A vector for expressing GAL4(1–147) fusions tn mammalian cells

Sadowski, Ivan; Ptashne, Mark

doi:10.1093/nar/17.18.7539

Cited by 556 publications

(374 citation statements)

References 2 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

402

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

402

301

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“…Plasmid pHLAB-luc was constructed by PCR amplification of the HLAB1 gene promoter from −284 to +1, which was then cloned into plasmid pGL3basic. For the Gal4 DNA-binding domain fusion proteins, the cDNA coding for the Gal4 DNA-binding domain was subcloned from plasmid pSG424 (Sadowski and Ptashne, 1989) into pCDNA3.1 (Invitrogen) to create pCGal4. The appropriate regions of ZXDC or CIITA were amplified by PCR, and subcloned into pCGal4, in frame with the Gal4 DNAbinding domain.…”

Section: Plasmid Dna Constructsmentioning

confidence: 99%

ZXDC, a novel zinc finger protein that binds CIITA and activates MHC gene transcription

Al-Kandari

Jambunathan

Navalgund

et al. 2007

Molecular Immunology

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The class II trans-activator (CIITA) is recognized as the master regulator of major histocompatibility complex (MHC) class II gene transcription and contributes to the transcription of MHC class I genes. To better understand the function of CIITA, we performed yeast two-hybrid with the C-terminal 807 amino acids of CIITA, and cloned a novel human cDNA named zinc finger, X-linked, duplicated family member C (ZXDC). The 858 amino acid ZXDC protein contains 10 zinc fingers and a transcriptional activation domain, and was found to interact with the region of CIITA containing leucine-rich repeats. Over-expression of ZXDC in human cell lines resulted in super-activation of MHC class I and class II promoters by CIITA. Conversely, silencing of ZXDC expression reduced the ability of CIITA to activate transcription of MHC class II genes. Given the specific interaction between the ZXDC and CIITA proteins, as well as the effect of ZXDC on MHC gene transcription, it appears that ZXDC is an important regulator of both MHC class I and class II transcription.

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“…The plasmid RSV-CREB [22] served as template. The PCR product was cloned into the BamHI and SacI sites of pSG424 [23]. All constructs were confirmed by sequencing.…”

Section: Plasmid Constructsmentioning

confidence: 99%

Inhibition of membrane depolarisation-induced transcriptional activity of cyclic AMP response element binding protein (CREB) by the dual-leucine-zipper-bearing kinase in a pancreatic islet beta cell line

et al. 2005

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Aims/hypothesis: The activation of the transcription factor cyclic AMP response element binding protein (CREB) by protein kinase A is inhibited by the human orthologue of the mitogen-activated protein kinase, dualleucine-zipper-bearing kinase (DLK) in teratocarcinoma cells. However, pancreatic beta cells are electrically excitable and a major pathway regulating CREB in these cells is membrane depolarisation, leading to calcium influx and activation of the calcium/calmodulin-dependent protein phosphatase calcineurin. Therefore, the effect of DLK on CREB activity induced by membrane depolarisation was investigated in the beta cell line HIT. Materials and methods: Reporter gene assays and biochemical techniques were used. Results: RT-PCR, Western blot analysis and immunohistochemistry demonstrated the expression of DLK in HIT cells and primary mouse islets. In transient transfection experiments, DLK inhibited both GAL4-CREB activity induced by membrane depolarisation, and transcription directed by the CREB binding site, the cyclic AMP response element. Furthermore, DLK inhibited the transcriptional activity conferred by the CREB coactivator, CREB binding protein, both under basal conditions and after membrane depolarisation. DLK was also effective in response to glucose, the most potent physiological stimulus and known to cause membrane depolarisation of beta cells. Inhibition of calcineurin enhanced DLK activity, whereas overexpression of calcineurin reduced the inhibition by DLK of transcription directed by cyclic AMP response element after membrane depolarisation. Conclusions/interpretation: These results demonstrate a calcineurin-sensitive inhibition by DLK of CREB activity after membrane depolarisation in pancreatic islet beta cells. This inhibition may, at least partially, be mediated at the coactivator level. The results thus suggest that DLK plays a role in the regulation of beta cell function, including insulin gene transcription and beta cell apoptosis.

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A vector for expressing GAL4(1–147) fusions tn mammalian cells

Cited by 556 publications

References 2 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

ZXDC, a novel zinc finger protein that binds CIITA and activates MHC gene transcription

Inhibition of membrane depolarisation-induced transcriptional activity of cyclic AMP response element binding protein (CREB) by the dual-leucine-zipper-bearing kinase in a pancreatic islet beta cell line

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