TFIIIB subunit Bdp1p is required for periodic integration of the Ty1 retrotransposon and targeting of Isw2p to <i>S. cerevisiae</i> tDNAs

Bachman, Nurjana; Gelbart, Marnie E.; Tsukiyama, Toshio; Boeke, Jef D.

doi:10.1101/gad.1299105

Cited by 78 publications

(63 citation statements)

References 42 publications

(71 reference statements)

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“…Much of Bdp1 can be deleted with retention of yeast viability (the N-terminal 240 amino acids, the C-terminal 108 amino acids, 68 amino acids between residues 312 and 372, and 17 amino acids between 253 and 269), leaving just 161 Bdp1 residues that are essential (26,28). Nevertheless, there are multiple sites of interaction between Bdp1 and the Brf1⅐TBP⅐DNA complex that independently suffice for TFIIIB-DNA complex formation and transcription, and this has been an impediment to identifying the individual sites within Brf1 that are involved in Bdp1 interaction.…”

Section: Discussionmentioning

confidence: 99%

Mapping the Principal Interaction Site of the Brf1 and Bdp1 Subunits of Saccharomyces cerevisiae TFIIIB

Kassavetis

Driscoll²,

Geiduschek

2006

Journal of Biological Chemistry

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The Brf1 subunit of the central RNA polymerase (pol) III transcription initiation factor TFIIIB is bipartite; its N-terminal TFIIBrelated half is principally responsible for recruiting pol III to the promoter and for promoter opening near the transcriptional start site, whereas its pol III-specific C-terminal half contributes most of the affinities that hold the three subunits of TFIIIB together. Here, the principal attachment site of Brf1 for the Bdp1 subunit of TFIIIB has been mapped by a combination of structure-informed, site-directed mutagenesis and photochemical protein-DNA cross-linking. A 66-amino acid segment of Brf1 is shown to serve as a two-sided adhesive surface, with the side chains projecting away from its extended interface with TATA-binding protein anchoring Bdp1 binding. An extensive collection of N-terminal, C-terminal, and internal deletion proteins has been used to demarcate the interacting Bdp1 domain to a 66-amino acid segment that includes the SANT domain of this subunit and is phylogenetically the most conserved region of Bdp1.The RNA polymerase (pol) 3 III transcription apparatus of yeast comprises, in addition to the polymerase itself, three transcription initiation factors, TFIIIA, -B, and -C.The three-subunit TFIIIB is the core initiation factor required and sufficient for recruiting pol III to the promoter and accurately initiating transcription. The large six-subunit TFIIIC places TFIIIB on DNA upstream of the transcriptional start site and protects the generally very small pol III transcription units from encroachment by repressive chromatin (1, 2). TFIIIA is the 5 S rRNA gene-specific factor that places TFIIIC on the transcription unit-internal promoter of these genes (reviewed in Refs. 3-5).The work that is presented here deals with the structure of TFIIIB, which is composed of three subunits: the TATA-binding protein (TBP), Brf1, and Bdp1. TBP and Brf1 bind tightly and co-purify, whereas Bdp1 dissociates during later steps of conventional column chromatography. (The two separated fractions of TFIIIB have been designated BЈ and BЉ, and B double prime has been adopted as the eponym of the Bdp1 subunit.) The 596-amino acid Brf1 is a fusion protein, with an N-terminal half that is related to the pol II transcription initiation factor TFIIB and a pol III-specific C-terminal half. A conserved segment of the C-terminal half (homology domain II; amino acids 439 -515) constitutes the Brf1 high affinity binding site for the evolutionarily highly conserved core of TBP (comprising all but the N-terminal 60 amino acids of the Saccharomyces cerevisiae protein); the N-terminal half of Brf1 engages TBP through a separate lower affinity interaction.The N-and C-terminal halves of Brf1 also provide separate Bdp1-docking sites, with the C-proximal homology domain II contributing the higher Bdp1 affinity. TBP makes only a minor direct contribution to bringing Bdp1 into TFIIIB. Thus, the pol III-specific half of Brf1 appears to contribute the interactions that principally hold TFIIIB together. Ind...

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Section: Discussionmentioning

confidence: 99%

Mapping the Principal Interaction Site of the Brf1 and Bdp1 Subunits of Saccharomyces cerevisiae TFIIIB

Kassavetis

Driscoll²,

Geiduschek

2006

Journal of Biological Chemistry

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“…These findings elucidate the importance of BET proteins for MLV integration efficiency and targeting and provide a route to developing safer MLV-based vectors for human gene therapy. (1)(2)(3)(4). The selection of chromosomal targets for retroviral integration varies markedly, tracking with the genus of the retrovirus studied (5)(6)(7).…”

mentioning

confidence: 99%

BET proteins promote efficient murine leukemia virus integration at transcription start sites

Sharma¹,

Larue²,

Plumb³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

185

267

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The selection of chromosomal targets for retroviral integration varies markedly, tracking with the genus of the retrovirus, suggestive of targeting by binding to cellular factors. γ-Retroviral murine leukemia virus (MLV) DNA integration into the host genome is favored at transcription start sites, but the underlying mechanism for this preference is unknown. Here, we have identified bromodomain and extraterminal domain (BET) proteins (Brd2, -3, -4) as cellular-binding partners of MLV integrase. We show that purified recombinant Brd4(1-720) binds with high affinity to MLV integrase and stimulates correct concerted integration in vitro. JQ-1, a small molecule that selectively inhibits interactions of BET proteins with modified histone sites impaired MLV but not HIV-1 integration in infected cells. Comparison of the distribution of BET protein-binding sites analyzed using ChIP-Seq data and MLV-integration sites revealed significant positive correlations. Antagonism of BET proteins, via JQ-1 treatment or RNA interference, reduced MLV-integration frequencies at transcription start sites. These findings elucidate the importance of BET proteins for MLV integration efficiency and targeting and provide a route to developing safer MLV-based vectors for human gene therapy. (1-4). The selection of chromosomal targets for retroviral integration varies markedly, tracking with the genus of the retrovirus studied (5-7). For example, the γ-retroviruses favor integration near transcription start sites, whereas lentiviruses favor integration within transcription units. These observations have suggested that different cellularbinding partners of retroviral integrases are likely to be responsible for integration target-site selection. However, to date, only one example has been reported: lens epithelium-derived growth factor (LEDGF/p75), which functions as a bimodal tether that engages HIV-1 intasomes and navigates them to active genes (8-14). Cellular cofactors of other retroviral genera are currently unknown.The molecular mechanisms of γ-retroviral murine leukemia virus (MLV) integration are of particular significance because MLV-based vectors are used for human gene therapy. In clinical trials, the use of γ-retroviral vectors to correct primary immunodeficiencies has been curative, but adverse events have occurred associated with insertion of MLV-based vectors near protooncogenes (reviewed in refs. 15-18). The identification of cellular factors for γ-retroviruses may provide mechanistic clues to facilitate the development of safer gene-therapy vectors.In this report, we have identified the bromodomain and extraterminal domain (BET) proteins (Brd2, -3, -4) as the cellularbinding partners of MLV IN and demonstrate their significance for stimulating and targeting MLV integration at transcription start sites. (Table 1, Table S1, and Fig. S1). Of these, Brd4 and Brd3 were the top hits in NIH 3T3 and Sup-T1 cells, respectively. Differential pull-down levels of these proteins (Table 1) could be attributable to the varying expression le...

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“…Because deletion of ISW2 does not affect chromatin structure at the rDNA, we cannot determine if rDNA silencing requires direct association of the Isw2 protein with the rDNA. Interestingly, recruitment of Isw2 to sequences upstream of tRNA genes requires Bdp1, a subunit of the Pol III transcription factor TFIIIB [40]. Thus, it is not unreasonable to speculate that Bdp1 recruits Isw2 to the Pol III-transcribed 5S rRNA gene in the rDNA, an association that may contribute to Isw2's role in rDNA silencing.…”

Section: Deletion Of Isw2 Does Not Alter Nucleosome Positioning At Thmentioning

confidence: 99%

Isw2 regulates gene silencing at the ribosomal DNA locus in Saccharomyces cerevisiae

Mueller

Bryk

2007

Biochemical and Biophysical Research Communications

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Three heterochromatin-like domains have been identified in S. cerevisiae that are refractory to transcription by Pol II, the silent mating-type loci, telomeres and the ribosomal DNA. Previous work has shown that chromatin remodelers can regulate silent chromatin. Here, we report the findings of an investigation into the role of ISW2 in transcriptional silencing at the rDNA. We show that the levels of retrotransposition and mRNA from a genetically marked Ty1 element located in the rDNA were increased significantly in isw2Δ cells, while transcript levels from Ty1 elements outside of the rDNA were not increased in cells lacking ISW2. Additionally, we show that Isw2 is not required for silencing at a telomere. Our findings demonstrate that Isw2 is required for transcriptional silencing at the rDNA and emphasize the differences in the regulation of transcriptional silencing at silent loci in S. cerevisiae.

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TFIIIB subunit Bdp1p is required for periodic integration of the Ty1 retrotransposon and targeting of Isw2p to S. cerevisiae tDNAs

Cited by 78 publications

References 42 publications

Mapping the Principal Interaction Site of the Brf1 and Bdp1 Subunits of Saccharomyces cerevisiae TFIIIB

Mapping the Principal Interaction Site of the Brf1 and Bdp1 Subunits of Saccharomyces cerevisiae TFIIIB

BET proteins promote efficient murine leukemia virus integration at transcription start sites

Isw2 regulates gene silencing at the ribosomal DNA locus in Saccharomyces cerevisiae

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