GAL4 fusion vectors for expression in yeast or mammalian cells

Sadowski, Ivan; Bell, Brendan; Broad, Peter; Hollis, Michael

doi:10.1016/0378-1119(92)90261-m

Cited by 214 publications

(195 citation statements)

References 17 publications

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“…Plasmids-The following plasmids have previously been described: pCEP4F, which contains the cytomegalovirus immediate early promoter and an ATG sequence followed by the FLAG epitope (23); pBJ5-HD1-F, which encodes a carboxyl-terminal FLAG epitope-tagged HDAC1 (10); pME18S-FLAG-HDAC2, which expresses FLAG epitopetagged HDAC2 (14); pM2, pM3, and pSG424, which contain the Gal4 DNA-binding domain under the control of the SV40 promoter/enhancer (24,25); pGal4-mRPD3 (pGal4-HDAC2), which expresses a Gal4-* This work was supported by Grant MCB-9631067 from the National Science Foundation (to E. S.) and Grant MT-9186 from the Medical Research Council of Canada (to J. R. D.). The costs of publication of this article were defrayed in part by the payment of page charges.…”

Section: Methodsmentioning

confidence: 99%

Isolation and Characterization of cDNAs Corresponding to an Additional Member of the Human Histone Deacetylase Gene Family

Yang

Yao

Sun

et al. 1997

Journal of Biological Chemistry

405

301

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Several human cDNAs encoding a histone deacetylase protein, HDAC3, have been isolated. Analysis of the predicted amino acid sequence of HDAC3 revealed an open reading frame of 428 amino acids with a predicted molecular mass of 49 kDa. The HDAC3 protein is 50% identical in DNA sequence and 53% identical in protein sequence compared with the previously cloned human HDAC1. Comparison of the HDAC3 sequence with human HDAC2 also yielded similar results, with 51% identity in DNA sequence and 52% identity in protein sequence. The expressed HDAC3 protein is functionally active because it possesses histone deacetylase activity, represses transcription when tethered to a promoter, and binds transcription factor YY1. Similar to HDAC1 and HDAC2, HDAC3 is ubiquitously expressed in many different cell types.The organization of chromatin structure is a fundamental and significant component of transcriptional regulation in all eukaryotic cells. Transcriptionally active or repressed chromatin is determined, at least in part, by the modification of histones. For example, hyperacetylation of histones generally leads to an increase in transcription, whereas hypoacetylation of histones appears to have the opposite effect (reviewed in Refs. 1-4). Nuclear histone acetyltransferases such as transcription factors GCN5, PCAF, p300/CBP, and TAF II 230/250 have been identified from different organisms (5-9).Several yeast and mammalian histone deacetylases have been identified, and their corresponding genes have been cloned (10 -12). In yeast, the HDA1 protein, which shares sequence similarity to RPD3, is a subunit of a large histone deacetylase complex HDA. RPD3 is also associated with another yeast histone deacetylase complex HDB. Using a trapoxin (an inhibitor of histone deacetylase) affinity matrix, Taunton et al. (10) purified and cloned a human 55-kDa protein related to the yeast protein RPD3. Immunoprecipitation of this 55-kDa protein, HDAC1 (also called HD1), showed that it contains histone deacetylase activity. A second human histone deacetylase protein, HDAC2, with high homology to yeast RPD3 was identified based on a yeast two-hybrid trap experiment with the YY1 transcription factor as a bait (11). YY1 negatively regulates transcription by tethering HDAC2 to DNA as a corepressor. Both HDAC1 and HDAC2 exist in a complex with the corepressor mSIN3 and mediate Mad transcriptional repression (13-15). In addition, HDAC1 and HDAC2 are essential components of two thyroid hormone receptor corepressors, N-CoR and SMRT (16 -18). HDAC1 is also an important factor that represses transactivation by progesterone receptor (19).The yeast RPD3 protein was originally identified in genetic screens for transcriptional repressors (20). Besides human and mouse, highly homologous yeast RPD3 sequences have been identified in Drosophila (21), Caenorhabditis elegans (X78454 and 1176665), and Xenopus laevis (gi:576995). Currently, it has not yet been established whether the C. elegans or X. laevis RPD3-related proteins have histone deacetylase activities o...

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Section: Methodsmentioning

confidence: 99%

Isolation and Characterization of cDNAs Corresponding to an Additional Member of the Human Histone Deacetylase Gene Family

Yang

Yao

Sun

et al. 1997

Journal of Biological Chemistry

405

301

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“…SUV39H1 SET (provided by T. Jenuwein) contains the SET domain downstream of an engineered nuclear localization sequence and localizes to the nucleus (30a). To construct GAL4 DNA binding domain (DBD) fusion proteins, SUV39H1 was cloned in frame with amino acids 1 to 147 of GAL4 in the pM3 vector (provided by R. Baer) (44). Reporter constructs for transient transcriptional assays contained a firefly luciferase gene with (pLUC/ GAL4) or without (pLUC) four GAL4 sites upstream of the myelomonocytic growth factor promoter (provided by R. Eisenman) (2).…”

Section: Methodsmentioning

confidence: 99%

Set Domain-Dependent Regulation of Transcriptional Silencing and Growth Control by SUV39H1, a Mammalian Ortholog of Drosophila Su(var)3-9

Firestein

Cui

Huie

et al. 2000

Molecular and Cellular Biology

101

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Mammalian SET domain-containing proteins define a distinctive class of chromatin-associated factors that are targets for growth control signals and oncogenic activation. SUV39H1, a mammalian ortholog of Drosophila Su(var)3-9, contains both SET and chromo domains, signature motifs for proteins that contribute to epigenetic control of gene expression through effects on the regional organization of chromatin structure. In this report we demonstrate that SUV39H1 represses transcription in a transient transcriptional assay when tethered to DNA through the GAL4 DNA binding domain. Under these conditions, SUV39H1 displays features of a long-range repressor capable of acting over several kilobases to silence basal promoters. A possible role in chromatin-mediated gene silencing is supported by the localization of exogenously expressed SUV39H1 to nuclear bodies with morphologic features suggestive of heterochromatin in interphase cells. In addition, we show that SUV39H1 is phosphorylated specifically at the G 1 /S cell cycle transition and when forcibly expressed suppresses cell growth. Growth suppression as well as the ability of SUV39H1 to form nuclear bodies and silence transcription are antagonized by the oncogenic antiphosphatase Sbf1 that when hyperexpressed interacts with the SET domain and stabilizes the phosphorylated form of SUV39H1. These studies suggest a phosphorylation-dependent mechanism for regulating the chromatin organizing activity of a mammalian su(var) protein and implicate the SET domain as a gatekeeper motif that integrates upstream signaling pathways to epigenetic regulation and growth control.The formation and propagation of higher-order chromatin states are dynamic processes that establish distinct domains that are either permissive or restrictive for transcription. The functions of such domains have been implicated in the epigenetic control of developmental gene expression in Drosophila (38), proper sister chromatid segregation during meiosis (11), and telomeric and centromeric silencing in yeast (22,35). The molecular mechanisms that regulate these and other properties of higher-order chromatin are essentially unknown.Genetic analyses in Drosophila and yeast have identified several genes that participate in the formation of euchromatin or heterochromatin states. Some of these encode proteins that contribute to either an enhancement [E(var)] or suppression [Su(var)] of position effect variegation (PEV) (42). PEV is a gene silencing mechanism that results from the spreading of heterochromatin, thus implicating E(var) and Su(var) proteins in the formation of euchromatic and heterochromatic domains, respectively. Several E(var) and Su(var) proteins share distinctive motifs that are important for their ability to organize chromatin domains. The Su(var)3-9 protein and its mammalian ortholog SUV39H1 are unique in being the only characterized PEV modifiers that share two of these consensus motifs, the chromo and SET domains (1, 50). Chromo domains are 40-amino-acid modular motifs that are implicated in...

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“…As a positive control bait construct, we used the KOX1-KRAB domain (aa 1 ± 122) fused to the LexA DNA-binding domain. We detected strong interaction between this KOX1-KRAB bait and the TIF1b target in yeast, as seen from the growth of the yeast transformants in histidine-de®cient media and the detection of b-galactosidase activity in these transfor- The backbone plasmid for expression of fusion proteins was pM3-VP16-FLAG, derived from mammalian expression vector pM3 (Sadowski et al, 1992). The expressed fusion protein contains the N-terminus (amino acids 1 ± 147) of GAL4 (the DNA binding domain), and four copies of the VP16 activation domain.…”

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confidence: 90%

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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GAL4 fusion vectors for expression in yeast or mammalian cells

Cited by 214 publications

References 17 publications

Isolation and Characterization of cDNAs Corresponding to an Additional Member of the Human Histone Deacetylase Gene Family

Isolation and Characterization of cDNAs Corresponding to an Additional Member of the Human Histone Deacetylase Gene Family

Set Domain-Dependent Regulation of Transcriptional Silencing and Growth Control by SUV39H1, a Mammalian Ortholog of Drosophila Su(var)3-9

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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