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. 2005 Dec 15;19(24):2969-78.
doi: 10.1101/gad.1362305. Epub 2005 Nov 30.

A role for the CPF 3'-end processing machinery in RNAP II-dependent gene looping

Athar Ansari  1 , Michael Hampsey
Affiliations

A role for the CPF 3'-end processing machinery in RNAP II-dependent gene looping

Athar Ansari et al. Genes Dev. .

Abstract

The prevailing view of the RNA polymerase II (RNAP II) transcription cycle is that RNAP II is recruited to the promoter, transcribes a linear DNA template, then terminates transcription and dissociates from the template. Subsequent rounds of transcription are thought to require de novo recruitment of RNAP II to the promoter. Several recent findings, including physical interaction of 3'-end processing factors with both promoter and terminator regions, challenge this concept. Here we report a physical association of promoter and terminator regions of the yeast BUD3 and SEN1 genes. These interactions are transcription-dependent, require the Ssu72 and Pta1 components of the CPF 3'-end processing complex, and require the phosphatase activity of Ssu72. We propose a model for RNAP II transcription in which promoter and terminator regions are juxtaposed, and that the resulting gene loops facilitate transcription reinitiation by the same molecule of RNAP II in a manner dependent upon Ssu72-mediated CTD dephosphorylation.

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Figures

Figure 1.
Figure 1.
Gene looping at the BUD3 and SEN1 genes. (A) Schematic depiction of mCCC analysis to detect gene looping. Under transcriptionally permissive conditions, formaldehyde is used to cross-link transiently interacting chromatin regions, followed by restriction digestion to cut DNA upstream of the promoter, within the ORF, and downstream of the terminator. DNA ends are then ligated in dilute solution to minimize intermolecular ligation. Cross-links are reversed, and PCR is performed with divergent primers P1 and T1. Accordingly, P1-T1 PCR products represent ligation of distal, divergent regions of the gene of interest and are therefore indicative of gene looping. If looping does not occur, then ligation of the DNA ends adjacent to P1 and T1 should occur infrequently, and the P1-T1 PCR products should be diminished relative to the signal observed under conditions that favor looping. (B) Schematic depiction of the BUD3 and SEN1 genes showing the positions of the XhoI or HindIII restriction sites and the P1 and T1 PCR primers used in mCCC analysis. (C) The rpb1-1 mutation diminishes loop formation. Chromatin was isolated from the rpb1-1 strain grown at 24°C (permissive temperature, transcription ON) or following a 1-h temperature shift to 37°C (non-permissive temperature, transcription OFF). The P1-T1 PCR products correspond to ligation of distal, divergent ends of the BUD3 and SEN1 genes that result from cross-linking of the respective promoter and terminator regions as depicted in A. Control PCR represents an intergenic region of chromosome V to ascertain that equal amounts of template DNA were present in all reactions. (D) Identical to C, except using the isogenic RPB1 wild-type strain.
Figure 2.
Figure 2.
(A) RNA slot blot analysis of total poly(A) RNA in rpb1-1, wild-type (WT), and ssu72-td strains following incubation of cells at 37°C for the indicated periods of time. (B) The sug1-1 mutant (KMY1171) does not affect loop formation. Identical to Figure 1C, except the mCCC assay was performed using a sug1-1 mutant, which exhibits rapid cell growth inhibition at 37°C, comparable to rpb1-1, but does not affect global transcription. In this case no effect on the P1-T1 PCR signal was observed for either BUD3 or SEN1. (C,D) Transcription-induced gene looping at BUD3 and SEN1 is dependent on formaldehyde cross-linking and ligation. Chromatin was isolated from the wild-type strain (FY23) and grown at 24°C, and mCCC analysis was performed as depicted in Figure 1, except without formaldehyde cross-linking (C) or ligation (D).
Figure 3.
Figure 3.
Galactose-induced transcription of GAL1p-BUD3 and GAL1p-SEN1 results in gene looping. (A) The GAL1 promoter (GAL1p) was cloned upstream of the BUD3 and SEN1 genes at their native chromosomal loci. (B) Strains were grown in dextrose (transcription repressed) or galactose (transcription induced), and gene looping was assayed by mCCC analysis as depicted in Figure 1.
Figure 4.
Figure 4.
Gene looping at BUD3 and SEN1 is dependent on the RNAP II CTD phosphatase activity of Ssu72. (A) The Ssu72 protein was depleted from the ssu72-td degron strain (XH-24) following a 1-h temperature shift from 24°C to 37°C as described previously (Krishnamurthy et al. 2004). Chromatin was isolated from cells and subjected to mCCC analysis as depicted in Figure 1. (B) Identical to A except that the ssu72-td strain carries the SSU72 wild-type plasmid (pM712), which restores the normal level of Ssu72 protein and its CTD phosphatase activity (Krishnamurthy et al. 2004). (C) Identical to A except that the ssu72-td strain carries the ssu72-C15S plasmid (pM698), which encodes a catalytically inactive form of the Ssu72 phosphatase (Krishnamurthy et al. 2004).
Figure 5.
Figure 5.
Gene looping at BUD3 and SEN1 is dependent upon the Pta1 subunit of the CPF 3′-end processing complex. (A) Pta1 protein was depleted in the pta1-td degron strain (XH-23) following a 1-h temperature shift from 24°C to 37°C as described previously (Krishnamurthy et al. 2004). Chromatin was isolated from cells and subjected to mCCC analysis as depicted in Figure 1. (B) Identical to A except that the experiment was done using an isogenic wild-type strain.
Figure 6.
Figure 6.
DNA looping juxtaposes the promoter and terminator regions of SEN1. mCCC analysis was performed as depicted in Figure 1 with modifications described in the Materials and Methods. (A) Schematic depiction of the SEN1 gene, showing HindIII sites (HIII) and the P, C, and T primers used in mCCC analysis. The relative positions of the HindIII sites are drawn approximately to scale. (B) PCR products derived from the indicated primer pairs. PCR signals are expected for the P1-C1 and T1-C6 primer pairs, as these correspond to intramolecular ligation products. As the number of HindIII sites between primer pairs increases, the PCR signals decrease (cf. P1-C1, P1-C3, P1-C5; and T1-C6, T1-C4, T1-C2). These results stand in marked contrast to P1-T1, which produces a strong PCR signal despite the presence of three intervening HindIII sites, indicating that DNA looping juxtaposes restriction fragments encompassing the promoter and terminator regions.
Figure 7.
Figure 7.
Detection and mapping of gene looping at SEN1 by LF-ChIP. (A) Schematic depiction of LF-ChIP to detect and map gene looping. Transiently interacting regions of chromatin are cross-linked in whole cells using formaldehyde. Chromatin is extracted, extensively sonicated, and digested with HindIII, which cuts SEN1 upstream of the promoter, downstream of the terminator, and at three sites within the ORF (Fig. 1B). Following fill-in of overhanging DNA ends, samples are ligated in dilute solution to minimize intermolecular ligation products, followed either by immunoprecipitation with anti-Ssu72 antibodies or by Pta1-TAP pull-down, and the DNA is amplified with the P2-T2 primer pair that flanks the SEN1 ORF. Ligated DNA is then assayed using primer pairs that define regions A-F. A critical control in this procedure is omission of the DNA ligation step, in which case PCR products simply reflect Ssu72 and Pta1 occupancy of the respective DNA fragments (A-F) by ChIP. (B) Promoter and terminator regions of SEN1 are physically linked during transcription. LF-ChIP was performed as described in A. The indicated PCR products for regions A-F of SEN1 represent input DNA prior to ChIP (upper panel), Ssu72 ChIP DNA products obtained without ligation of fragmented DNA (middle panel), and PCR products representing regions of DNA brought together by gene looping (bottom panel). (C) Identical to B, except ChIP was performed using Pta1-TAP.
Figure 8.
Figure 8.
A model for CPF-mediated promoter-terminator interactions. We propose that transient DNA looping occurs following a pioneer round of transcription and facilitates transcription reinitiation by RNAP II (see Discussion). Physical interaction between the promoter (P) and terminator (T) regions would be mediated, in part, by direct contact between Ssu72 and TFIIB. (Black line) Double-stranded DNA; (green lines) mRNA.
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