The regulation of gene expression depends critically upon chromatin structure. Transcription of protein-coding genes can be reconstituted on naked DNA with only the general transcription factors and RNA polymerase II. This minimal system cannot transcribe DNA packaged into chromatin, indicating that accessory factors may facilitate access to DNA. Two classes of accessory factor, ATP-dependent chromatin-remodelling enzymes and histone acetyltransferases, facilitate transcription initiation from chromatin templates. FACT (for facilitates chromatin transcription) is a chromatin-specific elongation factor required for transcription of chromatin templates in vitro. Here we show that FACT comprises a new human homologue of the Saccharomyces cerevisiae Spt16/Cdc68 protein and the high-mobility group-1-like protein structure-specific recognition protein-1. Yeast SPT16/CDC68 is an essential gene that has been implicated in transcription and cell-cycle regulation. Consistent with our biochemical analysis of FACT, we provide evidence that Spt16/Cdc68 is involved in transcript elongation in vivo. Moreover, FACT specifically interacts with nucleosomes and histone H2A/H2B dimers, indicating that it may work by promoting nucleosome disassembly upon transcription. In support of this model, we show that FACT activity is abrogated by covalently crosslinking nucleosomal histones.
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. The RNA polymerase II (RNAP II) transcription cycle involves distinct steps that include assembly of a preinitiation complex (PIC), initiation, elongation, termination, and reinitiation. Whereas initiation requires recruitment of RNAP II and a complete set of initiation factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) (Woychik and Hampsey 2002;Hahn 2004), reinitiation has been proposed to occur via a different pathway (Yudkovsky et al. 2000). Following initiation, a subset of the initiation factors and Mediator, which facilitates the interaction between gene-specific regulatory proteins and the general transcription factors, is retained at the promoter, forming a "scaffold" that functions as an intermediate for subsequent rounds of transcription in vitro. Accordingly, reinitiation can facilitate higher levels of transcription by bypassing recruitment of factors retained in the scaffold, requiring de novo recruitment of only TFIIB, TFIIF, and RNAP II (Yudkovsky et al. 2000). Whether transcription reinitiation involves terminationdependent recycling of RNAP II from the terminator to the promoter without release from template DNA is not known, although such facilitated recycling has been reported for yeast RNAP III (Dieci andSentenac 1996, 2003).Progression of RNAP II through the transcription cycle is accompanied by changes in the phosphorylation status of the CTD, a reiterated heptapeptide sequence (Y 1 -S 2 -P 3 -T 4 -S 5 -P 6 -S 7 ) present at the C terminus of the Rpb1 subunit of RNAP II (Kobor and Greenblatt 2002). RNAP II is recruited to the promoter in an unphosphorylated form (RNAP IIA) that becomes extensively phosphorylated (RNAP IIO) during transcription. In yeast, phosphorylation of Ser5 of the CTD is catalyzed by the Kin28 subunit of TFIIH, whereas Ser2 is phosphorylated by the Ctk1 subunit of the CTDK-I elongation complex (Cho et al. 2001). Dephosphorylation of Ser5-P and Ser2-P are, in turn, catalyzed by the Ssu72 and Fcp1 phosphatases, respectively (Cho et al. 2001;Krishnamurthy et al. ...
Phosphorylation of serine-2 (S2) and serine-5 (S5) of the C-terminal domain (CTD) of RNA polymerase II (RNAP II) is a dynamic process that regulates the transcription cycle and coordinates recruitment of RNA processing factors. The Fcp1 CTD phosphatase catalyzes dephosphorylation of S2-P. Here, we report that Ssu72, a component of the yeast cleavage/polyadenylation factor (CPF) complex, is a CTD phosphatase with specificity for S5-P. Ssu72 catalyzes CTD S5-P dephosphorylation in association with the Pta1 component of the CPF complex, although its essential role in 3' end processing is independent of catalytic activity. Depletion of Ssu72 impairs transcription in vitro, and this defect can be rescued by recombinant, catalytically active Ssu72. We propose that Ssu72 has a dual role in transcription, one as a CTD S5-P phosphatase that regenerates the initiation-competent, hypophosphorylated form of RNAP II and the other as a factor necessary for cleavage of pre-mRNA and efficient transcription termination.
Histone acetylation plays a key role in the regulation of eukaryotic gene expression. Recently, histone acetylation and deacetylation were found to be catalyzed by structurally distinct, multisubunit complexes that mediate, respectively, activation and repression of transcription. Here, we identify SAP30 as a novel component of the human histone deacetylase complex that includes Sin3, the histone deacetylases HDAC1 and HDAC2, histone binding proteins RbAp46 and RbAp48, as well as other polypeptides. Moreover, we describe a SAP30 homolog in yeast that is functionally related to Sin3 and the histone deacetylase Rpd3. The human SAP30 complex is active in deacetylating core histone octamers, but inactive in deacetylating nucleosomal histones due to the inability of the histone binding proteins RbAp46 and RbAp48 to gain access to nucleosomal histones. These results define SAP30 as a component of a histone deacetylase complex conserved among eukaryotic organisms.
Recent studies demonstrated the existence of gene loops that juxtapose the promoter and terminator regions of genes with exceptionally long ORFs in yeast. Here we report that looping is not idiosyncratic to long genes but occurs between the distal ends of genes with ORFs as short as 1 kb. Moreover, looping is dependent upon the general transcription factor TFIIB: the E62K (glutamic acid 62 --> lysine) form of TFIIB adversely affects looping at every gene tested, including BLM10, SAC3, GAL10, SEN1, and HEM3. TFIIB crosslinks to both the promoter and terminator regions of the PMA1 and BLM10 genes, and its association with the terminator, but not the promoter, is adversely affected by E62K and by depletion of the Ssu72 component of the CPF 3' end processing complex, and is independent of TBP. We propose a model suggesting that TFIIB binds RNAP II at the terminator, which in turn associates with the promoter scaffold.
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