Evidence for a Complex of Transcription Factor IIB (TFIIB) with Poly(A) Polymerase and Cleavage Factor 1 Subunits Required for Gene Looping

Department of Biological Science, Wayne State University, Detroit, Michigan 48202, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 08/2011; 286(39):33709-18. DOI: 10.1074/jbc.M110.193870
Source: PubMed


Gene looping, defined as the interaction of the promoter and the terminator regions of a gene during transcription, requires transcription factor IIB (TFIIB). We have earlier demonstrated association of TFIIB with the distal ends of a gene in an activator-dependent manner (El Kaderi, B., Medler, S., Raghunayakula, S., and Ansari, A. (2009) J. Biol. Chem. 284, 25015-25025). The presence of TFIIB at the 3' end of a gene required its interaction with cleavage factor 1 (CF1) 3' end processing complex subunit Rna15. Here, employing affinity chromatography and glycerol gradient centrifugation, we show that TFIIB associates with poly(A) polymerase and the entire CF1 complex in yeast cells. The factors required for general transcription such as TATA-binding protein, RNA polymerase II, and TFIIH are not a component of the TFIIB complex. This holo-TFIIB complex was resistant to MNase digestion. The complex was observed only in the looping-competent strains, but not in the looping-defective sua7-1 strain. The requirement of Rna15 in gene looping has been demonstrated earlier. Here we provide evidence that poly(A) polymerase (Pap1) as well as CF1 subunits Rna14 and Pcf11 are also required for loop formation of MET16 and INO1 genes. Accordingly, cross-linking of TFIIB to the 3' end of genes was abolished in the mutants of Pap1, Rna14, and Pcf11. We further show that in sua7-1 cells, where holo-TFIIB complex is not formed, the kinetics of activated transcription is altered. These results suggest that a complex of TFIIB, CF1 subunits, and Pap1 exists in yeast cells. Furthermore, TFIIB interaction with the CF1 complex and Pap1 is crucial for gene looping and transcriptional regulation.

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    • "Looped genes contain TFIIB at their 5 0 and 3 0 ends in yeast (Singh and Hampsey 2007). Gene-loop formation is impaired by defects in TFIIB and other transcription initiation proteins (Medler et al. 2011). Previous results showed the dependence of gene looping on Ssu72 and Pta1, which are components of the 3 0 end processing complex in yeast (Pappas and Hampsey 2000; Ansari and Hampsey 2005; Zhang et al. 2012). "
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    ABSTRACT: Gene looping, defined as the physical interaction between the promoter and terminator regions of a RNA polymerase II-transcribed gene, is widespread in yeast and mammalian cells. Gene looping has been shown to play important roles in transcription. Gene-loop formation is dependent on regulatory proteins localized at the 5' and 3' ends of genes, such as TFIIB. However, whether other factors contribute to gene looping remains to be elucidated. Here, we investigated the contribution of intrinsic DNA and chromatin structures to gene looping. We found that Saccharomyces cerevisiae looped genes show high DNA bendability around middle and 3/4 regions in open reading frames (ORFs). This bendability pattern is conserved between yeast species, whereas the position of bendability peak varies substantially among species. Looped genes in human cells also show high DNA bendability. Nucleosome positioning around looped ORF middle regions are unstable. We also present evidence indicating that this unstable nucleosome positioning is involved in gene looping. These results suggest a mechanism by which DNA bendability and unstable nucleosome positioning could assist in the formation of gene loops.
    Preview · Article · Oct 2013 · Molecular Biology and Evolution
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    • "In yeast, gene loop formation requires an interaction between general transcription factors and 3 0 RNA processing factors (Ansari and Hampsey, 2005; Medler et al, 2011; Tan- Wong et al, 2012). However, mutations in Arabidopsis polyadenylation factors FCA and FPA did not disrupt the FI–FV interaction (Figure 2A) despite increasing FLC transcript levels 20-fold (Figure 2B). "
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    ABSTRACT: Gene activation in eukaryotes frequently involves interactions between chromosomal regions. We have investigated whether higher-order chromatin structures are involved in the regulation of the Arabidopsis floral repressor gene FLC, a target of several chromatin regulatory pathways. Here, we identify a gene loop involving the physical interaction of the 5' and 3' flanking regions of the FLC locus using chromosome conformation capture. The FLC loop is unaffected by mutations disrupting conserved chromatin regulatory pathways leading to very different expression states. However, the loop is disrupted during vernalization, the cold-induced, Polycomb-dependent epigenetic silencing of FLC. Loop disruption parallels timing of the cold-induced FLC transcriptional shut-down and upregulation of FLC antisense transcripts, but does not need a cold-induced PHD protein required for the epigenetic silencing. We suggest that gene loop disruption is an early step in the switch from an expressed to a Polycomb-silenced state.
    Full-text · Article · Dec 2012 · The EMBO Journal
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    • "These include in mammals CPSF, CstF, and CFI (Glover-Cutter et al., 2008; Rozenblatt-Rosen et al., 2009; Venkataraman et al., 2005). PAP has long been known to associate only loosely with the other core polyadenylation factors (Takagaki et al., 1988), but recently it was reported to crosslink to both 5 0 and 3 0 end of genes in yeast, where it is necessary for gene looping (Medler et al., 2011). These data are consistent with our results showing that PAP can be recruited to the 3 0 ends of transcribed genes in human cells, implying that, even if polyadenylation occurs after release of the mRNA from RNAP II, PAP joins the 3 0 processing complex cotranscriptionally. "
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    ABSTRACT: The 3' ends of most eukaryotic mRNAs are produced by an endonucleolytic cleavage followed by synthesis of a poly(A) tail. Poly(A) polymerase (PAP), the enzyme that catalyzes the formation of the tail, is subject to tight regulation involving several posttranslational modifications. Here we show that the enzyme poly(ADP-ribose) polymerase 1 (PARP1) modifies PAP and regulates its activity both in vitro and in vivo. PARP1 binds to and modifies PAP by poly(ADP-ribosyl)ation (PARylation) in vitro, which inhibits PAP activity. In vivo we show that PAP is PARylated during heat shock, leading to inhibition of polyadenylation in a PARP1-dependent manner. The observed inhibition reflects reduced RNA binding affinity of PARylated PAP in vitro and decreased PAP association with non-heat shock protein-encoding genes in vivo. Our results provide direct evidence that PARylation can control processing of mRNA precursors, and also identify PARP1 as a regulator of polyadenylation during thermal stress.
    Preview · Article · Dec 2012 · Molecular cell
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