Characterization of a Silencer Element and Purification of a Silencer Protein That Negatively Regulates the Human Adenine Nucleotide Translocator 2 Promoter

Baráth, Péter; Albert-Fournier, Benjamin; Luciaková, Katarína; Nelson, Bruce R.

doi:10.1074/jbc.274.6.3378

Cited by 23 publications

(50 citation statements)

References 46 publications

(24 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…That the Go-1 and Go-2 elements described here are responsible for growth arrest repression of ANT2 is strengthened by experiments that exclude the participation of additional repressor or silencer regions reported to be present in the ANT2 promoter (26,41,42). One of these is an Sp1-binding element FIG.…”

Section: Discussionmentioning

confidence: 96%

“…We have also identified a putative silencer centered at nt Ϫ332 in the ANT2 promoter (41). To test the contribution of this region to growth arrest repression, promoter-driven reporter gene constructs were prepared with deletions between the Go element and the AB boxes, some of which (clones ⌬Ϫ692/Ϫ235 and ⌬Ϫ546/Ϫ235) (Fig.…”

Section: Mapping Of Proteins Bound To the Ant2 Promoter In Growth-mentioning

confidence: 99%

See 1 more Smart Citation

Repression of the Human Adenine Nucleotide Translocase-2 Gene in Growth-arrested Human Diploid Cells

Luciaková

Baráth

Poliakova

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

Adenine nucleotide translocase-2 (ANT2) catalyzes the exchange of ATP for ADP across the mitochondrial membrane, thus playing an important role in maintaining the cytosolic phosphorylation potential required for cell growth. Expression of ANT2 is activated by growth stimulation of quiescent cells and is down-regulated when cells become growth-arrested. In this study, we address the mechanism of growth arrest repression. Using a combination of transfection, in vivo dimethyl sulfate mapping, and in vitro DNase I mapping experiments, we identified two protein-binding elements (Go-1 and Go-2) that are responsible for growth arrest of ANT2 expression in human diploid fibroblasts. Proteins that bound the Go elements were purified and identified by matrix-assisted laser desorption ionization time-offlight mass spectrometry as members of the NF1 family of transcription factors. Chromatin immunoprecipitation analysis showed that NF1 was bound to both Go-1 and Go-2 in quiescent human diploid cells in vivo, but not in the same cells stimulated to growth by serum. NF1 binding correlated with the disappearance of ANT2 transcripts in quiescent cells. Furthermore, overexpression of NF1-A, -C, and -X in NIH3T3 cells repressed expression of an ANT2-driven reporter gene construct. Two additional putative repressor elements in the ANT2 promoter, an Sp1 element juxtaposed to the transcription start site and a silencer centered at nucleotide ؊332, did not appear to contribute to growth arrest repression. Thus, enhanced binding of NF1 is a key step in the growth arrest repression of ANT2 transcription. To our knowledge, this is the first report showing a role for NF1 in growth arrest.

show abstract

Section: Discussionmentioning

confidence: 96%

Section: Mapping Of Proteins Bound To the Ant2 Promoter In Growth-mentioning

confidence: 99%

Repression of the Human Adenine Nucleotide Translocase-2 Gene in Growth-arrested Human Diploid Cells

Luciaková

Baráth

Poliakova

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…21 In non-proliferating cells ANT2 expression appears to be negatively regulated by the general transcription factor Sp1, 22 as well as specific hexanucleotide silencer element in the ANT2 promoter. 23 All ANT isoforms are members of the family formed by the carriers of the mitochondrial IM. Proteins from this family are characterized by a tripartite structure with three repeated segments of about 100 amino acid residues each.…”

Section: Introductionmentioning

confidence: 99%

Permeabilization of the mitochondrial inner membrane during apoptosis: impact of the adenine nucleotide translocator

et al. 2000

View full text Add to dashboard Cite

Mitochondrial membrane permeabilization can be a rate limiting step of apoptotic as well as necrotic cell death. Permeabilization of the outer mitochondrial membrane (OM) and/or inner membrane (IM) is, at least in part, mediated by the permeability transition pore complex (PTPC). The PTPC is formed in the IM/OM contact site and contains the two most abundant IM and OM proteins, adenine nucleotide translocator (ANT, in the IM) and voltage-dependent anion channel (VDAC, in the OM), the matrix protein cyclophilin D, which can interact with ANT, as well as apoptosis-regulatory proteins from the Bax/Bcl-2 family. Here we discuss that ANT has two opposite functions. On the one hand, ANT is a vital, specific antiporter which accounts for the exchange of ATP and ADP on IM. On the other hand, ANT can form a non-specific pore, as this has been shown by electrophysiological characterization of purified ANT reconstituted into synthetic lipid bilayers or by measuring the permeabilization of proteoliposomes containing ANT. Pore formation by ANT is induced by a variety of different agents (e.g. Ca 2+ , atractyloside, thiol oxidation, the pro-apoptotic HIV-1 protein Vpr, etc.) and is enhanced by Bax and inhibited by Bcl-2, as well as by ADP. In isolated mitochondria, pore formation by ANT leads to an increase in IM permeability to solutes up to 1500 Da, swelling of the mitochondrial matrix, and OM permeabilization, presumably due to physical rupture of OM. Although alternative mechanisms of mitochondrial membrane permeabilization may exist, ANT emerges as a major player in the regulation of cell death.

show abstract

“…This region contains two DNA elements, Go-1 and Go-2, that bind nuclear factor 1 (NF1) in growth-arrested cells, but not in growthactivated cells [13]. NF1 binding is associated with growth arrest repression of ANT2 transcription.A third repressor (silencer) region, which does not participate in growth arrest repression of the gene [13], but confers repression on a heterologous herpes simplex virus thymidine kinase (HSVtk) reporter gene, has also been located in the ANT2 promoter between the AB activation boxes and the Go repressor region [14]. Two DNA sequences in the silencer region (Site-2 and Site-3) are strongly protected in the DNase I assay.…”

mentioning

confidence: 99%

“…A third repressor (silencer) region, which does not participate in growth arrest repression of the gene [13], but confers repression on a heterologous herpes simplex virus thymidine kinase (HSVtk) reporter gene, has also been located in the ANT2 promoter between the AB activation boxes and the Go repressor region [14]. Two DNA sequences in the silencer region (Site-2 and Site-3) are strongly protected in the DNase I assay.…”

mentioning

confidence: 99%

Identification of NF1 as a silencer protein of the human adenine nucleotide translocase‐2 gene

Baráth

Poliakova

Luciaková

et al. 2004

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

The human adenine nucleotide translocase-2 (ANT2) promoter contains a silencer region that confers partial repression on the heterologous herpes simplex virus thymidine kinase (HSVtk) promoter [Barath, P., Albert-Fournier, B., Luciakova, K., Nelson, B.D. (1999) J. Biol. Chem. 274, 3378-3384]. Two sequences in the silencer (Site-2 and Site-3) are protected in the DNase I assay in vitro, and one of these is a repeated GTCCTG element previously shown to act as the active repressor element. We have now purified the DNA binding protein, and identified it using MALDI-TOF MS as a 33-kDa member of the nuclear factor 1 (NF1) family of transcription factors. NF1 purified from rat liver and HeLa cell nuclei bind to both silencer Site-2 and Site-3, resulting in a DNase I footprint identical to that obtained with purified recombinant NF1. Furthermore, transient transfection experiments with reporter constructs containing mutated silencer Site-2 and/or Site-3 show that both sites contribute to repression of the HSVtk promoter. Finally, chromatin immunoprecipitation analysis reveals that NF1 is bound to both elements on the endogenous HeLa cell ANT2 promoter. Our data support the belief that NF1 acts as a repressor when bound to silencing Site-2 and Site-3 of the ANT2 gene.Keywords: adenine nucleotide translocase; NF1; promoter regulation; silencer protein; transcription.Two major isoforms of the adenine nucleotide translocase (ANT) are expressed in mammalian cells. Both catalyse the exchange of mitochondrial ATP for cytosolic ADP, thereby playing key roles in maintaining the cytosolic phosphorylation potential, adenylate charge, and the energy status of the cell. The two forms, ANT1 and ANT2 [1][2][3][4], are differentially expressed in mammalian tissue. ANT1 is expressed predominately in heart and skeletal muscle [5,6] whereas ANT2 is more widely expressed [5,7,8]. ANT2 is strongly growth regulated [9,10], and expression of the gene is downregulated in growth arrested cells [10] and re-activated in cells entering the G 1 growth phase. Activation of ANT2 expression is mediated at the level of transcription [10].To understand ANT2 expression, we have undertaken a study of its promoter. Constitutive ANT2 expression is maintained by two synergistically interacting Sp1 elements (the AB boxes) in the proximal promoter [11]. However, activation via the AB box Sp1 is modulated by three separate repressor regions. One of these is an Sp1 binding element (C box) juxtaposed to the transcriptional start site, which, when occupied, decreases ANT2 expression severalfold [11]. Repression appears to involve a direct interaction between Sp1 bound to the AB and C boxes [12]. A second repressor region, that is responsible for ANT2 down regulation in growth-arrested cells, has recently been identified in the distal promoter [10]. This region contains two DNA elements, Go-1 and Go-2, that bind nuclear factor 1 (NF1) in growth-arrested cells, but not in growthactivated cells [13]. NF1 binding is associated with growth arrest repression of ANT2 tr...

show abstract

Characterization of a Silencer Element and Purification of a Silencer Protein That Negatively Regulates the Human Adenine Nucleotide Translocator 2 Promoter

Cited by 23 publications

References 46 publications

Repression of the Human Adenine Nucleotide Translocase-2 Gene in Growth-arrested Human Diploid Cells

Repression of the Human Adenine Nucleotide Translocase-2 Gene in Growth-arrested Human Diploid Cells

Permeabilization of the mitochondrial inner membrane during apoptosis: impact of the adenine nucleotide translocator

Identification of NF1 as a silencer protein of the human adenine nucleotide translocase‐2 gene

Contact Info

Product

Resources

About