The C terminus of the synovial sarcoma-associated SSX proteins interacts with the LIM homeobox protein LHX4

Abstract

As a result of the synovial sarcoma-associated t(X;18) translocation, the SS18 gene on chromosome 18 is fused to either one of the three closely related SSX genes on the X chromosome. The SS18 protein is thought to act as a transcriptional co-activator, whereas the SSX proteins are thought to act as transcriptional corepressors. The main SSX-repression domain is located in its C terminus, a domain that is retained in the respective SS18-SSX fusion proteins. Both the SS18 and SSX proteins lack DNAbinding domains. Previously, we found that the SS18 and SS18-SSX fusion proteins may be tethered to DNA targets via the SS18-interacting protein AF10. Here, we set out to isolate proteins that interact with the SSX C-terminal repression domain using a yeast two-hybrid interaction trap. Of the positive clones isolated, two corresponded to the LIM homeobox protein LHX4, a DNA-binding protein that is involved in transcription regulation. An endogenous interaction was subsequently established in mammalian cells via colocalization and coimmunoprecipitation of the respective proteins. Interestingly, the LHX4 gene was previously found to be deregulated in various human leukemias. In addition, it was previously found that LIM homeobox proteins may bind to and activate the glycoprotein-a (CGA) promoter. Using LHX4 chromatin immunoprecipitation and CGApromoter assays, we found that endogenous LHX4 binds to the CGA promoter and that LHX4-mediated CGA activation is enhanced by the SS18-SSX protein, but not by the SSX protein. Taken together, we conclude that this novel protein -protein interaction may have direct consequences for the (de)regulation of SSX and/or SS18-SSX target genes and, thus, for the development of human synovial sarcomas.

Cancer/testis antigen SSX2 enhances invasiveness in MCF-7 cells by repressing ERα signaling

“…In addition, the conserved C-terminus of SSX2 named as SSXRD (SSX repression domain) is associated with transcriptional repressor regulation (19,20). The SSX KRAB domain interacts with the RAB3IP (RAB3A interacting protein) and SSX2IP (SSX2 interacting protein), while the SSXRD domain interacts with LHX4 (LIM homeobox 4) and PcG protein (21,22 Vector construction. Wild-type SSX2 ORF was isolated by PCR from human testis cDNA (forward primer is 5'-GGGGAATTC ATGAACGGAGACGACGCCTT-3' and reverse primer is 5'-CGCGGATCCTCACCAGCTGTTTTCTCTCA-3').…”

Synovial Sarcoma: From Genetics to Genetic-based Animal Modeling

“…The SSXRD domain is responsible for the nuclear localization of this protein, interaction with polycomb group proteins implicated in gene repression, and association with histones [18,19,35,73]. The C-terminal of SSX proteins also interacts with the Lim homeobox protein LHX4, a putative transcriptional factor that is deregulated in leukemias [15]. These suggest SSX, in association with other proteins, predominantly repress target genes.…”

Synovial sarcoma is a gateway to the role of chromatin remodeling in cancer

“…E-cadherin levels are lower in SS18-SSX1 compared to SS18-SSX2 cells which is believed to cause less epithelial differentiation [24]. The carboxy-termini of SSX1, SSX2, and SSX4 are retained in the fusion proteins and interact equally well in vivo with LHX4 (LIM homeobox protein 4) [111]. The LHX4 protein exhibits features typical of transcription factors.…”

“…The SSX proteins are rich in charged aa (about 40 %) and contain an acidic carboxy-terminal tail and several consensus motifs for tyrosine phosphorylation and N-glycosylation. SSX contains two major functional domains referred as KRAB (Krüppel-associated box) and SSXRD (SSX repression domain) [111] (Fig. 1).…”