From transient cotransfections to the transcription repressor assay (TRA)

Thiesen, Hans‐Jürgen

doi:10.1016/b978-012442710-5.50038-1

Immunology Methods Manual 1996

DOI: 10.1016/b978-012442710-5.50038-1

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From transient cotransfections to the transcription repressor assay (TRA)

Hans‐Jürgen Thiesen¹

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1998

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“…To date the SSX-KRAB domain itself has not been tested for repression activity. In this study we have used a highly sensitive transcription repressor assay (TRA) ®rst described by Thiesen (1997) to assess the potential dominant repression e ect of protein domains fused to a potent transcriptional activator GAL4-VP16 (Sadowski et al, 1988).…”

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“…The repression activity exerted by the KOX1 MLE mutant is reduced by speci®c point mutations in KRAB A that substitute the sequence MLE with KKK (MLE?KKK) as indicated in Figure 2 (Margolin et al, 1994;Thiesen, 1997). The leucine residue in the MLE sequence occurs in the context of the highly conserved VMLENY motif common to almost all KRAB A sub-domains, and is one of the heptad repeats of leucines (MX6LX6LX6L, where X is any amino acid) capable of forming an amphipathic helix (Thiesen, 1990).…”

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A KRAB-related domain and a novel transcription repression domain in proteins encoded by SSX genes that are disrupted in human sarcomas

et al. 1998

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SSX genes show extensive nucleotide sequence conservation but little is known of their function. Disruption of SSX1 or SSX2, by chromosome translocation and`inframe' fusion to SYT, is a consistent feature of synovial sarcomas. The resulting SYT-SSX1/SSX2 proteins are activators of transcription; transactivation function is located in SYT. Unrearranged SSX1 can repress transcription, and this has been attributed to a putative KruÈ ppel associated box (KRAB) repression domain at the N-terminus. Here we isolated SSX-KRAB domains to speci®cally measure repression activity, using a previously characterized KOX1-KRAB domain as a control. In our repressor assay SSX1-and SSX2-KRAB domains down-modulated the transactivation of a reporter gene by threefold, compared with 83-fold repression achieved by KOX1-KRAB in the assay. Yeast two-hybrid analysis showed that SSX1-KRAB, unlike KOX1-KRAB, fails to interact with the KRAB corepressor TIF1b. These results raise questions about the evolutionary and functional relationship of SSX-KRAB and typical KRAB domains of KruÈ ppel zinc ®nger genes. We found that full-length SSX1 showed potent (74-fold) repression in our repressor assay, indicating the existence of a repression domain distinct from SSX-KRAB. By assaying deletion constructs of SSX1 we localized repression activity to 33 amino acids at the C-terminus. This novel domain is conserved between SSX family members, and, unlike the KRAB-related domain, is retained on fusion with SYT. This has important implications in understanding the mechanism by which the SYT-SSX fusion protein could contribute to neoplasia.Keywords: sarcoma; SSX; KRAB; transcription Chromosome translocation t(X;18)(p11.2;q11.2) (Clark et al., 1994;Crew et al., 1995) is a diagnostic feature of human synovial sarcomas, in some cases representing the sole cytogenetic abnormality (Sandberg and Bridge, 1994). All the available evidence indicates that this translocation is a key event in tumorigenesis. In all tumours characterized, the SYT gene on chromosome 18 is juxtaposed`in-frame' with either the SSX1 gene or SSX2 gene on chromosome X (Figure 1). SSX1 and SSX2 are now known to be members of a highly conserved multigene family Gure et al., 1997), and the SSX loci that have been mapped are all located in chromosome band Xp11.2 (Crew et al., 1995;. In contrast to SYT, which is a widely expressed gene (de Bruijn et al., 1996; J Knight., unpublished data), SSX transcripts show a very restricted distribution in adult human tissues. So far, SSX1 and SSX2 expression has only been detected in testis and thyroid (Crew et al., 1995;Tureci et al., 1996;Gure et al., 1997).As yet, little is known about the normal biological functions of the SYT and SSX gene products. No DNA binding sequences are recognizable in the SYT or SSX proteins. However, when coupled to a GAL4 DNA binding domain in in vitro reporter assays, SYT can activate transcription (70-fold activation) and SSX1 can repress transcription (50-fold repression) from a minimal promoter in NIH3T3 ®broblasts (Brett ...

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A KRAB-related domain and a novel transcription repression domain in proteins encoded by SSX genes that are disrupted in human sarcomas

et al. 1998

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From transient cotransfections to the transcription repressor assay (TRA)

Cited by 1 publication

References 28 publications

A KRAB-related domain and a novel transcription repression domain in proteins encoded by SSX genes that are disrupted in human sarcomas

A KRAB-related domain and a novel transcription repression domain in proteins encoded by SSX genes that are disrupted in human sarcomas

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