysis of the human endogenous coregulator complexome

Abstract

Elucidation of endogenous cellular protein-protein interactions and their networks is most desirable for biological studies. Here we report our study of endogenous human coregulator protein complex networks obtained from integrative mass spectrometry-based analysis of 3290 affinity purifications. By preserving weak protein interactions during complex isolation and utilizing high levels of reciprocity in the large dataset, we identified many unreported protein associations, such as a transcriptional network formed by ZMYND8, ZNF687, and ZNF592. Furthermore, our work revealed a tiered interplay within networks that share common proteins, providing a conceptual organization of a cellular proteome composed of minimal endogenous modules (MEMOs), complex isoforms (uniCOREs), and regulatory complex-complex interaction networks (CCIs). This resource will effectively fill a void in linking correlative genomic studies with an understanding of transcriptional regulatory protein functions within the proteome for formulation and testing of future hypotheses.

Figure 1. HT-IP/MS Analysis of the Human Endogenous Complexome

A) Our HT-IP/MS workflow consists of IP/MS followed by filtering of non-specific identifications, definition of minimal core complex modules (MEMOs), and assignment of complex-complex interactions (CCIs). Data relationships are abbreviated as ‘exp-2-gene’, ‘ab-2-gene’, and ‘ab-2-memo’ for experiment-to-genes, antibody-to-genes, and antibody-2-MEMOs, respectively. B) Representative SDS-PAGE of NR coregulator complexes. IgG HC and LC are heavy and light chains of primary antibodies. C) Approximately 40% of the human gene products were recovered in our HT-IP/MS data in ~100,000 specific protein identifications. Majority of these were found in at least two or more antibody-different experiments, laying a foundation for finding reciprocally verified protein associations.

Figure 2. Protein Complex Heterogeneity in BRCA1 Network

Partial BRCA1-related CCI networks, where individual MEMOs are separated by horizontal black lines, are shown to highlight the relationships between major components of the BRCA1 interactome (see also Figure S2). (*) Column headers specify antibody names, not the intended antigens that were used to generate the antibody. Despite conventions, it is often misleading to label IP/MS experiments with 'intended' antigens, because majority of antibodies cross-react with 2 or more proteins and some do not IP the intended antigen at all (see Supplemental Table S1). A) Discernible ‘hierarchical’ organization of protein interactions illustrated by a selection of IPs containing BRCA1 uniCOREs with the highest SPC identifications for all precipitated proteins (compare TopAntibodies lanes with MaxSPC lane). Although all proteins are equally true interactors of BRCA1, extensive reciprocal evidence visually implicates exclusive patterns: First three lanes are BRCA1 and BARD1 IPs (1), where all BRCA1-containing complexes are shared, while experiments that target specific uniCOREs (2) show non-uniform distributions of BRCA1 interactors. To the right is a schematic interpretation of the findings. B) Top experiments for FAM175A reveal a stoichiometric complex between FAM175A, UIMC1, and BRE/BRCC36/C19orf62, but not with FAM175B (3) C) 3N analyses for FAM175B (C) and UIMC1 (D) show that all IPs that have FAM175A predominantly contain UIMC1 and BRCA1 (4), and thus reveal autonomous FAM175A/B uniCOREs. E) MORF4L1/2 MEMO interacts with BRCA1/BARD1/PALB2/BRCA2 uniCORE, and also forms uniCOREs with chromatin remodeling complexes SIN3B and BRD8.

Figure 3. Iterative Mining of HT-IP/MS Resource Reveals Topology of RNA Polymerase II Network

A–B) 3N analyses for POLR2A show three separate subnetworks containing RPAP2-GPN1/3, Integrator complex, and Mediator complex (see also Figures S3 and S6). The MEMOs in the POLR2A network were grouped by patterns of their distribution to show that Mediator and Integrator sub-networks are vividly independent of each other. C) Coregulator SPEN stands on crossroads of three seminal transcriptional processes. Its CCI network suggests an association with Pol-II through Mediator or HDAC3/NCOR complex. SPEN also co-precipitates with proteins that regulate splicing process, including multi-subunit SFRS and WTAP complexes. (*) Heatmap column headers specify antibody names, not the intended antigens that were used to generate the antibody (for antibody data, see Supplemental Table S1).

Figure 4. Coregulators Have Distinct Patterns of Protein Interaction Profiles

3N analyses broadly classify coregulator networks into having either stable preferential protein networks (Type I) or multiple transient interactions (Type II). A) Schematic illustrations of examples of Type I NR coregulators with previously unidentified subunits. Green: coregulators listed at NURSA.org; E1, D2, D1, B1, and D3 are SMARC subunits; B7A,B,C are BCL7A,B, and C; C20, C20orf11; C17, C17orf39; BP9 and BP10 are RANBP9 and RANBP10; Y5, YPEL5, respectively. B) 3N heatmap excerpt for SRC-3/NCOA3. This is a typical Type II coactivator that lacks stoichiometric steady-state complex while revealing a multitude of sub-stoichiometric interactions, such as CBP (CREBBP), p300 (EP300), REG-gamma (PSME3), and various transcription factors (TFs).

Figure 5. Z3 Complex is a Transcriptional Coregulator

A) Concise representation of the Z3 CCI network showing extensive interaction connections to transcriptional machinery (see also Figure S5A,B). Pol-II-Integrator network (*) is omitted due to space limitations. In addition to histone demethylases KDM5C, KDM5A and KDM1 (see text), Z3 also interacts with ASC1 (TRIP4) coregulator. (#) C112, C20orf112; P-BAF, Polybromo and Brg/Brahma-Associated Factor (see Figure 4A for BAF). Known NR coregulators are shown in green. B) Reciprocal IP/MS of Z3 proteins with ERa in MCF7 cells. C) Reciprocal IP/WB of overexpressed GFP-ZMYND8 and ERα in 293T cells; (1) and (2) are corresponding inputs. D) In vitro binding suggests that binding to ERα is via the N-terminal portion of ZMYND8 (Z8-F1). Asterisks indicate positions of ZMYND8 fragments. E) UCSC browser examples showing co-occupancy of ERα and ZMYND8 binding sites at E2-upregulated genes. F) ZMYND8 coactivates ERα in reporter luciferase assay. G) RNAi knockdown of ZMYND8 compromises upregulation of some E2-responsive target genes. p21 serves as a negative control.

Figure 6. Cancer Gene Alterations Group Within Protein Complex Modules

A) The P-BAF complex is significantly perturbed in lung cancers. Red: amplified; dark blue: deleted in NSCLC; light blue: deleted in lung lineages. B) The SIN3B uniCORE is a hub of proteins with genomic amplifications. All but two proteins of the SIN3B complex are significantly amplified in breast cancer lineages (dark red) or epithelial lineages (pink). All genes marked with an asterisk are listed in Cancer Gene Sensus (Futreal et al.) and/or have been implicated causally in cancer development.