DNA looping facilitates targeting of a chromatin remodeling enzyme.

ATP-dependent chromatin remodeling enzymes are highly abundant and play pivotal roles regulating DNA-dependent processes. The mechanisms by which they are targeted to specific loci have not been well understood on a genome-wide scale. Here, we present evidence that a major targeting mechanism for the Isw2 chromatin remodeling enzyme to specific genomic loci is through sequence-specific transcription factor (TF)-dependent recruitment. Unexpectedly, Isw2 is recruited in a TF-dependent fashion to a large number of loci without TF binding sites. Using the 3C assay, we show that Isw2 can be targeted by Ume6- and TFIIB-dependent DNA looping. These results identify DNA looping as a mechanism for the recruitment of a chromatin remodeling enzyme and define a function for DNA looping. We also present evidence suggesting that Ume6-dependent DNA looping is involved in chromatin remodeling and transcriptional repression, revealing a mechanism by which the three-dimensional folding of chromatin affects DNA-dependent processes.

Promoter-Terminator Gene Loops Affect Alternative 3'-End Processing in Yeast.

Many eukaryotic genes undergo alternative 3'-end poly(A)-site selection producing transcript isoforms with 3'-UTRs of different lengths and post-transcriptional fates. Gene loops are dynamic structures that juxtapose the 3'-ends of genes with their promoters. Several functions have been attributed to looping, including memory of recent transcriptional activity and polarity of transcription initiation. In this study, we investigated the relationship between gene loops and alternative poly(A)-site. Using the KlCYC1 gene of the yeast Kluyveromyces lactis, which includes a single promoter and two poly(A) sites separated by 394 nucleotides, we demonstrate in two yeast species the formation of alternative gene loops (L1 and L2) that juxtapose the KlCYC1 promoter with either proximal or distal 3'-end processing sites, resulting in the synthesis of short and long forms of KlCYC1 mRNA. Furthermore, synthesis of short and long mRNAs and formation of the L1 and L2 loops are growth phase-dependent. Chromatin immunoprecipitation experiments revealed that the Ssu72 RNA polymerase II carboxyl-terminal domain phosphatase, a critical determinant of looping, peaks in early log phase at the proximal poly(A) site, but as growth phase advances, it extends to the distal site. These results define a cause-and-effect relationship between gene loops and alternative poly(A) site selection that responds to different physiological signals manifested by RNA polymerase II carboxyl-terminal domain phosphorylation status.

A physiological role for gene loops in yeast.

DNA loops that juxtapose the promoter and terminator regions of RNA polymerase II-transcribed genes have been identified in yeast and mammalian cells. Loop formation is transcription-dependent and requires components of the pre-mRNA 3'-end processing machinery. Here we report that looping at the yeast GAL10 gene persists following a cycle of transcriptional activation and repression. Moreover, GAL10 and a GAL1p-SEN1 reporter undergo rapid reactivation kinetics following a cycle of activation and repression-a phenomenon defined as "transcriptional memory"-and this effect correlates with the persistence of looping. We propose that gene loops facilitate transcriptional memory in yeast.

The Ssu72 phosphatase mediates the RNA polymerase II initiation-elongation transition.

Transitions between the different stages of the RNAPII transcription cycle involve the recruitment and exchange of factors, including mRNA capping enzymes, elongation factors, splicing factors, 3'-end-processing complexes, and termination factors. These transitions are coordinated by the dynamic phosphorylation of the C-terminal domain (CTD) of the largest subunit of RNAPII (Rpb1). The CTD is composed of reiterated heptapeptide repeats (Y(1)S(2)P(3)T(4)S(5)P(6)S(7)) that undergo phosphorylation and dephosphorylation as RNAPII transitions through the transcription cycle. An essential phosphatase in this process is Ssu72, which exhibits catalytic specificity for Ser(P)(5) and Ser(P)(7). Ssu72 is unique in that it is specific for Ser(P)(5) in one orientation of the CTD and for Ser(P)(7) when bound in the opposite orientation. Moreover, Ssu72 interacts with components of the initiation machinery and affects start site selection yet is an integral component of the CPF 3'-end-processing complex. Here we provide a comprehensive view of the effects of Ssu72 with respect to its Ser(P)(5) phosphatase activity. We demonstrate that Ssu72 dephosphorylates Ser(P)(5) at the initiation-elongation transition. Furthermore, Ssu72 indirectly affects the levels of Ser(P)(2) during the elongation stage of transcription but does so independent of its catalytic activity.

The interaction of Pcf11 and Clp1 is needed for mRNA 3'-end formation and is modulated by amino acids in the ATP-binding site.

Polyadenylation of eukaryotic mRNAs contributes to stability, transport and translation, and is catalyzed by a large complex of conserved proteins. The Pcf11 subunit of the yeast CF IA factor functions as a scaffold for the processing machinery during the termination and polyadenylation of transcripts. Its partner, Clp1, is needed for mRNA processing, but its precise molecular role has remained enigmatic. We show that Clp1 interacts with the Cleavage-Polyadenylation Factor (CPF) through its N-terminal and central domains, and thus provides cross-factor connections within the processing complex. Clp1 is known to bind ATP, consistent with the reported RNA kinase activity of human Clp1. However, substitution of conserved amino acids in the ATP-binding site did not affect cell growth, suggesting that the essential function of yeast Clp1 does not involve ATP hydrolysis. Surprisingly, non-viable mutations predicted to displace ATP did not affect ATP binding but disturbed the Clp1-Pcf11 interaction. In support of the importance of this interaction, a mutation in Pcf11 that disrupts the Clp1 contact caused defects in growth, 3'-end processing and transcription termination. These results define Clp1 as a bridge between CF IA and CPF and indicate that the Clp1-Pcf11 interaction is modulated by amino acids in the conserved ATP-binding site of Clp1.

Mechanism of start site selection by RNA polymerase II: interplay between TFIIB and Ssl2/XPB helicase subunit of TFIIH.

TFIIB is essential for transcription initiation by RNA polymerase II. TFIIB also cross-links to terminator regions and is required for gene loops that juxtapose promoter-terminator elements in a transcription-dependent manner. The Saccharomyces cerevisiae sua7-1 mutation encodes an altered form of TFIIB (E62K) that is defective for both start site selection and gene looping. Here we report the isolation of an ssl2 mutant, encoding an altered form of TFIIH, as a suppressor of the cold-sensitive growth defect of the sua7-1 mutation. Ssl2 (Rad25) is orthologous to human XPB and is a member of the SF2 family of ATP-dependent DNA helicases. The ssl2 suppressor allele encodes an arginine replacement of the conserved histidine residue (H508R) located within the DEVH-containing helicase domain. In addition to suppressing the TFIIB E62K growth defect, Ssl2 H508R partially restores both normal start site selection and gene looping. Moreover, Ssl2, like TFIIB, associates with promoter and terminator regions, and the diminished association of TFIIB E62K with the PMA1 terminator is restored by the Ssl2 H508R suppressor. These results define a novel, functional interaction between TFIIB and Ssl2 that affects start site selection and gene looping.

Conformational coupling, bridge helix dynamics and active site dehydration in catalysis by RNA polymerase.

Molecular dynamics simulation of Thermus thermophilus (Tt) RNA polymerase (RNAP) in a catalytic conformation demonstrates that the active site dNMP-NTP base pair must be substantially dehydrated to support full active site closing and optimum conditions for phosphodiester bond synthesis. In silico mutant beta R428A RNAP, which was designed based on substitutions at the homologous position (Rpb2 R512) of Saccharomyces cerevisiae (Sc) RNAP II, was used as a reference structure to compare to Tt RNAP in simulations. Long range conformational coupling linking a dynamic segment of the bridge alpha-helix, the extended fork loop, the active site, and the trigger loop-trigger helix is apparent and adversely affected in beta R428A RNAP. Furthermore, bridge helix bending is detected in the catalytic structure, indicating that bridge helix dynamics may regulate phosphodiester bond synthesis as well as translocation. An active site "latch" assembly that includes a key trigger helix residue Tt beta' H1242 and highly conserved active site residues beta E445 and R557 appears to help regulate active site hydration/dehydration. The potential relevance of these observations in understanding RNAP and DNAP induced fit and fidelity is discussed.

Role for the Ssu72 C-terminal domain phosphatase in RNA polymerase II transcription elongation.

The RNA polymerase II (RNAP II) transcription cycle is accompanied by changes in the phosphorylation status of the C-terminal domain (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 largest RNAP II subunit. One of the enzymes involved in this process is Ssu72, a CTD phosphatase with specificity for serine-5-P. Here we report that the ssu72-2-encoded Ssu72-R129A protein is catalytically impaired in vitro and that the ssu72-2 mutant accumulates the serine-5-P form of RNAP II in vivo. An in vitro transcription system derived from the ssu72-2 mutant exhibits impaired elongation efficiency. Mutations in RPB1 and RPB2, the genes encoding the two largest subunits of RNAP II, were identified as suppressors of ssu72-2. The rpb1-1001 suppressor encodes an R1281A replacement, whereas rpb2-1001 encodes an R983G replacement. This information led us to identify the previously defined rpb2-4 and rpb2-10 alleles, which encode catalytically slow forms of RNAP II, as additional suppressors of ssu72-2. Furthermore, deletion of SPT4, which encodes a subunit of the Spt4-Spt5 early elongation complex, also suppresses ssu72-2, whereas the spt5-242 allele is suppressed by rpb2-1001. These results define Ssu72 as a transcription elongation factor. We propose a model in which Ssu72 catalyzes serine-5-P dephosphorylation subsequent to addition of the 7-methylguanosine cap on pre-mRNA in a manner that facilitates the RNAP II transition into the elongation stage of the transcription cycle.

Detection of gene loops by 3C in yeast.

"Chromosome conformation capture" (3C) is a powerful method to detect physical interaction between any two genomic loci. 3C involves formaldehyde crosslinking to stabilize transient interactions, followed by restriction digestion, ligation and locus-specific PCR. Accordingly, 3C reveals complex three-dimensional interactions between distal genetic elements within intact cells at high resolution. Here, we describe a modified 3C protocol designed for detection of transient chromatin interactions in the yeast Saccharomyces cerevisiae. Using this protocol, we are able to detect juxtaposition of promoter and terminator regions of genes with ORFs as short as 1kb in length. We anticipate that this method will be generally applicable to detect dynamic, short-range chromatin interactions and will facilitate the characterization of gene loops and their functional consequences.

Transcription activation: unveiling the essential nature of TFIID.

The TAF subunits of TFIID mediate activation of subsets of the eukaryotic genome. Recent results demonstrate that TFIID is recruited to promoters in an activator-specific manner involving functional interaction between upstream regulatory elements and the core promoter, thereby coordinating the expression of distinct sets of genes.

Connecting the DOTs: covalent histone modifications and the formation of silent chromatin.

Histone methylation has emerged as a significant regulator of chromatin structure and function. Two different classes of histone methyltransferase (HMT) have been described, which target either lysine or arginine residues in the histone N-terminal tails. A flurry of recent papers now describe a third class of HMT that affects chromatin silencing indirectly, not by methylation of histone tails, but instead by targeting a conserved lysine residue in the core domain of the nucleosome.

Functional interaction between TFIIB and the Rpb2 subunit of RNA polymerase II: implications for the mechanism of transcription initiation.

The general transcription factor TFIIB is required for accurate initiation, although the mechanism by which RNA polymerase II (RNAP II) identifies initiation sites is not well understood. Here we describe results from genetic and biochemical analyses of an altered form of yeast TFIIB containing an arginine-78 --> cysteine (R78C) replacement in the "B-finger" domain. TFIIB R78C shifts start site selection downstream of normal and confers a cold-sensitive growth defect (Csm(-)). Suppression of the R78C Csm(-) phenotype identified a functional interaction between TFIIB and the Rpb2 subunit of RNAP II and defined a novel role for Rpb2 in start site selection. The rpb2 suppressor encodes a glycine-369 --> serine (G369S) replacement, located in the "lobe" domain of Rpb2 and near the Rpb9 subunit, which was identified previously as an effector of start site selection. The Rpb2-Rpb9 "lobe-jaw" region of RNAP II is downstream of the catalytic center and distal to the site of RNAP II-TFIIB interaction. A TFIIB R78C mutant extract was defective for promoter-specific run-on transcription but yielded an altered pattern of abortive initiation products, indicating that the R78C defect does not preclude initiation. The sua7-3 rpb2-101 double mutant was sensitive to 6-azauracil in vivo and to nucleoside triphosphate substrate depletion in vitro. In the context of the recent X-ray structure of the yeast RNAP II-TFIIB complex, these results define a functional interaction between the B-finger domain of TFIIB and the distal lobe-jaw region of RNAP II and provide insight into the mechanism of start site selection.

Evidence that the Tfg1/Tfg2 dimer interface of TFIIF lies near the active center of the RNA polymerase II initiation complex.

The ssu71 alleles of the TFG1 gene, which encodes the largest subunit of TFIIF, were isolated as suppressors of a TFIIB defect that affects the accuracy of transcription start site selection in the yeast Saccharomyces cerevisiae. Here we report that ssu71-1 also suppresses the cell growth and start site defects associated with an altered form of the Rpb1 subunit of RNA polymerase II (RNAP II). The ssu71-1 and ssu71-2 alleles were cloned and found to encode single amino acid replacements of glycine-363, either glycine to aspartic acid (G363D) or glycine to arginine (G363R). Two other charged replacements, G363E and G363K, were constructed by site-directed mutagenesis and suppress both TFIIB E62K and Rpb1 N445S, whereas neither G363A nor G363P exhibited any effect. G363 is phylogenetically conserved and its counterpart in human TFIIF (RAP74 G112) is located within the RAP74/RAP30 dimerization domain. We propose that the TFIIF dimerization domain is located in proximity to the B-finger of TFIIB near the active center of RNAP II where the TFIIB-TFIIF-RNAP II interface plays a key role in start site selection.

Genetic analysis of the Warburg effect in yeast.

We recently discovered that the Warburg effect, defined by the dramatically enhanced metabolism of glucose to pyruvate, even in well-oxygenated cancer cells, can occur as a consequence of mutations that enhance lipid biosynthesis at the expense of respiratory capacity. Specifically, mutations in the E1 subunit of either of two respiratory enzymes, pyruvate dehydrogenase (PDC) or α-ketoglutarate dehydrogenase (KGDC), change substrate specificity from the 3-carbon α-ketoacid pyruvate, or the 5-carbon α-ketoacid α-ketoglutarate, to the 4-carbon α-ketoacid oxaloacetate (OADC). These mutations result in OADC-catalyzed synthesis of malonyl-CoA (MaCoA), the essential precursor of all fatty acids. These mutants arose as spontaneous suppressors of a yeast acc1(cs) cold-sensitive mutation encoding an altered form of AcCoA carboxylase (Acc1) that fails to produce MaCoA at the restrictive temperature (16 °C). Notably, these suppressors are respiratory defective as a result of the same nuclear mutations that suppress acc1(cs). These mutants also suppress sensitivity to Soraphen A, a potent inhibitor of Acc1 activity, at normal temperature (30 °C). To our knowledge, OADC activity has never been identified in eukaryotic cells. Our results offer a novel perspective on the Warburg effect: the reprogramming of energy metabolism in cancer cells as a consequence of mutational impairment of respiration to meet the fatty acid requirements of rapidly proliferating cells. We suggest OADC activity is a common feature of cancer cells and represents a novel target for the development of chemotherapeutics.

Detection of short-range chromatin interactions by chromosome conformation capture (3C) in yeast.

We describe a modified 3C ("chromosome conformation capture") protocol for detection of transient, short-range chromatin interactions in the yeast Saccharomyces cerevisiae. 3C was initially described by Job Dekker and involves formaldehyde cross-linking to stabilize transient chromatin interactions, followed by restriction digestion, ligation, and locus-specific PCR. As such, 3C reveals complex three-dimensional interactions between distal genetic elements within intact cells at high resolution. Using a modified version of Dekker's protocol, we are able to detect gene loops that juxtapose promoter and terminator regions of yeast genes with ORFs as short as 1 kb. We are using this technique to define the cis- and trans-acting requirements for the formation and maintenance of gene loops, and to elucidate their physiological consequences. We anticipate that this method will be generally applicable to detect dynamic, short-range chromatin interactions, not limited to gene loops.

Control of eukaryotic gene expression: gene loops and transcriptional memory.

Gene loops are dynamic structures that juxtapose promoter­terminator regions of Pol II-transcribed genes. Although first described in yeast, gene loops have now been identified in yeast and mammalian cells. Looping requires components of the transcription preinitiation complex, the pre-mRNA 30-end processing machinery, and subunits of the nuclear pore complex. Loop formation is transcription-dependent, but neither basal nor activated transcription requires looping. Rather, looping appears to affect cellular memory of recent transcriptional activity, enabling a more rapid response to subsequent stimuli. The nuclear pore has been implicated in both memory and looping. Our working model is that loops are formed and/or maintained at the nuclear pore to facilitate hand-off of Pol II form the terminator to the promoter, thereby bypassing Pol II recruitment as the rate-limiting step in reactivation of transcription. Involvement of the nuclear pore also suggests that looping might facilitate mRNA export to the cytoplasm. The technology now exists to test these ideas.

The essential N terminus of the Pta1 scaffold protein is required for snoRNA transcription termination and Ssu72 function but is dispensable for pre-mRNA 3'-end processing.

Saccharomyces cerevisiae Pta1 is a component of the cleavage/polyadenylation factor (CPF) 3'-end processing complex and functions in pre-mRNA cleavage, poly(A) addition, and transcription termination. In this study, we investigated the role of the N-terminal region of Pta1 in transcription and processing. We report that a deletion of the first 75 amino acids (pta1-Delta75) causes thermosensitive growth, while the deletion of an additional 25 amino acids is lethal. The pta1-Delta75 mutant is defective for snoRNA termination, RNA polymerase II C-terminal domain Ser5-P dephosphorylation, and gene looping but is fully functional for mRNA 3'-end processing. Furthermore, different regions of Pta1 interact with the CPF subunits Ssu72, Pti1, and Ysh1, supporting the idea that Pta1 acts as a scaffold to organize CPF. The first 300 amino acids of Pta1 are sufficient for interactions with Ssu72, which is needed for pre-mRNA cleavage. By the degron-mediated depletion of Pta1, we show that the removal of this essential region leads to a loss of Ssu72, yet surprisingly, in vitro cleavage and polyadenylation remain efficient. In addition, a fragment containing amino acids 1 to 300 suppresses 3'-end processing in wild-type extracts. These findings suggest that the amino terminus of Pta1 has an inhibitory effect and that this effect can be neutralized through the interaction with Ssu72.

Functional interaction of the Ess1 prolyl isomerase with components of the RNA polymerase II initiation and termination machineries.

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a reiterated heptad sequence (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7) that plays a key role in the transcription cycle, coordinating the exchange of transcription and RNA processing factors. The structure of the CTD is flexible and undergoes conformational changes in response to serine phosphorylation and proline isomerization. Here we report that the Ess1 peptidyl prolyl isomerase functionally interacts with the transcription initiation factor TFIIB and with the Ssu72 CTD phosphatase and Pta1 components of the CPF 3'-end processing complex. The ess1(A144T) and ess1(H164R) mutants, initially described by Hanes and coworkers (Yeast 5:55-72, 1989), accumulate the pSer5 phosphorylated form of Pol II; confer phosphate, galactose, and inositol auxotrophies; and fail to activate PHO5, GAL10, and INO1 reporter genes. These mutants are also defective for transcription termination, but in vitro experiments indicate that this defect is not caused by altering the processing efficiency of the cleavage/polyadenylation machinery. Consistent with a role in initiation and termination, Ess1 associates with the promoter and terminator regions of the PMA1 and PHO5 genes. We propose that Ess1 facilitates pSer5-Pro6 dephosphorylation by generating the CTD structural conformation recognized by the Ssu72 phosphatase and that pSer5 dephosphorylation affects both early and late stages of the transcription cycle.

The Rsp5 E3 ligase mediates turnover of low affinity phosphate transporters in Saccharomyces cerevisiae.

In an effort to identify novel components of the PHO regulon in Saccharomyces cerevisiae, we have isolated and characterized suppressors of the Pho(-) phenotype associated with deletion of the Pho4 transcriptional activator. Here we report that either a defective form of the Rsp5 E3 ubiquitin ligase or deletion of the End3 component of the endocytic pathway restores growth of the pho4 Delta mutant in the presence of limiting inorganic phosphate (P i). The spa1-1 suppressor allele of RSP5 encodes a phenylalanine-to-valine replacement at position 748 (F748V) within the catalytic HECT domain of Rsp5. Consistent with suppression due to impaired ubiquitin ligase activity, the heat-sensitive growth defect of the spa1-1 mutant is suppressed either by overexpression of ubiquitin or by osmotic stabilization. Western blot analyses revealed that the cellular levels of the Pho87 and Pho91 low affinity P i are markedly increased in the spa1-1 mutant, yet Pho84 high affinity P i transporter levels are unaffected. Furthermore, Pho87 and Pho91 are ubiquitinated in vivo in an Rsp5-dependent manner, and the Pho+ phenotype of the spa1-1 suppressor is dependent upon Pho87 and Pho91. We conclude that turnover of the low affinity P i transporters is initiated by Rsp5-mediated ubiquitination followed by internalization and degradation by the endocytic pathway.