The FUS-DDIT3 Interactome in Myxoid Liposarcoma

Abstract

Myxoid liposarcoma is a malignant lipogenic tumor that develops in deep soft tissues. While local control rates are good, current chemotherapy options remain ineffective against metastatic disease. Myxoid liposarcoma is characterized by the FUS-DDIT3 fusion oncoprotein that is proposed to function as an aberrant transcription factor, but its exact mechanism of action has remained unclear. To identify the key functional interacting partners of FUS-DDIT3, this study utilized immunoprecipitation-mass spectrometry (IP-MS) to identify the FUS-DDIT3 interactome in whole cell lysates of myxoid liposarcoma cells, and results showed an enrichment of RNA processing proteins. Further quantitative MS analyses of FUS-DDIT3 complexes isolated from nuclear lysates showed that members of several chromatin regulatory complexes were present in the FUS-DDIT3 interactome, including NuRD, SWI/SNF, PRC1, PRC2, and MLL1 COMPASS-like complexes. Co-immunoprecipitation validated the associations of FUS-DDIT3 with BRG1/SMARCA4, BAF155/SMARCC1, BAF57/SMARCE1, and KDM1A. Data from this study provides candidates for functional validation as potential therapeutic targets, particularly for emerging epigenetic drugs.

Figure 1

Structure and domains of FUS, DDIT3 and FUS-DDIT3. Wild type FUS contains the following protein domains: a low complexity serine/tyrosine/glycine/glutamine (SYGQ)-rich domain, three arginine- and glycine-rich RGG motif domains, a RNA recognition motif (RRM) domain, a zinc finger (ZnF) domain, and a non-classical proline-tyrosine (PY) nuclear localization signal. Wild type DDIT3 contains a transactivation/repression domain in the N-terminus followed by a basic leucine zipper in its C-terminus. The two FUS-DDIT3 fusion variants used in this study retain the SYGQ-rich and RGG1 domains of FUS, and also include the in frame amino acid sequence of a portion of the previously untranslated region (UTR) from DDIT3 exon 2. Schematic illustration of protein domain structure was generated with the tool Illustrator of Biological Sequences (IBS).

Figure 2

Functional classification of the whole cell FUS-DDIT3 interactome. (A) The largest class of proteins in the FUS-DDIT3 interactome from whole cell lysates are the nucleic acid binding proteins. The PANTHER protein classification of the FUS-DDIT3 interactome shows the percentage of each protein class against the total number of proteins with a class hit (n = 47). The nucleic acid binding protein class (n = 24) is further broken down into three different sub-classes (DNA-binding, RNA-binding and nuclease). PC numbers refer to PANTHER class ID. (B) The FUS-DDIT3 interactome is enriched for RNA processing proteins, based on the Gene Ontology classification of biological processes enriched in the FUS-DDIT3 whole cell lysate interactome (n = 75).

Figure 3

Reciprocal co-immunoprecipitation and proximity ligation assays validate FUS-DDIT3’s association with NONO, PSPC1 and SFPQ. (A) Whole cell lysates from two myxoid liposarcoma cell lines, 402-91 and 1765-92, each harboring a different variant of the FUS-DDIT3 fusion oncoprotein, were used to test for reciprocal co-immunoprecipitation of the Drosophila behavior/human splicing proteins NONO, PSPC1, and SFPQ with the FUS-DDIT3 oncoprotein. For color blots, FUS was detected in the green channel and DDIT3 in the red channel, and the FUS-DDIT3 band is indicated by an overlap of FUS and DDIT3 signal to form a merged yellow-colored band. Larger images of the same blots with visible IgG bands from each IP antibody are in Figure S4 to visualize the amount of antibody used. (B) Proximity ligation assay, a technique that amplifies a red signal when two proteins colocalize within 40 nm of each other in situ, was performed in myxoid liposarcoma cell lines 402-91, 1765-92, DL-221, and negative control cell line HeLa. Red signals indicate proximity between proteins of interest. Nuclei were counter-stained with DAPI (blue). Scale bar = 5 μm.

Figure 4

The FUS-DDIT3 nuclear interactome. (A) Scatterplot of the FUS-DDIT3 nuclear interactome showing proteins with an average enrichment ratio of >1 in DDIT3 vs control IPs (n = 174). Each circle represents a putative FUS-DDIT3 nuclear interactor, with circle size corresponding to the number of peptides detected in the mass spectrometry analysis. Each protein is plotted by the log2 values of its average enrichment ratio (log2(Protein Ratio)) and the transformed coefficient of variation for the average ratio across triplicate IPs. The protein enrichment score (PES, see Methods) is calculated to take into account the abundance measurement for each protein (enrichment ratio) and the reproducibility of this measurement through the CV values (Table S3). A cutoff PES z-score ≥ 0.5 was applied to identify 43 top ranking-proteins in the FUS-DDIT3 nuclear interactome (highlighted in blue). (B) The same 174 proteins from the FUS-DDIT3 nuclear interactome are plotted by their z-scores and ranking of their PES values (higher PES value = higher rank). The seven proteins previously validated from the whole cell lysate IP-MS screen are highlighted in red. The PES z-score = 0.5 is indicated by a dotted line.

Figure 5

The FUS-DDIT3 nuclear interactome contains mRNA processing proteins and chromatin regulators. Enriched gene ontology (A) biological processes or (B) cellular components in the FUS-DDIT3 nuclear interactome (n = 174). Numbers after each bar indicate the number of group members found in interactome / total number of group members in database. For enriched cellular components, only chromatin regulatory complexes are presented here; the full list can be found in Table S4. (C) Proteins with an average enrichment ratio of >1 in DDIT3 vs control IPs are presented (n = 174). Components of various chromatin regulatory complexes are indicated. Each circle represents a putative FUS-DDIT3 nuclear interactor, with circle size corresponding to the number of peptides detected in the mass spectrometry analysis. Each protein is plotted by the log2 values of its average enrichment ratio (log2(Protein Ratio)) and the transformed coefficient of variation for the average ratio across triplicate IPs. (D) The same proteins are plotted by the z-scores and rank of their PES values (higher PES value = higher rank). The PES z-score = 0.5 is indicated by a dotted line.

Figure 6

Co-immunoprecipitation (co-IP) validates FUS-DDIT3’s interactions with KDM1A, BRG1, BAF155 and BAF57 in 402-91 and 1765-92 myxoid liposarcoma cells. FUS was detected in the green channel, DDIT3 in the red channel. A merged yellow band represents FUS-DDIT3. Black arrows on blots point to proteins detected by the indicated Western blot antibodies. Grey arrows point to IgG chains from IP antibody. (A) Reciprocal co-IPs of FUS-DDIT3 and KDM1A were carried out in whole cell lysates. (B) Co-immunoprecipitation with FUS-DDIT3 was observed for SWI/SNF components BRG1 (SMARCA4), BAF155 (SMARCC1) and BAF57 (SMARCE1) in nuclear lysates. White bands indicate scan signal saturation for IgG heavy chain of IP antibody.