Nuclear Factor Binding to a DNA Sequence Element That Represses MMTV Transcription Induces a Structural Transition and Leads to the Contact of Single-Stranded Binding Proteins with DNA

Giffin, Ward; Haché, Robert J.G.

doi:10.1089/dna.1995.14.1025

Cited by 10 publications

(13 citation statements)

References 59 publications

(44 reference statements)

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“…KMnO 4 and dimethylsulfate sequence reactions were performed as previously described (27) on the same plasmids in linear, denatured form, and primer extension was also used to position sites of KMnO 4 modification.…”

Section: Methodsmentioning

confidence: 99%

“…1, lanes 1 and 2). Under these stringent binding conditions, which included the addition of 2 g of highly sheared calf thymus DNA competitor DNA, no Ku binding was detected on the double-stranded DNA ends of a nonspecific oligonucleotide (27). Incubation of dsNRE1 and the upper, single strand with recombinant Ku resulted in the formation of a similar complex (lanes 5 and 6).…”

Section: Repression Of Mmtv Transcription By Dna-pk Correlates With Tmentioning

confidence: 97%

“…Ku induces a structural transition in the MMTV LTR upstream of NRE1. We have, however, previously reported that sequences upstream from NRE1 in the MMTV LTR between positions Ϫ395 and Ϫ420 undergo a Mg 2ϩ -dependent structural transition in the presence of crude nuclear T-cell extract that is detected by single-stranded-DNA-specific cleavage agents, including KMnO 4 (27). KMnO 4 is a reagent that specifically modifies T's in single-stranded DNA or DNAs containing double-to single-strand transitions (25).…”

Section: Repression Of Mmtv Transcription By Dna-pk Correlates With Tmentioning

confidence: 99%

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Ku Antigen-DNA Conformation Determines the Activation of DNA-Dependent Protein Kinase and DNA Sequence-Directed Repression of Mouse Mammary Tumor Virus Transcription

Giffin¹,

Gong²,

Schild-Poulter³

et al. 1999

Molecular and Cellular Biology

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Mouse mammary tumor virus (MMTV) transcription is repressed by DNA-dependent protein kinase (DNA-PK) through a DNA sequence element, NRE1, in the viral long terminal repeat that is a sequence-specific DNA binding site for the Ku antigen subunit of the kinase. While Ku is an essential component of the active kinase, how the catalytic subunit of DNA-PK (DNA-PK cs ) is regulated through its association with Ku is only beginning to be understood. We report that activation of DNA-PK cs and the repression of MMTV transcription from NRE1 are dependent upon Ku conformation, the manipulation of DNA structure by Ku, and the contact of Ku80 with DNA. Truncation of one copy of the overlapping direct repeat that comprises NRE1 abrogated the repression of MMTV transcription by Ku-DNA-PK cs . Remarkably, the truncated element was recognized by Ku-DNA-PK cs with affinity similar to that of the full-length element but was unable to promote the activation of DNA-PK cs . Analysis of Ku-DNA-PK cs interactions with DNA ends, double-and single-stranded forms of NRE1, and the truncated NRE1 element revealed striking differences in Ku conformation that differentially affected the recruitment of DNA-PK cs and the activation of kinase activity.The long terminal repeat (LTR) of mouse mammary tumor virus (MMTV) includes a complex transcriptional regulatory region that is strongly responsive to steroid hormones and prolactin (5, 6, 13-15, 18, 37, 39, 67, 73, 83). Tumorigenesis is mediated through the insertional activation of cellular protooncogenes (12,42,84). In addition to the promoter-proximal hormone response element and distal prolactin-responsive region, MMTV also contains several DNA sequence elements in the central portion of the LTR that repress or limit virally induced transcription and the response of the virus to steroid hormones (3,51,55,56,76,77,90). At least some of these elements appear to function to restrict virally induced gene expression and tumorigenesis to the lactating mammary gland, where the effects of prolactin and steroid converge to overcome the negative regulatory elements to promote a strong induction of virally induced transcription. Deletion of portions of the viral LTR that include one or more of the negative regulatory sequences deregulates MMTV-induced transcription and leads to virally induced tumors at sites not normally observed with wild-type virus, most notably T-cell lymphoma (40,77,90,91).Studies by several groups have shown that one of the negative regulatory sequences in the MMTV LTR that acts to repress viral transcription occurs in a region between 350 and 400 bp upstream of the viral transcriptional initiation site (21,40,77,85,90). We previously delimited the repressor element within this region, NRE1, to 23 bp of DNA centered over a sequence containing an overlapping direct repeat of the sequence GAGAAAGA (31). NRE1 was shown to inhibit transcription from the MMTV promoter-proximal regulatory region in T cells and in transformed mammary fibroblasts derived from an MMTV-induced tumor (31). De...

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Section: Methodsmentioning

confidence: 99%

Section: Repression Of Mmtv Transcription By Dna-pk Correlates With Tmentioning

confidence: 97%

Section: Repression Of Mmtv Transcription By Dna-pk Correlates With Tmentioning

confidence: 99%

See 1 more Smart Citation

Ku Antigen-DNA Conformation Determines the Activation of DNA-Dependent Protein Kinase and DNA Sequence-Directed Repression of Mouse Mammary Tumor Virus Transcription

Giffin¹,

Gong²,

Schild-Poulter³

et al. 1999

Molecular and Cellular Biology

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“…Similarly, the shift in PARP-1 mobility in SDS-PAGE that marks its automodification was the same in MO59J/Fus1 cells that express DNA-PK and MO59J/Fus9 cells that are DNA-PK deficient (Figure 4b). Thus DNA-PK signaling expressing WT Ku (V79), clone V15B lacking Ku80 (V15B) and V15B cells supplemented with a cDNA expressing human Ku80 (V15B/Ku80) that reconstitutes Ku levels and function (Giffin & Hache´, 1995;Schild-Poulter et al, 2003). Bleomycin (bleo) treatment (1 h, 30 mg/ml) was as indicated.…”

Section: Resultsmentioning

confidence: 99%

Activation of PARP-1 in response to bleomycin depends on the Ku antigen and protein phosphatase 5

Fang¹,

Soubeyrand²,

Haché

2010

Oncogene

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Poly (ADP-ribose) polymerase-1 (PARP-1) has an important role in the cellular response to a broad spectrum of DNA lesions. PARP-1 is strongly activated in response to double-stranded DNA breaks (DSBs), yet its contribution to the DSB response is poorly understood. Here we used bleomycin, a radiomimetic that generates DSBs with high specificity to focus on the response of PARP-1 to DSBs. We report that the induction of PARP-1 activity by bleomycin depends on the Ku antigen, a nonhomologous-DNA-End-Joining factor and protein phosphatase 5 (PP5). PARP-1 activation in response to bleomycin was reduced over 10-fold in Ku-deficient cells, whereas its activation in response to U.V. was unaffected. PARP-1 activation was rescued by reexpression of Ku, but was refractory to manipulation of DNA-dependent protein kinase or ATM. Similarly, PARP-1 activation subsequent to bleomycin was reduced 2-fold on ablation of PP5 and was increased 5-fold when PP5 was overexpressed. PP5 seemed to act directly on PARP-1, as its basal phosphorylation was reduced on overexpression of PP5, and PP5 dephosphorylated PARP-1 in vitro. These results highlight the functional importance of Ku antigen and PP5 for PARP-1 activity subsequent to DSBs.

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“…The link between proliferation, promoter activity, S1 sensitivity, and expression or activity of the single-stranded binding proteins, suggest that single-stranded proteins identified in this work exert a positive role in transcription. A growing number of single-stranded DNA-binding proteins have now been found to interact with regulatory elements and influence transcription by altering the DNA topology or conformation (33)(34)(35)(36)(37). Extensive studies of the c-myc promoter (37)(38)(39)(40) are suggestive of a model in which the cellular conditions control the interaction of double-stranded or single-stranded DNAbinding factors to specific promoter elements; the binding of proteins to single DNA strands is thought to induce structural transitions that are sensed as transcriptional signals and mediate c-myc response to cell cycle regulators, growth factors, and other inducing stimuli.…”

Section: Discussionmentioning

confidence: 99%

Interactions with Single-stranded and Double-stranded DNA-binding Factors and Alternative Promoter Conformation upon Transcriptional Activation of the Htf9-a/RanBP1 and Htf9-c Genes

Matteo¹,

Salerno²,

Guarguaglini³

et al. 1998

Journal of Biological Chemistry

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The murine Htf9-a/RanBP1 and Htf9-c genes are divergently transcribed from a shared TATA-less promoter. Transcription of both genes is initiated on complementary DNA strands and is controlled by cell cycledependent mechanisms. The bidirectional promoter harbors a genomic footprint flanking the major transcription start site of both genes. Transient promoter assays showed that the footprinted element is important for transcription of both genes. Protein-binding experiments and antibody assays indicated that members of the retinoid X receptor family interact with the doublestranded site. In addition, distinct factors interact with single DNA strands of the element. Double-stranded binding factors were highly expressed in liver cells, in which neither gene is transcribed, while single-stranded binding proteins were abundant in cycling cells, in which transcription of both genes is efficient. In vivo S1 analysis of the promoter depicted an S1-sensitive organization in cells in which transcription of both genes is active; S1 sensitivity was not detected in conditions of transcriptional repression. Thus, the same element is a target for either retinoid X receptor factors, or for single-stranded binding proteins, and form distinct complexes in different cellular conditions depending on the DNA conformation in the binding site.The murine Htf9 locus was isolated by virtue of its association with a CG-rich genomic sequence (1) and mapped to mouse chromosome 16 (2). The locus contains two transcriptional units, Htf9-a and Htf9-c, that are transcribed with opposite polarity from complementary DNA strands (see map in Fig. 1A). The lower strand gene, called Htf9-a, encodes Ran-binding protein 1 (RanBP1) 1 (3, 4), an interacting partner of the Ran GTPase, which is thought to cooperate in control of several processes regulated by the Ran network and including DNA replication, cell cycle progression, mitotic entry and exit, nuclear structure, and nucleo/cytoplasmic transport (reviewed in Refs. 5 and 6). The Htf9-c gene codes for a protein sharing extensive homologies with nucleic acid-modifying enzymes, including bacterial tRNA methyltransferases and yeast DNA endo-exonucleases (7); the evolutionary conservation of the protein sequence in the nucleic acid-interacting domain suggests that the murine Htf9-c gene product may also be implicated in nucleic acid metabolism and/or modifications. Both Htf9 divergently transcribed genes are expressed at low levels in many tissues and cell types (1, 8) and are up-regulated in proliferating cells: transcription is activated at the G 1 /S transition of the cell cycle and peaks in S phase, while being repressed in quiescent tissues and growth-arrested cells (7, 9). Promoter deletion and site-directed mutagenesis assays have revealed that cell cycle expression of both divergently transcribed genes is controlled by separate E2F and Sp1 promoter elements active in each orientation (7).Both the RanBP1 and Htf9-c transcriptional units are initiated in a genomic sequence that has typical features...

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Nuclear Factor Binding to a DNA Sequence Element That Represses MMTV Transcription Induces a Structural Transition and Leads to the Contact of Single-Stranded Binding Proteins with DNA

Cited by 10 publications

References 59 publications

Ku Antigen-DNA Conformation Determines the Activation of DNA-Dependent Protein Kinase and DNA Sequence-Directed Repression of Mouse Mammary Tumor Virus Transcription

Ku Antigen-DNA Conformation Determines the Activation of DNA-Dependent Protein Kinase and DNA Sequence-Directed Repression of Mouse Mammary Tumor Virus Transcription

Activation of PARP-1 in response to bleomycin depends on the Ku antigen and protein phosphatase 5

Interactions with Single-stranded and Double-stranded DNA-binding Factors and Alternative Promoter Conformation upon Transcriptional Activation of the Htf9-a/RanBP1 and Htf9-c Genes

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