A Transcription-Independent Role for TFIIB in Gene Looping Badri Nath Singh, Michael Hampsey  Molecular Cell  Volume 27, Issue 5, Pages 806-816 (September 2007) DOI: 10.1016/j.molcel.2007.07.013 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Gene Looping Is Not Idiosyncratic to Long Genes but Occurs at Four Different Genes Ranging in Length from 1.0 to 6.4 kb (A–D) Panels depict the BLM10, SAC3, GAL10, and HEM3 genes, respectively. The restriction sites (H, HindIII; E, EcoRI) and the divergent P1 and T1 primer pairs used in 3C analysis are indicated, along with flanking genes, all drawn to scale. ORF lengths are indicated under each gene. PCR primer sequences and their corresponding Saccharomyces genome database coordinates are listed in Table S1. The polarity of transcription is denoted by arrows. Gene looping was assayed prior to or following a 1 hr shift from the permissive (24°C) to the restrictive (37°C) temperature using the wild-type strain FY23 (lanes 1 and 2), the rpb1-1 mutant RS420 (lanes 3 and 4), and the ssu72-td degron strain XH-24 (lanes 5 and 6). Control PCR represents an intergenic region of chromosome V, generated using convergent primers. P1-T1 PCR products were quantified by dividing P1-T1 PCR signals by control PCR signals for each sample; these ratios were then divided by the ratio of the WT 24°C sample for each gene to yield the number depicted beneath each lane. Molecular Cell 2007 27, 806-816DOI: (10.1016/j.molcel.2007.07.013) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Convergent PCR Confirms that the BLM10 and HEM3 Genes Are Extensively Digested by HindIII during 3C Analysis (A) Schematic depiction of the BLM10 gene, depicting the positions of the HindIII sites and the F2 and R1 convergent PCR primers. (B) BLM10 F2-R1 PCR products using 3C samples either immediately prior to (uncut) or following restriction digestion (HindIII) from the same strains depicted in Figure 1 that had been incubated at either permissive (24°C) or restrictive (37°C) temperature (lanes 1–8), or from the same strains depicted in Figure 4 that had been incubated at 30°C (lanes 9 and 10). (C) Schematic depiction of the HEM3 gene, depicting the positions of the HindIII sites and the F1 and R1 convergent PCR primers. (D) HEM3 F1-R1 PCR products using 3C samples, as described in (B). The BLM10 F2 and R1 and the HEM3 F1 and R1 primers are identical to those depicted in Figure 3 and are defined in Table S1. Molecular Cell 2007 27, 806-816DOI: (10.1016/j.molcel.2007.07.013) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Gene Looping Juxtaposes the Promoter and Terminator Regions of BLM10 and HEM3 (A) General strategy for mapping juxtaposed regions in gene loops. 3C primer pairs are designed to detect promoter-ORF and terminator-ORF interactions. (B) Schematic depiction of the BLM10 gene, showing the positions of the HindIII sites and the promoter (P), terminator (T), forward (F), and reverse (R) primers used in the 3C analysis. (C) PCR products derived from the indicated primer pairs by 3C analysis. As the number of HindIII restriction sites between the divergent primer pairs increase, the PCR signals decrease, except for the P1-T1 primer pair, which yields a strong signal despite the presence of seven intervening HindIII sites. (D) Schematic depiction of the HEM3 gene, labeled as described for the BLM10 gene in (B). (E) PCR products derived from the indicated primer pairs by 3C analysis. The presence of a single HindIII restriction site between the P1 and F2 or T1 and R2 primer pairs diminishes the PCR signal, yet the P1-T1 primer pairs yield a strong signal despite the presence of both ORF HindIII sites. PCR products were quantified by normalizing each signal to the P1-T1 PCR signal. Molecular Cell 2007 27, 806-816DOI: (10.1016/j.molcel.2007.07.013) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 TFIIB Is Required for DNA Looping (A–E) Gene loops detected by 3C analysis using P1 and T1 PCR primers specific to the SEN1, BLM10, SAC3, GAL10, and HEM3 genes. Isogenic wild-type (YMH14) and sua7-1 (YMH124) strains were grown in YPD medium to mid-log phase. The sua7-1 allele encodes the E62K replacement in TFIIB (Pinto et al., 1994). P1-T1 PCR products were quantified as described in the Figure 1 legend. (F) The sua7-1 allele does not affect SEN1, BLM10, SAC3, GAL10, and HEM3 transcript levels, as detected by RT-PCR analysis. Transcript levels of ACT1 and 18S rRNA are included as controls. Molecular Cell 2007 27, 806-816DOI: (10.1016/j.molcel.2007.07.013) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 TFIIB Crosslinks to the Terminator Regions of the PMA1 and BLM10 Genes, Independent of TBP (A) Schematic depiction of the PMA1 gene showing the positions of the promoter (black box) and the two 3′ end processing/polyadenylation sites. The regions probed by ChIP are denoted 1–4 and 6–9. (B) ChIP analysis of TFIIB crosslinking to PMA1 using isogenic strains YMH14 (WT) and YMH124 (sua7-1). Chromatin was immunoprecipitated using polyclonal α-TFIIB antibody. Lanes correspond to the regions depicted in (A); lane V denotes an intergenic region of chromosome V. All PCR primer pairs used in this analysis are identical to those described previously (Komarnitsky et al., 2000). The input signal represents DNA prior to immunoprecipitation. (C) Quantification of the data shown in (B). (D) Schematic depiction of the BLM10 gene. The regions probed by ChIP are denoted 1–6. The 3′ end processing and poly(A) sites have not been reported for BLM10. The BLM10 ChIP primers are defined in Table S1. (E) ChIP analysis of TFIIB crosslinking to BLM10 using the same strains and antibody as in (B). (F) Quantification of the data shown in (E). (G) Identical to (B), except chromatin was immunoprecipitated using polyclonal α-TBP antibody. (H) Quantification of the data shown in (G). (I) Identical to (E), except chromatin was immunoprecipitated using polyclonal α-TBP antibody. (J) Quantification of the data shown in (I). For all ChIP experiments, factor association was quantified by dividing the IP:input signal for each region by the IP:input signal for region V. Molecular Cell 2007 27, 806-816DOI: (10.1016/j.molcel.2007.07.013) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 TFIIB Crosslinking to the Terminator Region Is Ssu72 Dependent (A) Schematic depiction of the PMA1 gene. (B) ChIP analysis of TFIIB crosslinking to PMA1, as described in Figure 5A, except using isogenic strains FY23 (WT) and XH-24 (ssu72-td) that had been incubated at either the permissive (24°C) or restrictive (37°C) temperature for 1 hr prior to crosslinking. Incubation of XH-24 at 37°C results in depletion of the Ssu72 CTD phosphatase (Krishnamurthy et al., 2004). (C) Quantification of the data shown in (B). (D) Schematic depiction of the BLM10 gene. (E) ChIP analysis of TFIIB crosslinking to BLM10, as depicted in Figure 5D. (F) Quantification of the data shown in (E). Results indicate that depletion of Ssu72 (ssu72-td, 37°C) eliminates crosslinking of TFIIB to the terminator regions of PMA1 (region 8) and BLM10 (region 5), but not to the promoter regions (region 1) of either gene. Factor association was quantified as described in the Figure 5 legend. These effects of ssu72-td are essentially identical to those associated with the sua7-1 TFIIB E62K replacement (Figure 5). Molecular Cell 2007 27, 806-816DOI: (10.1016/j.molcel.2007.07.013) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 7 Model for TFIIB-Dependent Gene Looping (1) Following PIC assembly, RNAP II initiates transcription. TFIIB and TFIIF (not shown) dissociate from the promoter, leaving behind the scaffold complex, which includes TFIIA, TFIID, TFIIE, TFIIH, and the Mediator (Yudkovsky et al., 2000). (2) Subsequent to transcription of the 3′ end processing signals, RNA is endonucleolytically cleaved (arrow) and polyadenylated, leaving behind a 3′ uncapped transcript in association with RNAP II. (3) We suggest that subsequent to mRNA processing, TFIIB can bind RNAP II at the terminator. RNAP II-TFIIB binding would be mediated, in part, by the B finger domain such that the sua7-1-encoded E62K replacement within the B finger precludes TFIIB binding. (4) The terminator-RNAP II-TFIIB complex (in association with TFIIF) binds the scaffold, thereby forming a gene loop that juxtaposes the promoter and terminator and potentially creating a functional transcription reinitiation complex (RIC). Molecular Cell 2007 27, 806-816DOI: (10.1016/j.molcel.2007.07.013) Copyright © 2007 Elsevier Inc. Terms and Conditions