RNA Polymerase II Regulates Topoisomerase 1 Activity to Favor Efficient Transcription.

We report a mechanism through which the transcription machinery directly controls topoisomerase 1 (TOP1) activity to adjust DNA topology throughout the transcription cycle. By comparing TOP1 occupancy using chromatin immunoprecipitation sequencing (ChIP-seq) versus TOP1 activity using topoisomerase 1 sequencing (TOP1-seq), a method reported here to map catalytically engaged TOP1, TOP1 bound at promoters was discovered to become fully active only after pause-release. This transition coupled the phosphorylation of the carboxyl-terminal-domain (CTD) of RNA polymerase II (RNAPII) with stimulation of TOP1 above its basal rate, enhancing its processivity. TOP1 stimulation is strongly dependent on the kinase activity of BRD4, a protein that phosphorylates Ser2-CTD and regulates RNAPII pause-release. Thus the coordinated action of BRD4 and TOP1 overcame the torsional stress opposing transcription as RNAPII commenced elongation but preserved negative supercoiling that assists promoter melting at start sites. This nexus between transcription and DNA topology promises to elicit new strategies to intercept pathological gene expression.

An RNA polymerase II-associated TFIIF-like complex is indispensable for SL RNA gene transcription in Trypanosoma brucei.

Trypanosomes are protistan parasites that diverged early in evolution from most eukaryotes. Their streamlined genomes are packed with arrays of tandemly linked genes that are transcribed polycistronically by RNA polymerase (pol) II. Individual mRNAs are processed from pre-mRNA by spliced leader (SL) trans splicing and polyadenylation. While there is no strong evidence that general transcription factors are needed for transcription initiation at these gene arrays, a RNA pol II transcription pre-initiation complex (PIC) is formed on promoters of SLRNA genes, which encode the small nuclear SL RNA, the SL donor in trans splicing. The factors that form the PIC are extremely divergent orthologues of the small nuclear RNA-activating complex, TBP, TFIIA, TFIIB, TFIIH, TFIIE and Mediator. Here, we functionally characterized a heterodimeric complex of unannotated, nuclear proteins that interacts with RNA pol II and is essential for PIC formation, SL RNA synthesis in vivo, SLRNA transcription in vitro, and parasite viability. These functional attributes suggest that the factor represents TFIIF although the amino acid sequences are too divergent to firmly make this conclusion. This work strongly indicates that early-diverged trypanosomes have orthologues of each and every general transcription factor, requiring them for the synthesis of SL RNA.

Direct visualization of cardiac transcription factories reveals regulatory principles of nuclear architecture during pathological remodeling.

Heart failure is associated with hypertrophying of cardiomyocytes and changes in transcriptional activity. Studies from rapidly dividing cells in culture have suggested that transcription may be compartmentalized into factories within the nucleus, but this phenomenon has not been tested in vivo and the role of nuclear architecture in cardiac gene regulation is unknown. While alterations to transcription have been linked to disease, little is known about the regulation of the spatial organization of transcription and its properties in the pathological setting. In the present study, we investigate the structural features of endogenous transcription factories in the heart and determine the principles connecting chromatin structure to transcriptional regulation in vivo. Super-resolution imaging of endogenous RNA polymerase II clusters in neonatal and adult cardiomyocytes revealed distinct properties of transcription factories in response to pathological stress: neonatal nuclei demonstrated changes in number of clusters, with parallel increases in nuclear area, while the adult nuclei underwent changes in size and intensity of RNA polymerase II foci. Fluorescence in situ hybridization-based labeling of genes revealed locus-specific relationships between expression change and anatomical localization-with respect to nuclear periphery and heterochromatin regions, both sites associated with gene silencing-in the nuclei of cardiomyocytes in hearts (but not liver hepatocytes) of mice subjected to pathologic stimuli that induce heart failure. These findings demonstrate a role for chromatin organization and rearrangement of nuclear architecture for cell type-specific transcription in vivo during disease. RNA polymerase II ChIP and chromatin conformation capture studies in the same model system demonstrate formation and reorganization of distinct nuclear compartments regulating gene expression. These findings reveal locus-specific compartmentalization of stress-activated, housekeeping and silenced genes in the anatomical context of the endogenous nucleus, revealing basic principles of global chromatin structure and nuclear architecture in the regulation of gene expression in healthy and diseased conditions.

smFRET experiments of the RNA polymerase II transcription initiation complex.

Single-molecule fluorescence and in particular single-molecule Förster Resonance Energy Transfer (smFRET) is a powerful tool to provide real-time information on the dynamic architecture of large macromolecular structures such as eukaryotic transcription initiation complexes. In contrast to other structural biology methods, not only structural details, but dynamics transitions are revealed thus closing in on the underlying molecular mechanisms. Here, we describe a comprehensive quantitative biophysical toolbox which can be used for rigorous analysis of dynamic protein-nucleic acid complexes and is applied to the study of eukaryotic transcription initiation. We present detailed protocols for the purification of all essential protein components of the minimal eukaryotic transcription initiation complex. Moreover, we demonstrate how elaborate strategies for site-specific protein labeling can be used to produce complexes with dye molecules attached to arbitrary desired positions. These complexes are then used for smFRET measurements. Moreover, we describe the Nano-Positioning System (NPS) which allows us to quantitatively use the results from a network of smFRET measurements to obtain structural information. With this we provide a toolbox to answer open questions which could not be addressed using methods like X-ray crystallography or cryo-electron microscopy.

Punctuation and syntax of the RNA polymerase II CTD code in fission yeast.

The primary structure and phosphorylation pattern of the tandem Y(1)S(2)P(3)T(4)S(5)P(6)S(7) repeats of the RNA polymerase II carboxyl-terminal domain (CTD) convey information about the transcription apparatus--a CTD code--to a large ensemble of CTD-binding receptor proteins. Four of the seven coding "letters" of the fission yeast CTD (Tyr1, Pro3, Ser5, Pro6) are essential in vivo, but the grammatical rules of the code are obscure. Here we show that the minimal fission yeast CTD coding unit is a decapeptide Y(1)S(2)P(3)T(4)S(5)P(6)S(7)Y(1)S(2)P(3) and the spacing between coding units is flexible; the coding unit must contain two Tyr1 residues and the spacing between consecutive tyrosines is important; Ser5-PO(4)-Pro6 comprises an essential two-letter code "word" that is read by the mRNA capping apparatus; and a threshold number of Ser5-PO(4)-Pro6 words are needed to comprise a readable "sentence" of CTD information. Bypassing the essentiality of the Ser5 and Pro6 letters by fusion of capping enzymes to the CTD helped reveal how CTD phosphorylation circuits are wired in vivo. We found that the Ser2-PO(4) mark is independent of Ser5, Pro6, Ser7, and Thr4, whereas the Ser5-PO(4) mark is independent of Ser2, Ser7, and Thr4. These results provide unique insights to the reading and writing of the CTD code.

Proteomic analysis of mitotic RNA polymerase II reveals novel interactors and association with proteins dysfunctional in disease.

RNA polymerase II (RNAPII) transcribes protein-coding genes in eukaryotes and interacts with factors involved in chromatin remodeling, transcriptional activation, elongation, and RNA processing. Here, we present the isolation of native RNAPII complexes using mild extraction conditions and immunoaffinity purification. RNAPII complexes were extracted from mitotic cells, where they exist dissociated from chromatin. The proteomic content of native complexes in total and size-fractionated extracts was determined using highly sensitive LC-MS/MS. Protein associations with RNAPII were validated by high-resolution immunolocalization experiments in both mitotic cells and in interphase nuclei. Functional assays of transcriptional activity were performed after siRNA-mediated knockdown. We identify >400 RNAPII associated proteins in mitosis, among these previously uncharacterized proteins for which we show roles in transcriptional elongation. We also identify, as novel functional RNAPII interactors, two proteins involved in human disease, ALMS1 and TFG, emphasizing the importance of gene regulation for normal development and physiology.

Site-directed mutagenesis, purification and assay of Saccharomyces cerevisiae RNA polymerase II.

In order to analyze the structure-function of multi-subunit RNA polymerases (RNAPs), it is necessary to make site-directed mutations in key residues. Because Saccharomyces cerevisiae RNAP II is isolated as a 12 subunit enzyme that has not been amenable to in vitro reconstitution, making site-directed mutations in a particular subunit presents technical issues. In this work, we demonstrate a method to generate and purify site-directed mutants in the second largest (Rpb2) RNAP II subunit from yeast, using a tandem affinity purification tag. Mutants are analyzed for growth defects in vivo and for defects in transcriptional elongation in vitro. We show that Rpb2 R512A/C located just C-terminal to fork loop 2 (Rpb2 500-511) has transcriptional defects that are distinct from surrounding fork loop 2 region mutants. Rpb2 E529A/D replacements are faster and E529Q is slower than wild type RNAP II in elongation. E529 appears to form an ion pair with K987, an essential active site residue. Mutations are also analyzed within the active site region indicating key residues for catalysis and the importance of a Rpb2 R983-E1028 ion pair. Rpb2 R983Q and E1028Q are defective in escape from a transcriptional stall.

Strategies for crystallization and structure determination of very large macromolecular assemblies.

High-resolution structures of macromolecular assemblies are pivotal for our understanding of their biological functions in fundamental cellular processes. In the field of X-ray crystallography, recent methodological and instrumental advances have led to the structure determinations of macromolecular assemblies of increased size and complexity, such as those of ribosomal complexes, RNA polymerases, and large multifunctional enzymes. These advances include the use of robotic screening techniques that maximize the chances of obtaining well-diffracting crystals of large complexes through the fine sampling of crystallization space. Sophisticated crystal optimization and cryoprotection techniques and the use of highly brilliant X-ray beams from third-generation synchrotron light sources now allow data collection from weakly diffracting crystals with large asymmetric units. Combined approaches are used to derive phase information, including phases calculated from electron microscopy (EM) models, heavy atom clusters, and density modification protocols. New crystallographic software tools prove valuable for structure determination and model refinement of large macromolecular complexes.

Protein characterization of Saccharomyces cerevisiae RNA polymerase II after in vivo cross-linking.

To characterize proteins associated with active transcription complexes, we purified RNA polymerase II (pol II) from Saccharomyces cerevisiae after fixing live cells with formaldehyde. The approach mimics ChIP and requires solubilizing cross-linked complexes with sonication. Pol II was affinity-purified, and associated proteins were identified by MS. Several classes of proteins depended on cross-linking, including Mediator, general transcription factors, elongation factors, ribonucleoprotein particle (RNP) proteins, and histones. A tagged RNP protein reciprocally purified pol II under identical cross-linking conditions, and the association between RNP proteins and pol II was largely RNase-sensitive. The data indicate that the cross-linked Pol II purification contains elongating pol II with associated nascent RNP. Consistent with this view, some elongation factors no longer associate with pol II after inactivation of transcription in the temperature-sensitive pol II mutant, rpb1-1. Taken together, our data suggest that the cross-linked pol II purification contains a mixed population of pol II, including initiating pol II and elongating pol II.

Transcription of hepatitis delta virus RNA by RNA polymerase II.

Previous studies have indicated that the replication of the RNA genome of hepatitis delta virus (HDV) involves redirection of RNA polymerase II (Pol II), a host enzyme that normally uses DNA as a template. However, there has been some controversy about whether in one part of this HDV RNA transcription, a polymerase other than Pol II is involved. The present study applied a recently described cell system (293-HDV) of tetracycline-inducible HDV RNA replication to provide new data regarding the involvement of host polymerases in HDV transcription. The data generated with a nuclear run-on assay demonstrated that synthesis not only of genomic RNA but also of its complement, the antigenome, could be inhibited by low concentrations of amanitin specific for Pol II transcription. Subsequent studies used immunoprecipitation and rate-zonal sedimentation of nuclear extracts together with double immunostaining of 293-HDV cells, in order to examine the associations between Pol II and HDV RNAs, as well as the small delta antigen, an HDV-encoded protein known to be essential for replication. Findings include evidence that HDV replication is somehow able to direct the available delta antigen to sites in the nucleoplasm, almost exclusively colocalized with Pol II in what others have described as transcription factories.

Transcription-dependent redistribution of the large subunit of RNA polymerase II to discrete nuclear domains.

A subpopulation of the largest subunit of RNA polymerase II (Pol II LS) is located in 20-50 discrete subnuclear domains that are closely linked to speckle domains, which store splicing proteins. The speckle-associated fraction of Pol II LS is hyperphosphorylated on the COOH-terminal domain (CTD), and it is highly resistant to extraction by detergents. A diffuse nucleoplasmic fraction of Pol II LS is relatively hypophosphorylated on the CTD, and it is easily extracted by detergents. In transcriptionally active nuclei, speckle bound hyperphosphorylated Pol II LS molecules are distributed in irregularly shaped speckle domains, which appear to be interconnected via a reticular network. When transcription is inhibited, hyperphosphorylated Pol II LS and splicing protein SC35 accumulate in speckle domains, which are transformed into enlarged, dot-like structures lacking interconnections. When cells are released from transcriptional inhibition, Pol IIO and SC35 redistribute back to the interconnected speckle pattern of transcriptionally active cells. The redistribution of Pol II and SC35 is synchronous, reversible, and temperature dependent. It is concluded that: (a) hyperphosphorylation of Pol II LS's CTD is a better indicator of its tight association to discrete subnuclear domains than its transcriptional activity; (b) during states of transcriptional inhibition, hyperphosphorylated Pol II LS can be stored in enlarged speckle domains, which under the light microscope appear to coincide with the storage sites for splicing proteins; and (c) Pol II and splicing proteins redistribute simultaneously according to the overall transcriptional activity of the nucleus.

Structural and functional characterization of PC2 and RNA polymerase II-associated subpopulations of metazoan Mediator.

The coactivator complexes TRAP/SMCC and PC2 represent two forms of Mediator. To further understand the implications of the heterogeneity of the cellular Mediator populations for regulation of RNA polymerase II (Pol II) transcription, we used a combination of affinity and conventional chromatographic methods. Our analysis revealed a spectrum of complexes, including some containing significant proportions of Pol II. Interestingly, the subunit composition of the Pol II-associated Mediator population resembled that of PC2 more closely than that of the larger TRAP/SMCC complex. In in vitro transcription assays reconstituted from homogeneous preparations of general transcription factors, Mediator-associated Pol II displayed a greater specific activity (relative to that of standard Pol II) in activator-independent (basal) transcription in addition to the previously described effects of Mediator on activator-dependent transcription. Purified PC2 complex also stimulated basal activity under these conditions. Immobilized template assays in which activator-recruited preinitiation complexes were allowed to undergo one cycle of transcription revealed partial disruption of Mediator that resulted in a PC2-like complex being retained in the scaffold. This result implies that PC2 could originate as a result of a normal cellular process. Our results are thus consistent with a dynamic nature of the Mediator complex and further extend the functional similarities between Saccharomyces cerevisiae and metazoan Mediator complexes.

An RNA polymerase II transcription factor inactivated in poliovirus-infected cells copurifies with transcription factor TFIID.

Inhibition of host cell RNA polymerase II-mediated transcription by poliovirus infection was studied in vitro. Whole-cell extracts prepared from poliovirus-infected HeLa cells at 3 h postinfection were shown to be deficient in a factor required for specific transcription from the adenovirus major late promoter. Three lines of evidence suggest that transcription factor TFIID is deficient in poliovirus-infected cells. First, the activity required to specifically restore transcription in poliovirus-infected cell extracts was shown to copurify with TFIID through three chromatographic steps. Second, transcription reactions reconstituted with phosphocellulose-derived chromatographic fractions revealed a fourfold decrease in the specific activity of the TFIID-containing fraction prepared from poliovirus-infected cells compared with that of the same fraction prepared from mock-infected cells. Finally, TFIID and the activity required to specifically restore transcription in virus-infected cell extracts were shown to have the same kinetics of heat inactivation. Together, these results suggest that inactivation of TFIID is an early event in the inhibition of host cell RNA polymerase II transcription by poliovirus.

A portion of RNA polymerase II molecules has a component essential for stress responses and stress survival.

Cells respond to stress by altering gene expression, and these adjustments facilitate stress tolerance. Although transcriptional changes are integral to most stress responses, little is known about the mechanisms that permit the transcription apparatus itself to tolerate stress. Here we report that a major role of the RNA polymerase II subunit RPB4 is to permit appropriate transcriptional responses during stress. Yeast cells lacking RPB4 have essentially wild-type growth rates at moderate temperatures (18 to 22 degrees C), but their growth rates are substantially reduced at temperatures outside this range. When subjected to a heat shock, cells lacking RPB4 rapidly lose the ability to transcribe genes and experience a dramatic loss in viability. When cells lacking RPB4 are subjected to the nutrient stress that accompanies entry into stationary phase, they also exhibit a substantial decline in mRNA synthesis and in viability relative to wild-type cells. Interestingly, the portion of RNA polymerase II molecules that contain RPB4 is small in log phase but increases substantially as cells enter stationary phase. We propose that the association of RPB4 with the other RNA polymerase II subunits increases the tolerance of the enzyme to stress.

Factors involved in specific transcription by mammalian RNA polymerase II: role of transcription factors IIA, IID, and IIB during formation of a transcription-competent complex.

Human transcription factor TFIID, the TATA-binding protein, was partially purified to a form capable of associating stably with the TATA motif of the adenovirus major late promoter. Binding of the human and yeast TFIID to the TATA motif was stimulated by TFIIA. TFIIA is an integral part of a complex capable of binding other transcription factors. A complex formed with human TFIID and TFIIA (DA complex) was specifically recognized by TFIIB. We found that TFIIB activity was contained in a single polypeptide of 32 kDa and that this polypeptide participated in transcription and was capable of binding to the DA complex to form the DAB complex. Formation of the DAB complex required TFIIA, TFIID, and sequences downstream of the transcriptional start site; however, the DA complex could be formed on an oligonucleotide containing only the adenovirus major late promoter TATA motif. Using anti-TFIIB antibodies and reagents that affect the stability of a transcription-competent complex, we found that yeast and human TFIID yielded DAB complexes with different stabilities.

The HIP1 initiator element plays a role in determining the in vitro requirement of the dihydrofolate reductase gene promoter for the C-terminal domain of RNA polymerase II.

We examined the ability of purified RNA polymerase (RNAP) II lacking the carboxy-terminal heptapeptide repeat domain (CTD), called RNAP IIB, to transcribe a variety of promoters in HeLa extracts in which endogenous RNAP II activity was inhibited with anti-CTD monoclonal antibodies. Not all promoters were efficiently transcribed by RNAP IIB, and transcription did not correlate with the in vitro strength of the promoter or with the presence of a consensus TATA box. This was best illustrated by the GC-rich, non-TATA box promoters of the bidirectional dihydrofolate reductase (DHFR)-REP-encoding locus. Whereas the REP promoter was transcribed by RNAP IIB, the DHFR promoter remained inactive after addition of RNAP IIB to the antibody-inhibited reactions. However, both promoters were efficiently transcribed when purified RNAP with an intact CTD was added. We analyzed a series of promoter deletions to identify which cis elements determine the requirement for the CTD of RNAP II. All of the promoter deletions of both DHFR and REP retained the characteristics of their respective full-length promoters, suggesting that the information necessary to specify the requirement for the CTD is contained within approximately 65 bp near the initiation site. Furthermore, a synthetic minimal promoter of DHFR, consisting of a single binding site for Sp1 and a binding site for the HIP1 initiator cloned into a bacterial vector sequence, required RNAP II with an intact CTD for activity in vitro. Since the synthetic minimal promoter of DHFR and the smallest REP promoter deletion are both activated by Sp1, the differential response in this assay does not result from upstream activators. However, the sequences around the start sites of DHFR and REP are not similar and our data suggest that they bind different proteins. Therefore, we propose that specific initiator elements are important for determination of the requirement of some promoters for the CTD.

RNA polymerase II elongation factors of Saccharomyces cerevisiae: a targeted proteomics approach.

To physically characterize the web of interactions connecting the Saccharomyces cerevisiae proteins suspected to be RNA polymerase II (RNAPII) elongation factors, subunits of Spt4/Spt5 and Spt16/Pob3 (corresponding to human DSIF and FACT), Spt6, TFIIF (Tfg1, -2, and -3), TFIIS, Rtf1, and Elongator (Elp1, -2, -3, -4, -5, and -6) were affinity purified under conditions designed to minimize loss of associated polypeptides and then identified by mass spectrometry. Spt16/Pob3 was discovered to associate with three distinct complexes: histones; Chd1/casein kinase II (CKII); and Rtf1, Paf1, Ctr9, Cdc73, and a previously uncharacterized protein, Leo1. Rtf1 and Chd1 have previously been implicated in the control of elongation, and the sensitivity to 6-azauracil of strains lacking Paf1, Cdc73, or Leo1 suggested that these proteins are involved in elongation by RNAPII as well. Confirmation came from chromatin immunoprecipitation (ChIP) assays demonstrating that all components of this complex, including Leo1, cross-linked to the promoter, coding region, and 3' end of the ADH1 gene. In contrast, the three subunits of TFIIF cross-linked only to the promoter-containing fragment of ADH1. Spt6 interacted with the uncharacterized, essential protein Iws1 (interacts with Spt6), and Spt5 interacted either with Spt4 or with a truncated form of Spt6. ChIP on Spt6 and the novel protein Iws1 resulted in the cross-linking of both proteins to all three regions of the ADH1 gene, suggesting that Iws1 is likely an Spt6-interacting elongation factor. Spt5, Spt6, and Iws1 are phosphorylated on consensus CKII sites in vivo, conceivably by the Chd1/CKII associated with Spt16/Pob3. All the elongation factors but Elongator copurified with RNAPII.

A human RNA polymerase II complex containing factors that modify chromatin structure.

We have isolated a human RNA polymerase II complex that contains chromatin structure remodeling activity and histone acetyltransferase activity. This complex contains the Srb proteins, the Swi-Snf complex, and the histone acetyltransferases CBP and PCAF in addition to RNA polymerase II. Notably, the general transcription factors are absent from this complex. The complex was purified by two different methods: conventional chromatography and affinity chromatography using antibodies directed against CDK8, the human homolog of the yeast Srb10 protein. Protein interaction studies demonstrate a direct interaction between RNA polymerase II and the histone acetyltransferases p300 and PCAF. Importantly, p300 interacts specifically with the nonphosphorylated, initiation-competent form of RNA polymerase II. In contrast, PCAF interacts with the elongation-competent, phosphorylated form of RNA polymerase II.

Functional substitution of an essential yeast RNA polymerase subunit by a highly conserved mammalian counterpart.

We isolated the cDNA encoding the homolog of the Saccharomyces cerevisiae nuclear RNA polymerase common subunit RPB6 from hamster CHO cells. Alignment of yeast RPB6 with its mammalian counterpart revealed that the subunits have nearly identical carboxy-terminal halves and a short acidic region at the amino terminus. Remarkably, the length and amino acid sequence of the hamster RPB6 are identical to those of the human RPB6 subunit. The conservation in sequence from lower to higher eukaryotes also reflects conservation of function in vivo, since hamster RPB6 supports normal wild-type yeast cell growth in the absence of the essential gene encoding RPB6.

Ctr9, Rtf1, and Leo1 are components of the Paf1/RNA polymerase II complex.

The Saccharomyces cerevisiae Paf1-RNA polymerase II (Pol II) complex is biochemically and functionally distinct from the Srb-mediator form of Pol II holoenzyme and is required for full expression of a subset of genes. In this work we have used tandem affinity purification tags to isolate the Paf1 complex and mass spectrometry to identify additional components. We have established that Ctr9, Rtf1, and Leo1 are factors that associate with Paf1, Cdc73, and Pol II, but not with the Srb-mediator. Deletion of either PAF1 or CTR9 leads to similar severe pleiotropic phenotypes, which are unaltered when the two mutations are combined. In contrast, we found that deletion of LEO1 or RTF1 leads to few obvious phenotypes, although mutation of RTF1 suppresses mutations in TATA-binding protein, alters transcriptional start sites, and affects elongation. Remarkably, deletion of LEO1 or RTF1 suppresses many paf1Delta phenotypes. In particular, an rtf1Delta paf1Delta double mutant grew faster, was less temperature sensitive, and was more resistant to caffeine and hydroxyurea than a paf1Delta single mutant. In addition, expression of the G(1) cyclin CLN1, reduced nearly threefold in paf1Delta, is restored to wild-type levels in the rtf1Delta paf1Delta double mutant. We suggest that lack of Paf1 results in a defective complex and a block in transcription, which is relieved by removal of Leo1 or Rtf1.