STAT3β, a Splice Variant of Transcription Factor STAT3, Is a Dominant Negative Regulator of Transcription

Secondary cell wall (SCW) biosynthesis is the biological process that generates wood, an important renewable feedstock for materials and energy. NAC domain transcription factors, particularly Vascular-Related NAC-Domain (VND) and Secondary Wall-Associated NAC Domain (SND) proteins, are known to regulate SCW differentiation. The regulation of VND and SND is important to maintain homeostasis for plants to avoid abnormal growth and development. We previously identified a splice variant, PtrSND1-A2IR, derived from PtrSND1-A2 as a dominant-negative regulator, which suppresses the transactivation of all PtrSND1 family members. PtrSND1-A2IR also suppresses the self-activation of the PtrSND1 family members except for its cognate transcription factor, PtrSND1-A2, suggesting the existence of an unknown factor needed to regulate PtrSND1-A2. Here, a splice variant, PtrVND6-C1IR, derived from PtrVND6-C1 was discovered that suppresses the protein functions of all PtrVND6 family members. PtrVND6-C1IR also suppresses the expression of all PtrSND1 members, including PtrSND1-A2, demonstrating that PtrVND6-C1IR is the previously unidentified regulator of PtrSND1-A2. We also found that PtrVND6-C1IR cannot suppress the expression of its cognate transcription factor, PtrVND6-C1. PtrVND6-C1 is suppressed by PtrSND1-A2IR. Both PtrVND6-C1IR and PtrSND1-A2IR cannot suppress their cognate transcription factors but can suppress all members of the other family. The results indicate that the splice variants from the PtrVND6 and PtrSND1 family may exert reciprocal cross-regulation for complete transcriptional regulation of these two families in wood formation. This reciprocal cross-regulation between families suggests a general mechanism among NAC domain proteins and likely other transcription factors, where intron-retained splice variants provide an additional level of regulation.

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Trp63 is regulated by STAT5 in mammary tissue and subject to differentiation in cancer

Endocrine Related Cancer ◽

10.1530/erc-14-0032 ◽

2014 ◽

Vol 21 (3) ◽

pp. 443-457 ◽

Cited By ~ 7

Author(s):

Shahin Assefnia ◽

Keunsoo Kang ◽

Svenja Groeneveld ◽

Daisuke Yamaji ◽

Sarah Dabydeen ◽

...

Keyword(s):

Transcription Factor ◽

Triple Negative ◽

Cultured Cells ◽

Dominant Negative ◽

Negative Regulator ◽

Proximal Promoter ◽

Mammary Tissue ◽

Mrna Levels ◽

Rna Seq ◽

Mesenchymal Transition

Transformation-related protein 63 (Trp63), the predominant member of the Trp53 family, contributes to epithelial differentiation and is expressed in breast neoplasia.Trp63features two distinct promoters yielding specific mRNAs encoding two major TRP63 isoforms, a transactivating transcription factor and a dominant negative isoform. Specific TRP63 isoforms are linked to cell cycle arrest, apoptosis, survival, and epithelial mesenchymal transition (EMT). Although TRP63 overexpression in cultured cells is used to elucidate functions, little is known aboutTrp63regulation in normal and cancerous mammary tissues. This study used ChIP-seq to interrogate transcription factor binding and histone modifications of theTrp63locus in mammary tissue and RNA-seq and immunohistochemistry to gauge gene expression. H3K4me2 and H3K4me3 marks coincided only with the proximal promoter, supporting RNA-seq data showing the predominance of the dominant negative isoform. STAT5 bound specifically to theTrp63proximal promoter andTrp63mRNA levels were elevated upon deletingStat5from mammary tissue, suggesting its role as a negative regulator. The dominant negative TRP63 isoform was localized to nuclei of basal mammary epithelial cells throughout reproductive cycles and retained in a majority of the triple-negative cancers generated from loss of full-lengthBrca1. Increased expression of dominant negative isoforms was correlated with developmental windows of increased progesterone receptor binding to the proximalTrp63promoter and decreased expression during lactation was correlated with STAT5 binding to the same region. TRP63 is present in the majority of triple-negative cancers resulting from loss ofBrca1but diminished in less differentiated cancer subtypes and in cancer cells undergoing EMT.

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Bcl-6 May Be a Survival Factor in Pre-B Cell Ph+ Blast Crisis Cell Lines.

Blood ◽

10.1182/blood.v110.11.4536.4536 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4536-4536

Author(s):

Ana Batlle ◽

Eric W. Lam ◽

Simon D. Wagner

Keyword(s):

Transcription Factor ◽

Tyrosine Kinase ◽

Cell Lines ◽

Blast Crisis ◽

Chronic Phase ◽

Negative Control ◽

Dominant Negative ◽

Negative Regulator ◽

Mammalian Expression ◽

Survival Factor

Abstract The constitutively active tyrosine kinase Bcr-Abl fusion protein is essential for the development of chronic myeloid leukaemia. Inhibitors of its tyrosine kinase activity e.g. Imatinib, are highly effective in treating chronic phase disease but are only transiently useful in blast crisis, especially lymphoid blast crisis. Bcl-6 is a transcriptional repressor that is required for the formation and maintenance of germinal centre B-cells. Following reports that Imatinib increases expression of Bcl-6 in Ph+ cell lines representative of lymphoid blast crisis we have investigated the regulation of this molecule and its functional importance. We utilised BV173 and Z119. Basal Bcl-6 protein expression was detectable in Z119, but not BV173. As anticipated Imatinib increased Bcl-6 expression in both cell lines. STAT5 is an important target of Bcr-Abl and has been implicated as both a positive and negative regulator of Bcl-6 transcription. Transient transfection with a mammalian expression plasmid bearing a dominant negative STAT5 reduced Bcl-6 expression in Z119 implying that activated STAT5 promotes Bcl-6 expression in this cell line, but also that STAT5 cannot be the main target of Bcr-Abl because Imatinib and dominant negative STAT5 have opposing effects. Next we considered the possibility that phosphorylation status of the transcription factor FoxO3a, which has been shown to bind to the Bcl-6 promoter, correlated with Bcl-6 expression. We found that decreased phosphorylation of Foxo3a, activating this transcription factor, was highly associated with increased Bcl-6 expression suggesting that it is an significant Bcr-Abl target. Next we wished to find out the functional implications of increased Bcl-6 expression. We found that a low concentration of Imatinib (0.25μM) was sufficient to induce Bcl-6 in BV173 and Z119, and caused death of these cell lines over several days. Next we combined Imatinib with a previously reported peptide antagonist of the Bcl-6/SMRT co-repressor interaction. Neither this peptide nor a mutated negative control peptide had an effect on cell numbers or apoptosis over 48 hours of culture when used alone. However, when combined with Imatinib the wild-type peptide, but not the control, reduced cell growth in Z119. We conclude that Bcl-6 may be a survival factor in Ph+ lymphoid blast crisis cell lines following treatment with Imatinib and that specific treatments to abrogate Bcl-6 function may find a place in the treatment of lymphoid blast crisis of CML.

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Mutation P291fsinsC in the transcription factor hepatocyte nuclear factor-1alpha is dominant negative

Diabetes ◽

10.2337/diabetes.47.8.1231 ◽

1998 ◽

Vol 47 (8) ◽

pp. 1231-1235 ◽

Cited By ~ 19

Author(s):

K. Yamagata ◽

Q. Yang ◽

K. Yamamoto ◽

H. Iwahashi ◽

J. Miyagawa ◽

...

Keyword(s):

Transcription Factor ◽

Dominant Negative ◽

Nuclear Factor ◽

Hepatocyte Nuclear Factor

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Protein kinase C-theta isoenzyme selective stimulation of the transcription factor complex AP-1 in T lymphocytes.

Molecular and Cellular Biology ◽

10.1128/mcb.16.4.1842 ◽

1996 ◽

Vol 16 (4) ◽

pp. 1842-1850 ◽

Cited By ~ 193

Author(s):

G Baier-Bitterlich ◽

F Uberall ◽

B Bauer ◽

F Fresser ◽

H Wachter ◽

...

Keyword(s):

Transcription Factor ◽

Transcriptional Activation ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Kinase C ◽

Pkc Isoforms ◽

Pkc Alpha ◽

Transcriptional Complex ◽

Induced Signal ◽

Transcription Factor Complex

T-lymphocyte stimulation requires activation of several protein kinases, including the major phorbol ester receptor protein kinase C (PKC), ultimately leading to induction of lymphokines, such as interleukin-2 (IL-2). The revelant PKC isoforms which are involved in the activation cascades of nuclear transcription factors involved in IL-2 production have not yet been clearly defined. We have examined the potential role of two representative PKC isoforms in the induction of the IL-2 gene, i.e., PKC-alpha and PKC-theta, the latter being expressed predominantly in hematopoietic cell lines, particularly T cells. Similar to that of PKC-alpha, PKC-theta overexpression in murine EL4 thymoma cells caused a significant increase in phorbol 12-myristate 13-acetate (PMA)-induced transcriptional activation of full-length IL-2-chloramphenicol acetyltransferase (CAT) and NF-AT-CAT but not of NF-IL2A-CAT or NF-kappaB promoter-CAT reporter gene constructs. Importantly, the critical AP-1 enhancer element was differentially modulated by these two distinct PKC isoenzymes, since only PKC-theta but not PKC-alpha overexpression resulted in an approximately 2.8-fold increase in AP-1-collagenase promoter CAT expression in comparison with the vector control. Deletion of the AP-1 enhancer site in the collagenase promoter rendered it unresponsive to PKC-theta. Expression of a constitutively active mutant PKC-theta A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-theta K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-RasS17N completely inhibited the PKC-O A148E-induced signal, PKC-O. Expression of a constitutively active mutant PKC-O A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-O K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-enRasS17N completely inhibited in the PKC-O A148E-induced signal, identifying PKC-theta as a specific constituent upstream of or parallel to Ras in the signaling cascade leading to AP transcriptional activation.

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CHOP Enhancement of Gene Transcription by Interactions with Jun/Fos AP-1 Complex Proteins

Molecular and Cellular Biology ◽

10.1128/mcb.19.11.7589 ◽

1999 ◽

Vol 19 (11) ◽

pp. 7589-7599 ◽

Cited By ~ 95

Author(s):

Mariano Ubeda ◽

Mario Vallejo ◽

Joel F. Habener

Keyword(s):

Transcription Factor ◽

Gene Transcription ◽

Stress Responses ◽

Leucine Zipper ◽

Target Genes ◽

Cellular Stress ◽

Dominant Negative ◽

Dimerization Domain ◽

Mobility Shift

ABSTRACT The transcription factor CHOP (C/EBP homologous protein 10) is a bZIP protein induced by a variety of stimuli that evoke cellular stress responses and has been shown to arrest cell growth and to promote programmed cell death. CHOP cannot form homodimers but forms stable heterodimers with the C/EBP family of activating transcription factors. Although initially characterized as a dominant negative inhibitor of C/EBPs in the activation of gene transcription, CHOP-C/EBP can activate certain target genes. Here we show that CHOP interacts with members of the immediate-early response, growth-promoting AP-1 transcription factor family, JunD, c-Jun, and c-Fos, to activate promoter elements in the somatostatin, JunD, and collagenase genes. The leucine zipper dimerization domain is required for interactions with AP-1 proteins and transactivation of transcription. Analyses by electrophoretic mobility shift assays and by an in vivo mammalian two-hybrid system for protein-protein interactions indicate that CHOP interacts with AP-1 proteins inside cells and suggest that it is recruited to the AP-1 complex by a tethering mechanism rather than by direct binding of DNA. Thus, CHOP not only is a negative or a positive regulator of C/EBP target genes but also, when tethered to AP-1 factors, can activate AP-1 target genes. These findings establish the existence of a new mechanism by which CHOP regulates gene expression when cells are exposed to cellular stress.

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Promoter-Proximal Regulatory Elements Involved in oriP-EBNA1-Independent and -Dependent Activation of the Epstein-Barr Virus C Promoter in B-Lymphoid Cell Lines

Journal of Virology ◽

10.1128/jvi.75.13.5796-5811.2001 ◽

2001 ◽

Vol 75 (13) ◽

pp. 5796-5811 ◽

Cited By ~ 31

Author(s):

Tina Nilsson ◽

Henrik Zetterberg ◽

Yuyan Camilla Wang ◽

Lars Rymo

Keyword(s):

Transcription Factor ◽

Epstein Barr Virus ◽

Regulatory Elements ◽

Negative Regulator ◽

Regulatory Sequences ◽

Barr Virus ◽

Group I ◽

Epstein Barr ◽

B Lymphoid ◽

Virus C

ABSTRACT The identification of the cellular factors that control the transcription regulatory activity of the Epstein-Barr virus C promoter (Cp) is fundamental to the understanding of the molecular mechanisms that control virus latent gene expression. Using transient transfection of reporter plasmids in group I phenotype B-lymphoid cells, we have previously shown that the −248 to −55 region (−248/−55 region) of Cp contains elements that are essential fororiPI-EBNA1-dependent as well asoriPI-EBNA1-independent activation of the promoter. We now establish the importance of this region by a detailed mutational analysis of reporter plasmids carrying Cp regulatory sequences together with or without oriPI. The reporter plasmids were transfected into group I phenotype Rael cells and group III phenotype cbc-Rael cells, and the Cp activity measured was correlated with the binding of candidate transcription factors in electrophoretic mobility shift assays and further assessed in cotransfection experiments. We show that the NF-Y transcription factor interacts with the previously identified CCAAT box in the −71/−63 Cp region (M. T. Puglielli, M. Woisetschlaeger, and S. H. Speck, J. Virol. 70:5758–5768, 1996). We also show that members of the C/EBP transcription factor family interact with a C/EBP consensus sequence in the −119/−112 region of Cp and that this interaction is important for promoter activity. A central finding is the identification of a GC-rich sequence in the −99/−91 Cp region that is essential fororiPI-EBNA1-independent as well asoriPI-EBNA1-dependent activity of the promoter. This region contains overlapping binding sites for Sp1 and Egr-1, and our results suggest that Sp1 is a positive and Egr-1 is a negative regulator of Cp activity. Furthermore, we demonstrate that a reporter plasmid that in addition to oriPI contains only the −111/+76 region of Cp still retains the ability to be activated by EBNA1.

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