Proteomic analyses reveal distinct chromatin-associated and soluble transcription factor complexes.

The current knowledge on how transcription factors (TFs), the ultimate targets and executors of cellular signalling pathways, are regulated by protein-protein interactions remains limited. Here, we performed proteomics analyses of soluble and chromatin-associated complexes of 56 TFs, including the targets of many signalling pathways involved in development and cancer, and 37 members of the Forkhead box (FOX) TF family. Using tandem affinity purification followed by mass spectrometry (TAP/MS), we performed 214 purifications and identified 2,156 high-confident protein-protein interactions. We found that most TFs form very distinct protein complexes on and off chromatin. Using this data set, we categorized the transcription-related or unrelated regulators for general or specific TFs. Our study offers a valuable resource of protein-protein interaction networks for a large number of TFs and underscores the general principle that TFs form distinct location-specific protein complexes that are associated with the different regulation and diverse functions of these TFs

A scoring system for the follow up study of nuclear receptor coactivator complexes

We have systematically isolated a variety of coactivator complexes from HeLa S3 cells using proteomic approaches. In the present report, we have evaluated twelve coactivator complexes involved in nuclear receptor-dependent gene transcription that have been purified by using an immunoprecipitation method. The twelve purified coactivator complexes are SRC-1, SRC-2, SRC-3, CBP, p300, CAPER, E6-AP, ASC-1, CoREST, CRSP3, CRSP2, and CDK7 containing complexes. We have identified 153 protein components associated with these coactivator complexes using mass spectrometry. In order to systematically characterize the functional roles for these components in nuclear receptor-dependent gene transcription and their investigative potential, we have developed a scoring system. This scoring system is comprised of biological and experimental parameters. The biological evaluation considers aspects such as intrinsic enzymatic activity of a protein component, cellular signaling processes in which protein components may be involved, associations with human disease, specific protein motifs, and the known biological roles of other interacting partners of the identified protein. In the experimental evaluation, we include parameters, such as the availability of research materials for the functional study of the identified protein component; such as full-length cDNA clones, antibodies, and commercially available knock-out embryonic stem (ES) cells. Each scoring parameter has been assigned an arbitrary number of points according to perceived relative importance. On the basis of this scoring system, we prioritized each of the protein components in terms of the likelihood of their importance for coactivator complex networking in nuclear receptor-dependent gene transcription

Recovering Protein-Protein and Domain-Domain Interactions from Aggregation of IP-MS Proteomics of Coregulator Complexes

Coregulator proteins (CoRegs) are part of multi-protein complexes that transiently assemble with transcription factors and chromatin modifiers to regulate gene expression. In this study we analyzed data from 3,290 immuno-precipitations (IP) followed by mass spectrometry (MS) applied to human cell lines aimed at identifying CoRegs complexes. Using the semi-quantitative spectral counts, we scored binary protein-protein and domain-domain associations with several equations. Unlike previous applications, our methods scored prey-prey protein-protein interactions regardless of the baits used. We also predicted domain-domain interactions underlying predicted protein-protein interactions. The quality of predicted protein-protein and domain-domain interactions was evaluated using known binary interactions from the literature, whereas one protein-protein interaction, between STRN and CTTNBP2NL, was validated experimentally; and one domain-domain interaction, between the HEAT domain of PPP2R1A and the Pkinase domain of STK25, was validated using molecular docking simulations. The scoring schemes presented here recovered known, and predicted many new, complexes, protein-protein, and domain-domain interactions. The networks that resulted from the predictions are provided as a web-based interactive application at http://maayanlab.net/HT-IP-MS-2-PPI-DDI/

Computational and informatics strategies for identification of specific protein interaction partners in affinity purification mass spectrometry experiments

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/92060/1/pmic7070.pd

The Methyltransferase WBSCR22/Merm1 Enhances Glucocorticoid Receptor Function and Is Regulated in Lung Inflammation and Cancer

Glucocorticoids (GC) regulate cell fate and immune function. We identified the metastasis-promoting methyltransferase, metastasis-related methyltransferase 1 (WBSCR22/Merm1) as a novel glucocorticoid receptor (GR) regulator relevant to human disease. Merm1 binds the GR co-activator GRIP1 but not GR. Loss of Merm1 impaired both GR transactivation and transrepression by reducing GR recruitment to its binding sites. This was accompanied by loss of GR-dependent H3K4Me3 at a well characterized promoter. Inflammation promotes GC resistance, in part through the actions of TNFα and IFNγ. These cytokines suppressed Merm1 protein expression by driving ubiquitination of two conserved lysine residues. Restoration of Merm1 expression rescued GR transactivation. Cytokine suppression of Merm1 and of GR function was also seen in human lung explants. In addition, striking loss of Merm1 protein was observed in both inflammatory and neoplastic human lung pathologies. In conclusion, Merm1 is a novel regulator of chromatin structure affecting GR recruitment and function, contributing to loss of GC sensitivity in inflammation, with suppressed expression in pulmonary disease

Proteogenomic integration reveals therapeutic targets in breast cancer xenografts

Recent advances in mass spectrometry (MS) have enabled extensive analysis of cancer proteomes. Here, we employed quantitative proteomics to profile protein expression across 24 breast cancer patient-derived xenograft (PDX) models. Integrated proteogenomic analysis shows positive correlation between expression measurements from transcriptomic and proteomic analyses; further, gene expression-based intrinsic subtypes are largely re-capitulated using non-stromal protein markers. Proteogenomic analysis also validates a number of predicted genomic targets in multiple receptor tyrosine kinases. However, several protein/phosphoprotein events such as overexpression of AKT proteins and ARAF, BRAF, HSP90AB1 phosphosites are not readily explainable by genomic analysis, suggesting that druggable translational and/or post-translational regulatory events may be uniquely diagnosed by MS. Drug treatment experiments targeting HER2 and components of the PI3K pathway supported proteogenomic response predictions in seven xenograft models. Our study demonstrates that MS-based proteomics can identify therapeutic targets and highlights the potential of PDX drug response evaluation to annotate MS-based pathway activities

Validation Studies for Single Circulating Trophoblast Genetic Testing as a Form of Noninvasive Prenatal Diagnosis

It has long been appreciated that genetic analysis of fetal or trophoblast cells in maternal blood could revolutionize prenatal diagnosis. We implemented a protocol for single circulating trophoblast (SCT) testing using positive selection by magnetic-activated cell sorting and single-cell low-coverage whole-genome sequencing to detect fetal aneuploidies and copy-number variants (CNVs) at ∼1 Mb resolution. In 95 validation cases, we identified on average 0.20 putative trophoblasts/mL, of which 55% were of high quality and scorable for both aneuploidy and CNVs. We emphasize the importance of analyzing individual cells because some cells are apoptotic, in S-phase, or otherwise of poor quality. When two or more high-quality trophoblast cells were available for singleton pregnancies, there was complete concordance between all trophoblasts unless there was evidence of confined placental mosaicism. SCT results were highly concordant with available clinical data from chorionic villus sampling (CVS) or amniocentesis procedures. Although determining the exact sensitivity and specificity will require more data, this study further supports the potential for SCT testing to become a diagnostic prenatal test

Diverse Roles and Interactions of the SWI/SNF Chromatin Remodeling Complex Revealed Using Global Approaches

A systems understanding of nuclear organization and events is critical for determining how cells divide, differentiate, and respond to stimuli and for identifying the causes of diseases. Chromatin remodeling complexes such as SWI/SNF have been implicated in a wide variety of cellular processes including gene expression, nuclear organization, centromere function, and chromosomal stability, and mutations in SWI/SNF components have been linked to several types of cancer. To better understand the biological processes in which chromatin remodeling proteins participate, we globally mapped binding regions for several components of the SWI/SNF complex throughout the human genome using ChIP-Seq. SWI/SNF components were found to lie near regulatory elements integral to transcription (e.g. 5′ ends, RNA Polymerases II and III, and enhancers) as well as regions critical for chromosome organization (e.g. CTCF, lamins, and DNA replication origins). Interestingly we also find that certain configurations of SWI/SNF subunits are associated with transcripts that have higher levels of expression, whereas other configurations of SWI/SNF factors are associated with transcripts that have lower levels of expression. To further elucidate the association of SWI/SNF subunits with each other as well as with other nuclear proteins, we also analyzed SWI/SNF immunoprecipitated complexes by mass spectrometry. Individual SWI/SNF factors are associated with their own family members, as well as with cellular constituents such as nuclear matrix proteins, key transcription factors, and centromere components, implying a ubiquitous role in gene regulation and nuclear function. We find an overrepresentation of both SWI/SNF-associated regions and proteins in cell cycle and chromosome organization. Taken together the results from our ChIP and immunoprecipitation experiments suggest that SWI/SNF facilitates gene regulation and genome function more broadly and through a greater diversity of interactions than previously appreciated

SS18 Together with Animal-Specific Factors Defines Human BAF-Type SWI/SNF Complexes

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