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Nucleosome dynamics in the coding region of a transcriptionally active locus is critical for understanding how RNA polymerase II progresses through the gene body. Histone acetylation and deacetylation critically influence nucleosome accessibility during DNA metabolic processes like transcription. Effect of such histone modifications is context and residue dependent. Rather than effect of individual histone residues, the network of modifications of several histone residues in combination generates a chromatin landscape that is conducive for transcription. Here we show that in Saccharomyces cerevisiae , crosstalk between deacetylation of the H4 N-terminal tail residue H4K16 and acetylation of the H3 core domain residue H3K56, promotes RNA polymerase II progression through the gene body. Results indicate that deacetylation of H4K16 precedes and in turn induces H3K56 acetylation. Effectively, recruitment of Rtt109, the HAT responsible for H3K56 acetylation is essentially dependent on H4K16 deacetylation. In Hos2 deletion strains, where H4K16 deacetylation is abolished, both H3K56 acetylation and RNA polymerase II recruitment gets significantly impaired. Notably, H4K16 deacetylation and H3K56 acetylation are found to be essentially dependent on active transcription. In summary, H4K16 deacetylation promotes H3K56 acetylation and the two modifications together work towards successful functioning of RNA polymerase II during active transcription.

The roles of the MYC transcription factor in transcriptional regulation have been studied intensively. However, the general mechanism underlying the recruitment of MYC to chromatin is less clear. Here, we found that the Krüppel-like transcription factor ZFP281 plays important roles in recruiting MYC to active promoters in mouse embryonic stem cells. At the genome scale, ZFP281 is broadly associated with MYC, and the depletion of ZFP281 significantly reduces the levels of MYC and RNA polymerase II at the ZFP281- and MYC-cobound genes. Specially, we found that recruitment is required for the regulation of the Lin28a oncogene and pri-let-7 transcription. Our results therefore suggest a major role of ZFP281 in recruiting MYC to chromatin and the integration of ZFP281 and the MYC/LIN28A/Let-7 loop into a multilevel circuit., (Copyright © 2019 American Society for Microbiology.)

Eukaryotic RNA polymerase II (RNAPII) transcribes mRNA genes and non-protein-coding RNA (ncRNA) genes, including those encoding small nuclear and nucleolar RNAs (sn/snoRNAs). In metazoans, RNAPII transcription of sn/snoRNAs is facilitated by a number of specialized complexes, but no such complexes have been discovered in yeast. It has been proposed that yeast sn/snoRNA and mRNA expression relies on a set of common factors, but the extent to which regulators of mRNA genes function at yeast sn/snoRNA genes is unclear. Here, we investigated a potential role for the Mediator complex, essential for mRNA gene transcription, in sn/snoRNA gene transcription. We found that Mediator maps to sn/snoRNA gene regulatory regions and that rapid depletion of the essential structural subunit Med14 strongly reduces RNAPII and TFIIB occupancy as well as nascent transcription of sn/snoRNA genes. Deletion of Med3 and Med15, subunits of the activator-interacting Mediator tail module, does not affect Mediator recruitment to or RNAPII and TFIIB occupancy of sn/snoRNA genes. Our analyses suggest that Mediator promotes PIC formation and transcription at sn/snoRNA genes, expanding the role of this critical regulator beyond its known functions in mRNA gene transcription and demonstrating further mechanistic similarity between the transcription of mRNA and sn/snoRNA genes., (Copyright © 2018 American Society for Microbiology.)

RNA polymerase II (RNAPII) is one of the central enzymes in cell growth and organizational development. It is a large macromolecular complex consisting of 12 subunits. Relative to the clear definition of RNAPII structure and biological function, the molecular mechanism of how RNAPII is assembled is poorly understood, and thus the key assembly factors acting for the assembly of RNAPII remain elusive. In this study, we identified two factors, Gpn2 and Rba50, that directly participate in the assembly of RNAPII. Gpn2 and Rba50 were demonstrated to interact with Rpb12 and Rpb3, respectively. An interaction between Gpn2 and Rba50 was also demonstrated. When Gpn2 and Rba50 are functionally defective, the assembly of the Rpb3 subcomplex is disrupted, leading to defects in the assembly of RNAPII. Based on these results, we conclude that Gpn2 and Rba50 directly participate in the assembly of the Rpb3 subcomplex and subsequently the biogenesis of RNAPII., (Copyright © 2018 American Society for Microbiology.)

The cellular response to DNA damage is an intricate mechanism that involves the interplay among several pathways. In this study, we provide evidence of the roles of the polyadenylation factor cleavage stimulation factor 50 (CstF-50) and the ubiquitin (Ub) escort factor p97 as cofactors of BRCA1/BARD1 E3 Ub ligase, facilitating chromatin remodeling during the DNA damage response (DDR). CstF-50 and p97 formed complexes with BRCA1/BARD1, Ub, and some BRCA1/BARD1 substrates, such as RNA polymerase (RNAP) II and histones. Furthermore, CstF-50 and p97 had an additive effect on the activation of the ubiquitination of these BRCA1/BARD1 substrates during DDR. Importantly, as a result of these functional interactions, BRCA1/BARD1/CstF-50/p97 had a specific effect on the chromatin structure of genes that were differentially expressed. This study provides new insights into the roles of RNA processing, BRCA1/BARD1, the Ub pathway, and chromatin structure during DDR., (Copyright © 2018 Fonseca et al.)

A yeast in vitro system was developed that is active for transcription at both TATA-containing and TATA-less promoters. Transcription with extracts made from cells depleted of TFIID subunit Taf1 demonstrated that promoters of both classes are TFIID dependent, in agreement with recent in vivo findings. TFIID depletion can be complemented in vitro by additional recombinant TATA binding protein (TBP) at only the TATA-containing promoters. In contrast, high levels of TBP did not complement Taf1 depletion in vivo and instead repressed transcription from both promoter types. We also demonstrate the importance of the TATA-like sequence found at many TATA-less promoters and describe how the presence or absence of the TATA element is likely not the only feature that distinguishes these two types of promoters., (Copyright © 2017 American Society for Microbiology.)

Termination of Saccharomyces cerevisiae RNA polymerase II (Pol II) transcripts occurs through two alternative pathways. Termination of mRNAs is coupled to cleavage and polyadenylation while noncoding transcripts are terminated through the Nrd1-Nab3-Sen1 (NNS) pathway in a process that is linked to RNA degradation by the nuclear exosome. Some mRNA transcripts are also attenuated through premature termination directed by the NNS complex. In this paper we present the results of nuclear depletion of the NNS component Nab3. As expected, many noncoding RNAs fail to terminate properly. In addition, we observe that nitrogen catabolite-repressed genes are upregulated by Nab3 depletion., (Copyright © 2017 American Society for Microbiology.)

We have recently demonstrated that an mRNA capping enzyme, Cet1, impairs promoter-proximal accumulation/pausing of RNA polymerase II (Pol II) independently of its capping activity in Saccharomyces cerevisiae to control transcription. However, it is still unknown how Pol II pausing is regulated by Cet1. Here, we show that Cet1's N-terminal domain (NTD) promotes the recruitment of FACT ( fa cilitates c hromatin t ranscription that enhances the engagement of Pol II into transcriptional elongation) to the coding sequence of an active gene, ADH1 , independently of mRNA-capping activity. Absence of Cet1's NTD decreases FACT targeting to ADH1 and consequently reduces the engagement of Pol II in transcriptional elongation, leading to promoter-proximal accumulation of Pol II. Similar results were also observed at other genes. Consistently, Cet1 interacts with FACT. Collectively, our results support the notion that Cet1's NTD promotes FACT targeting to the active gene independently of mRNA-capping activity in facilitating Pol II's engagement in transcriptional elongation, thus deciphering a novel regulatory pathway of gene expression., (Copyright © 2017 American Society for Microbiology.)

Expression of the genome requires RNA polymerase II (RNAPII) to transcribe across many natural and unnatural barriers, and this transcription across barriers is facilitated by protein complexes called elongation factors (EFs). Genetic studies in Saccharomyces cerevisiae yeast suggest that multiple EFs collaborate to assist RNAPII in completing the transcription of genes, but the molecular mechanisms of how they cooperate to promote elongation are not well understood. The Ccr4-Not complex participates in multiple steps of mRNA metabolism and has recently been shown to be an EF. Here we describe how Ccr4-Not and TFIIS cooperate to stimulate elongation. We find that Ccr4-Not and TFIIS mutations show synthetically enhanced phenotypes, and biochemical analyses indicate that Ccr4-Not and TFIIS work synergistically to reactivate arrested RNAPII. Ccr4-Not increases the recruitment of TFIIS into elongation complexes and enhances the cleavage of the displaced transcript in backtracked RNAPII. This is mediated by an interaction between Ccr4-Not and the N terminus of TFIIS. In addition to revealing insights into how these two elongation factors cooperate to promote RNAPII elongation, our study extends the growing body of evidence suggesting that the N terminus of TFIIS acts as a docking/interacting site that allows it to synergize with other EFs to promote RNAPII transcription., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Mediator is a large, multisubunit complex that is required for essentially all mRNA transcription in eukaryotes. In spite of the importance of Mediator, the range of its targets and how it is recruited to these is not well understood. Previous work showed that in Saccharomyces cerevisiae, Mediator contributes to transcriptional activation by two distinct mechanisms, one depending on the tail module triad and favoring SAGA-regulated genes, and the second occurring independently of the tail module and favoring TFIID-regulated genes. Here, we use chromatin immunoprecipitation sequencing (ChIP-seq) to show that dependence on tail module subunits for Mediator recruitment and polymerase II (Pol II) association occurs preferentially at SAGA-regulated over TFIID-regulated genes on a genome-wide scale. We also show that recruitment of tail module subunits to active gene promoters continues genome-wide when Mediator integrity is compromised in med17 temperature-sensitive (ts) yeast, demonstrating the modular nature of the Mediator complex in vivo. In addition, our data indicate that promoters exhibiting strong and stable occupancy by Mediator have a wide range of activity and are enriched for targets of the Tup1-Cyc8 repressor complex. We also identify a number of strong Mediator occupancy peaks that overlap dubious open reading frames (ORFs) and are likely to include previously unrecognized upstream activator sequences., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

What has been will be again, what has been done will be done again; there is nothing new under the sun. -Ecclesiastes 1:9 (New International Version) Posttranscriptional regulation of gene expression has an important role in defining the phenotypic characteristics of an organism. Well-defined steps in mRNA metabolism that occur in the nucleus-capping, splicing, and polyadenylation-are mechanistically linked to the process of transcription. Recent evidence suggests another link between RNA polymerase II (Pol II) and a posttranscriptional process that occurs in the cytoplasm-mRNA decay. This conclusion appears to represent a conundrum. How could mRNA synthesis in the nucleus and mRNA decay in the cytoplasm be mechanistically linked? After a brief overview of mRNA processing, we will review the recent evidence for transcription-coupled mRNA decay and the possible involvement of Snf1, the Saccharomyces cerevisiae ortholog of AMP-activated protein kinase, in this process., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The C-terminal domain of the RNA polymerase II largest subunit (the Rpb1 CTD) is composed of tandem heptad repeats of the consensus sequence Y(1)S(2)P(3)T(4)S(5)P(6)S(7). We reported previously that Thr 4 is phosphorylated and functions in histone mRNA 3'-end formation in chicken DT40 cells. Here, we have extended our studies on Thr 4 and to other CTD mutations by using these cells. We found that an Rpb1 derivative containing only the N-terminal half of the CTD, as well as a similar derivative containing all-consensus repeats (26r), conferred full viability, while the C-terminal half, with more-divergent repeats, did not, reflecting a strong and specific defect in snRNA 3'-end formation. Mutation in 26r of all Ser 2 (S2A) or Ser 5 (S5A) residues resulted in lethality, while Ser 7 (S7A) mutants were fully viable. While S2A and S5A cells displayed defects in transcription and RNA processing, S7A cells behaved identically to 26r cells in all respects. Finally, we found that Thr 4 was phosphorylated by cyclin-dependent kinase 9 in cells and dephosphorylated both in vitro and in vivo by the phosphatase Fcp1., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The role of the negative elongation factor (NELF) in maintaining HIV latency was investigated following small hairpin RNA (shRNA) knockdown of the NELF-E subunit, a condition that induced high levels of proviral transcription in latently infected Jurkat T cells. Chromatin immunoprecipitation (ChIP) assays showed that latent proviruses accumulate RNA polymerase II (RNAP II) on the 5' long terminal repeat (LTR) but not on the 3' LTR. NELF colocalizes with RNAP II, and its level increases following proviral induction. RNAP II pause sites on the HIV provirus were mapped to high resolution by ChIP with high-throughput sequencing (ChIP-Seq). Like cellular promoters, RNAP II accumulates at around position +30, but HIV also shows additional pausing at +90, which is immediately downstream of a transactivation response (TAR) element and other distal sites on the HIV LTR. Following NELF-E knockdown or tumor necrosis factor alpha (TNF-α) stimulation, promoter-proximal RNAP II levels increase up to 3-fold, and there is a dramatic increase in RNAP II levels within the HIV genome. These data support a kinetic model for proviral transcription based on continuous replacement of paused RNAP II during both latency and productive transcription. In contrast to most cellular genes, HIV is highly activated by the combined effects of NELF-E depletion and activation of initiation by TNF-α, suggesting that opportunities exist to selectively activate latent HIV proviruses.

Facioscapulohumeral muscular dystrophy (FSHD) is linked to epigenetic dysregulation of the chromosome 4q35 D4Z4 macrosatellite. However, this does not account for the tissue specificity of FSHD pathology, which requires stable expression of an alternative full-length mRNA splice form of DUX4 (DUX4-fl) from the D4Z4 array in skeletal muscle. Here, we describe the identification of two enhancers, DUX4 myogenic enhancer 1 (DME1) and DME2 which activate DUX4-fl expression in skeletal myocytes but not fibroblasts. Analysis of the chromatin revealed histone modifications and RNA polymerase II occupancy consistent with DME1 and DME2 being functional enhancers. Chromosome conformation capture analysis confirmed association of DME1 and DME2 with the DUX4 promoter in vivo. The strong interaction between DME2 and the DUX4 promoter in both FSHD and unaffected primary myocytes was greatly reduced in fibroblasts, suggesting a muscle-specific interaction. Nucleosome occupancy and methylome sequencing analysis indicated that in most FSHD myocytes, both enhancers are associated with nucleosomes but have hypomethylated DNA, consistent with a permissive transcriptional state, sporadic occupancy, and the observed DUX4 expression in rare myonuclei. Our data support a model in which these myogenic enhancers associate with the DUX4 promoter in skeletal myocytes and activate transcription when epigenetically derepressed in FSHD, resulting in the pathological misexpression of DUX4-fl.

Topoisomerase IIβ (Top2β)-DNA cleavage complexes are known to arrest elongating RNA polymerase II (RNAPII), triggering a proteasomal degradation of the RNAPII large subunit (RNAPII LS) and Top2β itself as a prelude to DNA repair. Here, we demonstrate that the degradation of Top2β occurs through a novel ubiquitin-independent mechanism that requires only 19S AAA ATPases and 20S proteasome. Our results suggest that 19S AAA ATPases play a dual role in sensing the Top2β cleavage complex and coordinating its degradation by 20S proteasome when RNAPII is persistently stalled by the Top2β protein roadblock. Clarification of this transcription-associated proteasome pathway could shed light on a general role of 19S AAA ATPases in processing tight protein-DNA complexes during transcription elongation.

Recent papers have provided insight into the cytoplasmic assembly of RNA polymerase II (RNA pol II) and its transport to the nucleus. However, little is known about the mechanisms governing its nuclear assembly, stability, degradation, and recycling. We demonstrate that the foot of RNA pol II is crucial for the assembly and stability of the complex, by ensuring the correct association of Rpb1 with Rpb6 and of the dimer Rpb4-Rpb7 (Rpb4/7). Mutations at the foot affect the assembly and stability of the enzyme, a defect that is offset by RPB6 overexpression, in coordination with Rpb1 degradation by an Asr1-independent mechanism. Correct assembly is a prerequisite for the proper maintenance of several transcription steps. In fact, assembly defects alter transcriptional activity and the amount of enzyme associated with the genes, affect C-terminal domain (CTD) phosphorylation, interfere with the mRNA-capping machinery, and possibly increase the amount of stalled RNA pol II. In addition, our data show that TATA-binding protein (TBP) occupancy does not correlate with RNA pol II occupancy or transcriptional activity, suggesting a functional relationship between assembly, Mediator, and preinitiation complex (PIC) stability. Finally, our data help clarify the mechanisms governing the assembly and stability of RNA pol II.

In spite of the important regulatory functions of antisense transcripts in gene expression, it remains unknown how antisense transcription is initiated. Recent studies implicated RNA polymerase II in initiation of antisense transcription. However, how RNA polymerase II is targeted to initiate antisense transcription has not been elucidated. Here, we have analyzed the association of RNA polymerase II with the antisense initiation site at the 3' end of the GAL10 coding sequence in dextrose-containing growth medium that induces antisense transcription. We find that RNA polymerase II is targeted to the antisense initiation site at GAL10 by Reb1p activator as well as general transcription factors (e.g., TFIID, TFIIB, and Mediator) for antisense transcription initiation. Intriguingly, while GAL10 antisense transcription is dependent on TFIID, its sense transcription does not require TFIID. Further, the Gal4p activator that promotes GAL10 sense transcription is dispensable for antisense transcription. Moreover, the proteasome that facilitates GAL10 sense transcription does not control its antisense transcription. Taken together, our results reveal that GAL10 sense and antisense transcriptions are regulated differently and shed much light on the mechanisms of antisense transcription initiation.

Conserved from yeast to mammals, phosphorylation of the heptad repeat sequence Tyr(1)-Ser(2)-Pro(3)-Thr(4)-Ser(5)-Pro(6)-Ser(7) in the carboxy-terminal domain (CTD) of the largest RNA polymerase II (RNA Pol II) subunit, RPB1, mediates the enzyme's promoter escape and binding of RNA-processing factors, such as the m(7)G capping enzymes. The first critical step, Ser(5) phosphorylation, is carried out by cyclin-dependent kinase 7 (CDK7), a subunit of the basal transcription factor TFIIH. Many early-diverged protists, such as the lethal human parasite Trypanosoma brucei, however, lack the heptad repeats and, apparently, a CDK7 ortholog. Accordingly, characterization of trypanosome TFIIH did not identify a kinase component. The T. brucei CTD, however, is phosphorylated and essential for transcription. Here we show that silencing the expression of T. brucei cdc2-related kinase 9 (CRK9) leads to a loss of RPB1 phosphorylation. Surprisingly, this event did not impair RNA Pol II transcription or cotranscriptional m(7)G capping. Instead, we observed that CRK9 silencing led to a block of spliced leader (SL) trans splicing, an essential step in trypanosome mRNA maturation, that was caused by hypomethylation of the SL RNA's unique cap4.

KMT2B (MLL2/WBP7) is a member of the MLL subfamily of H3K4-specific histone lysine methyltransferases (KMT2) and is vital for normal embryonic development in the mouse. To gain insight into the molecular mechanism underlying KMT2B function, we focused on MagohB, which is controlled by a CpG island promoter. We show that in cells lacking Mll2-the gene encoding KMT2B-the MagohB promoter resides in inaccessible chromatin and is methylated. To dissect the molecular events leading to the establishment of silencing, we performed kinetic studies in Mll2-conditional-knockout embryonic stem cells. KMT2B depletion was followed by the loss of the active chromatin marks and progressive loss of RNA polymerase II binding with a concomitant downregulation of MagohB expression. Once the active chromatin marks were lost, the MagohB promoter was rapidly methylated. We demonstrate that in the presence of KMT2B, neither transcription elongation nor RNA polymerase II binding is required to maintain H3K4 trimethylation at the MagohB promoter and protect it from DNA methylation. Reexpression of KMT2B was sufficient to reinstate an active MagohB promoter. Our study provides a paradigm for the idea that KMT2 proteins are crucial components for establishing and maintaining the transcriptionally active and unmethylated state of CpG island promoters.

Adr1 and Cat8 are nutrient-regulated transcription factors in Saccharomyces cerevisiae that coactivate genes necessary for growth in the absence of a fermentable carbon source. Transcriptional activation by Adr1 is dependent on the AMP-activated protein kinase Snf1 and is inhibited by binding of Bmh, yeast 14-3-3 proteins, to the phosphorylated Adr1 regulatory domain. We show here that Bmh inhibits transcription by binding to Adr1 at promoters that contain a preinitiation complex, demonstrating that Bmh-mediated inhibition is not due to nuclear exclusion, inhibition of DNA binding, or RNA polymerase II (Pol II) recruitment. Adr1-dependent mRNA levels under repressing growth conditions are synergistically enhanced in a mutant lacking Bmh and the two major histone deacetylases (HDACs), suggesting that Bmh and HDACs inhibit gene expression independently. The synergism requires Snf1 and Adr1 but not Cat8. Inactivating Bmh or preventing it from binding to Adr1 suppresses the normal requirement for Cat8 at codependent promoters, suggesting that Bmh modulates combinatorial control of gene expression in addition to having an inhibitory role in transcription. Activating Snf1 by deleting Reg1, a Glc7 protein phosphatase regulatory subunit, is lethal in combination with defective Bmh in strain W303, suggesting that Bmh and Snf1 have opposing roles in an essential cellular process.

While the functions of hypoxia-inducible factor 1α (HIF1α)/aryl hydrocarbon receptor nuclear translocator (ARNT) and HIF2α/ARNT (HIF2) proteins in activating hypoxia-inducible genes are well established, the role of other transcription factors in the hypoxic transcriptional response is less clear. We report here for the first time that the basic helix-loop-helix-leucine-zip transcription factor upstream stimulatory factor 2 (USF2) is required for the hypoxic transcriptional response, specifically, for hypoxic activation of HIF2 target genes. We show that inhibiting USF2 activity greatly reduces hypoxic induction of HIF2 target genes in cell lines that have USF2 activity, while inducing USF2 activity in cells lacking USF2 activity restores hypoxic induction of HIF2 target genes. Mechanistically, USF2 activates HIF2 target genes by binding to HIF2 target gene promoters, interacting with HIF2α protein, and recruiting coactivators CBP and p300 to form enhanceosome complexes that contain HIF2α, USF2, CBP, p300, and RNA polymerase II on HIF2 target gene promoters. Functionally, the effect of USF2 knockdown on proliferation, motility, and clonogenic survival of HIF2-dependent tumor cells in vitro is phenocopied by HIF2α knockdown, indicating that USF2 works with HIF2 to activate HIF2 target genes and to drive HIF2-depedent tumorigenesis.

Initiation of transcription of RNA polymerase II (RNAPII)-dependent genes requires the participation of a host of basal transcription factors. Among genes requiring RNAPII for transcription, small nuclear RNAs (snRNAs) display a further requirement for a factor known as snRNA-activating protein complex (SNAPc). The scope of the biological function of SNAPc and its requirement for transcription of protein-coding genes has not been elucidated. To determine the genome-wide occupancy of SNAPc, we performed chromatin immunoprecipitation followed by high-throughput sequencing using antibodies against SNAPC4 and SNAPC1 subunits. Interestingly, while SNAPC4 occupancy was limited to snRNA genes, SNAPC1 chromatin residence extended beyond snRNA genes to include a large number of transcriptionally active protein-coding genes. Notably, SNAPC1 occupancy on highly active genes mirrored that of elongating RNAPII extending through the bodies and 3' ends of protein-coding genes. Inhibition of transcriptional elongation resulted in the loss of SNAPC1 from the 3' ends of genes, reflecting a functional association between SNAPC1 and elongating RNAPII. Importantly, while depletion of SNAPC1 had a small effect on basal transcription, it diminished the transcriptional responsiveness of a large number of genes to two distinct extracellular stimuli, epidermal growth factor (EGF) and retinoic acid (RA). These results highlight a role for SNAPC1 as a general transcriptional coactivator that functions through elongating RNAPII.

CrkRS (Cdc2-related kinase, Arg/Ser), or cyclin-dependent kinase 12 (CKD12), is a serine/threonine kinase believed to coordinate transcription and RNA splicing. While CDK12/CrkRS complexes were known to phosphorylate the C-terminal domain (CTD) of RNA polymerase II (RNA Pol II), the cyclin regulating this activity was not known. Using immunoprecipitation and mass spectrometry, we identified a 65-kDa isoform of cyclin K (cyclin K1) in endogenous CDK12/CrkRS protein complexes. We show that cyclin K1 complexes isolated from mammalian cells contain CDK12/CrkRS but do not contain CDK9, a presumed partner of cyclin K. Analysis of extensive RNA-Seq data shows that the 65-kDa cyclin K1 isoform is the predominantly expressed form across numerous tissue types. We also demonstrate that CDK12/CrkRS is dependent on cyclin K1 for its kinase activity and that small interfering RNA (siRNA) knockdown of CDK12/CrkRS or cyclin K1 has similar effects on the expression of a luciferase reporter gene. Our data suggest that cyclin K1 is the primary cyclin partner for CDK12/CrkRS and that cyclin K1 is required to activate CDK12/CrkRS to phosphorylate the CTD of RNA Pol II. These properties are consistent with a role of CDK12/CrkRS in regulating gene expression through phosphorylation of RNA Pol II.

The Ess1 prolyl isomerase in Saccharomyces cerevisiae regulates RNA polymerase II (pol II) by isomerizing peptide bonds within the pol II carboxy-terminal domain (CTD) heptapeptide repeat (YSPTSPS). Ess1 preferentially targets the Ser5-Pro6 bond when Ser5 is phosphorylated. Conformational changes in the CTD induced by Ess1 control the recruitment of essential cofactors to the pol II complex and may facilitate the ordered transition between initiation, elongation, termination, and RNA processing. Here, we show that Ess1 associates with the phospho-Ser5 form of polymerase in vivo, is present along the entire length of coding genes, and is critical for regulating the phosphorylation of Ser7 within the CTD. In addition, Ess1 represses the initiation of cryptic unstable transcripts (CUTs) and is required for efficient termination of mRNA transcription. Analysis using strains lacking nonsense-mediated decay suggests that as many as half of all yeast genes depend on Ess1 for efficient termination. Finally, we show that Ess1 is required for trimethylation of histone H3 lysine 4 (H3K4). Thus, Ess1 has direct effects on RNA polymerase transcription by controlling cofactor binding via conformationally induced changes in the CTD and indirect effects by influencing chromatin modification.

The integral telomerase RNA subunit templates the synthesis of telomeric repeats. The biological accumulation of human telomerase RNA (hTR) requires hTR H/ACA domain assembly with the same proteins that assemble on other human H/ACA RNAs. Despite this shared RNP composition, hTR accumulation is particularly sensitized to disruption by disease-linked H/ACA protein variants. We show that contrary to expectation, hTR-specific sequence requirements for biological accumulation do not act at an hTR-specific step of H/ACA RNP biogenesis; instead, they enhance hTR binding to the shared, chaperone-bound scaffold of H/ACA core proteins that mediates initial RNP assembly. We recapitulate physiological H/ACA RNP assembly with a preassembled NAF1/dyskerin/NOP10/NHP2 scaffold purified from cell extract and demonstrate that distributed sequence features of the hTR 3' hairpin synergize to improve scaffold binding. Our findings reveal that the hTR H/ACA domain is distinguished from other human H/ACA RNAs not by a distinct set of RNA-protein interactions but by an increased efficiency of RNP assembly. Our findings suggest a unifying mechanism for human telomerase deficiencies associated with H/ACA protein variants.

In fission yeast, discrete steps in mRNA maturation and synthesis depend on a complex containing the 5'-cap methyltransferase Pcm1 and Cdk9, which phosphorylates the RNA polymerase II (Pol II) carboxyl-terminal domain (CTD) and the processivity factor Spt5 to promote transcript elongation. Here we show that a Cdk9 carboxyl-terminal extension, distinct from the catalytic domain, mediates binding to both Pcm1 and the Pol II CTD. Removal of this segment diminishes Cdk9/Pcm1 chromatin recruitment and Spt5 phosphorylation in vivo and leads to slow growth and hypersensitivity to cold temperature, nutrient limitation, and the IMP dehydrogenase inhibitor mycophenolic acid (MPA). These phenotypes, and the Spt5 phosphorylation defect, are suppressed by Pcm1 overproduction, suggesting that normal transcript elongation and gene expression depend on physical linkage between Cdk9 and Pcm1. The extension is dispensable, however, for recognition of CTD substrates "primed" by Mcs6 (Cdk7). On defined peptide substrates in vitro, Cdk9 prefers CTD repeats phosphorylated at Ser7 over unmodified repeats. In vivo, Ser7 phosphorylation depends on Mcs6 activity, suggesting a conserved mechanism, independent of chromatin recruitment, to order transcriptional CDK functions. Therefore, fission yeast Cdk9 comprises a catalytic domain sufficient for primed substrate recognition and a multivalent recruitment module that couples transcription with capping.

The yeast Saccharomyces differentiates into filamentous pseudohyphae when exposed to a poor source of nitrogen in a process involving a collection of transcription factors regulated by nutrient signaling pathways. Phd1 is important for this process in that it regulates expression of most other transcription factors involved in differentiation and can induce filamentation on its own when overproduced. In this article, we show that Phd1 is an unstable protein whose degradation is initiated through phosphorylation by Cdk8 of the RNA polymerase II mediator subcomplex. Phd1 is stabilized by cdk8 disruption, and the naturally filamenting Σ1278b strain was found to have a sequence polymorphism that eliminates a Cdk8 phosphorylation site, which both stabilizes the protein and contributes to enhanced differentiation. In nitrogen-starved cells, PHD1 expression is upregulated and the Phd1 protein becomes stabilized, which causes its accumulation during differentiation. PHD1 expression is partially dependent upon Ste12, which was also previously shown to be destabilized by Cdk8-dependent phosphorylations, but to a significantly smaller extent than Phd1. These observations demonstrate the central role that Cdk8 plays in initiation of differentiation. Cdk8 activity is inhibited in cells shifted to limiting nutrient conditions, and we argue that this effect drives the initiation of differentiation through stabilization of multiple transcription factors, including Phd1, that cause activation of genes necessary for filamentous response.

To investigate the function and architecture of the open complex state of RNA polymerase II (Pol II), Saccharomyces cerevisiae minimal open complexes were assembled by using a series of heteroduplex HIS4 promoters, TATA binding protein (TBP), TFIIB, and Pol II. The yeast system demonstrates great flexibility in the position of active open complexes, spanning 30 to 80 bp downstream from TATA, consistent with the transcription start site scanning behavior of yeast Pol II. TFIIF unexpectedly modulates the activity of the open complexes, either repressing or stimulating initiation. The response to TFIIF was dependent on the sequence of the template strand within the single-stranded bubble. Mutations in the TFIIB reader and linker region, which were inactive on duplex DNA, were suppressed by the heteroduplex templates, showing that a major function of the TFIIB reader and linker is in the initiation or stabilization of single-stranded DNA. Probing of the architecture of the minimal open complexes with TFIIB-FeBABE [TFIIB-p-bromoacetamidobenzyl-EDTA-iron(III)] derivatives showed that the TFIIB core domain is surprisingly positioned away from Pol II, and the addition of TFIIF repositions the TFIIB core domain to the Pol II wall domain. Together, our results show an unexpected architecture of minimal open complexes and the regulation of activity by TFIIF and the TFIIB core domain.

The flap domain of multisubunit RNA polymerases (RNAPs), also called the wall, forms one side of the RNA exit channel. In bacterial RNAP, the mobile part of the flap is called the flap tip and makes essential contacts with initiation and elongation factors. Cocrystal structures suggest that the orthologous part of eukaryotic RNAPII, called the flap loop, contacts transcription factor IIB (TFIIB), but the function of the flap loop has not been assessed. We constructed and tested a deletion of the flap loop in human RNAPII (subunit RPB2 Δ873-884) that removes the flap loop interaction interface with TFIIB. Genome-wide analysis of the distribution of the RNAPII with the flap loop deletion expressed in a human embryonic kidney cell line (HEK 293) revealed no effect of the flap loop on global transcription initiation, RNAPII occupancy within genes, or the efficiency of promoter escape and productive elongation. In vitro, the flap loop deletion had no effect on promoter binding, abortive initiation or promoter escape, TFIIS-stimulated transcript cleavage, or inhibition of transcript elongation by the complex of negative elongation factor (NELF) and 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF). A modest effect on transcript elongation and pausing was suppressed by TFIIF. Although similar to the flap tip of bacterial RNAP, the RNAPII flap loop is not equivalently essential.

Nucleosomes containing histone variant H2A.Z (Htz1) serve to poise quiescent genes for activation and transcriptional initiation. However, little is known about their role in transcription elongation. Here we show that dominant mutations in the elongation genes SPT5 and SPT16 suppress the hypersensitivity of htz1Δ strains to drugs that inhibit elongation, indicating that Htz1 functions at the level of transcription elongation. Direct kinetic measurements of RNA polymerase II (Pol II) movement across the 9.5-kb GAL10p-VPS13 gene revealed that the elongation rate of polymerase is 24% slower in the absence of Htz1. We provide evidence for two nonexclusive mechanisms. First, we observed that both the phospho-Ser2 levels in the elongating isoform of Pol II and the loading of Spt5 and Elongator over the GAL1 open reading frame (ORF) depend on Htz1. Second, in the absence of Htz1, the density of nucleosome occupancy is increased over the GAL10p-VPS13 ORF and the chromatin is refractory to remodeling during active transcription. These results establish a mechanistic role for Htz1 in transcription elongation and suggest that Htz1-containing nucleosomes facilitate Pol II passage by affecting the correct assembly and modification status of Pol II elongation complexes and by favoring efficient nucleosome remodeling over the gene.

The conserved RECQ5 DNA helicase is a tumor suppressor in mammalian cells. Defects in RECQ5 lead to the accumulation of spontaneous DNA double-stranded breaks (DSBs) during replication, despite the fact that these cells are proficient in DSB repair by homologous recombination (HR). The reason for this is unknown. Here, we demonstrate that these DSBs are linked to RNA polymerase II (RNAPII)-dependent transcription. In human RECQ5-depleted cells, active RNAPII accumulates on chromatin, and DNA breaks are associated with an RNAPII-dependent transcribed locus. Hence, transcription inhibition eliminates both active RNAPII and spontaneous DSB formation. In addition, the regulatory effect of RECQ5 on transcription and its interaction with RNAPII are enhanced in S-phase cells, supporting a role for RECQ5 in preventing transcription-associated DSBs during replication. Finally, we show that the SET2-RPB1 interaction (SRI) domain of human RECQ5 is important for suppressing spontaneous DSBs and the p53-dependent transcription stress response caused by the stalling of active RNAPII on DNA. Thus, our studies provide novel insights into a mechanism by which RECQ5 regulates the transcription machinery via its dynamic interaction with RNAPII, thereby preventing genome instability.

Many human genes exhibit evidence of initiated RNA polymerase II (Pol II) at their promoters, despite a lack of significant full-length transcript. Such genes exhibit promoter-proximal "pausing," wherein initiated Pol II accumulates just downstream of the transcription start site due to a rate-limiting step mediating the transition to elongation. The mechanisms that regulate the escape of Pol II from pausing and the relationship to chromatin structure remain incompletely understood. Recently, we showed that CpG island hypermethylation and epigenetic silencing of TMS1/ASC in human breast cancers are accompanied by a local shift from histone H4 lysine 16 acetylation (H4K16Ac) to H4 lysine 20 trimethylation (H4K20me3). Here, we show that hMOF-mediated H4K16Ac and SUV420H2-mediated H4K20me3 play opposing roles in the regulation of Pol II pausing. We found that H4K16Ac promoted the release of Pol II from pausing through the recruitment of BRD4 and pTEFb. Aberrant methylation of CpG island DNA blocked Pol II recruitment to gene promoters. Whereas the inhibition of DNA methylation allowed for the reassociation and initiation of Pol II at the TMS1 promoter, Pol II remained paused in the presence of H4K20me3. Combined inhibition of H4K20me3 and DNA methylation resulted in the rerecruitment of hMOF and subsequent H4K16Ac, release of Pol II into active elongation, and synergistic reactivation of TMS1 expression. Marking by H4K20me3 was not restricted to TMS1 but also occurred at other genes independently of DNA methylation, where it similarly imposed a block to Pol II promoter escape through a mechanism that involved the local inhibition of H4K16Ac. These data indicate that H4K20me3 invokes gene repression by antagonizing hMOF-mediated H4K16Ac and suggest that overcoming Pol II pausing might be a rate-limiting step in achieving tumor suppressor gene reactivation in cancer therapy.

Proper gene expression relies on a class of ubiquitously expressed, uridine-rich small nuclear RNAs (snRNAs) transcribed by RNA polymerase II (RNAPII). Vertebrate snRNAs are transcribed from a unique promoter, which is required for proper 3'-end formation, and cleavage of the nascent transcript involves the activity of a poorly understood set of proteins called the Integrator complex. To examine 3'-end formation in Drosophila melanogaster, we developed a cell-based reporter that monitors aberrant 3'-end formation of snRNA through the gain in expression of green fluorescent protein (GFP). We used this reporter in Drosophila S2 cells to determine requirements for U7 snRNA 3'-end formation and found that processing was strongly dependent upon nucleotides located within the 3' stem-loop as well as sequences likely to comprise the Drosophila equivalent of the vertebrate 3' box. Substitution of the actin promoter for the snRNA promoter abolished proper 3'-end formation, demonstrating the conserved requirement for an snRNA promoter in Drosophila. We tested the requirement for all Drosophila Integrator subunits and found that Integrators 1, 4, 9, and 11 were essential for 3'-end formation and that Integrators 3 and 10 may be dispensable for processing. Depletion of cleavage and polyadenylation factors or of histone pre-mRNA processing factors did not affect U7 snRNA processing efficiency, demonstrating that the Integrator complex does not share components with the mRNA 3'-end processing machinery. Finally, flies harboring mutations in either Integrator 4 or 7 fail to complete development and accumulate significant levels of misprocessed snRNA in the larval stages.

The transcriptional coactivator Sub1 has been implicated in several aspects of mRNA metabolism in yeast, such as activation of transcription, termination, and 3'-end formation. Here, we present evidence that Sub1 plays a significant role in controlling phosphorylation of the RNA polymerase II large subunit C-terminal domain (CTD). We show that SUB1 genetically interacts with the genes encoding all four known CTD kinases, SRB10, KIN28, BUR1, and CTK1, suggesting that Sub1 acts to influence CTD phosphorylation at more than one step of the transcription cycle. To address this directly, we first used in vitro kinase assays, and we show that, on the one hand, SUB1 deletion increased CTD phosphorylation by Kin28, Bur1, and Ctk1 but, on the other, it decreased CTD phosphorylation by Srb10. Second, chromatin immunoprecipitation assays revealed that SUB1 deletion decreased Srb10 chromatin association on the inducible GAL1 gene but increased Kin28 and Ctk1 chromatin association on actively transcribed genes. Taken together, our data point to multiple roles for Sub1 in the regulation of CTD phosphorylation throughout the transcription cycle.

Short repetitive sequences are common in the human genome, and many fall within transcription units. We have previously shown that transcription through CAG repeat tracts destabilizes them in a way that depends on transcription-coupled nucleotide excision repair and mismatch repair. Recent observations that antisense transcription accompanies sense transcription in many human genes led us to test the effects of antisense transcription on triplet repeat instability in human cells. Here, we report that simultaneous sense and antisense transcription (convergent transcription) initiated from two inducible promoters flanking a CAG95 tract in a nonessential gene enhances repeat instability synergistically, arrests the cell cycle, and causes massive cell death via apoptosis. Using chemical inhibitors and small interfering RNA (siRNA) knockdowns, we identified the ATR (ataxia-telangiectasia mutated [ATM] and Rad3 related) signaling pathway as a key mediator of this cellular response. RNA polymerase II, replication protein A (RPA), and components of the ATR signaling pathway accumulate at convergently transcribed repeat tracts, accompanied by phosphorylation of ATR, CHK1, and p53. Cell death depends on simultaneous sense and antisense transcription and is proportional to their relative levels, it requires the presence of the repeat tract, and it occurs in both proliferating and nonproliferating cells. Convergent transcription through a CAG repeat represents a novel mechanism for triggering a cellular stress response, one that is initiated by events at a single locus in the genome and resembles the response to DNA damage.

The heptad repeat of the RNA polymerase II (RNAPII) C-terminal domain is phosphorylated at serine 5 near gene 5' ends and serine 2 near 3' ends in order to recruit pre-mRNA processing factors. Ser-5(P) is associated with gene 5' ends to recruit capping enzymes, whereas Ser-2(P) is associated with gene 3' ends to recruit cleavage and polyadenylation factors. In the gene clusters called operons in Caenorhabditis elegans, there is generally only a single promoter, but each gene in the operon forms a 3' end by the usual mechanism. Although downstream operon genes have 5' ends, they receive their caps by trans splicing rather than by capping enzymes. Thus, they are predicted to not need Ser-5 phosphorylation. Here we show by RNAPII chromatin immunoprecipitation (ChIP) that internal operon gene 5' ends do indeed lack Ser-5(P) peaks. In contrast, Ser-2(P) peaks occur at each mRNA 3' end, where the 3'-end formation machinery binds. These results provide additional support for the idea that the serine phosphorylation of the C-terminal domain (CTD) serves to bring RNA-processing enzymes to the transcription complex. Furthermore, these results provide a novel demonstration that genes in operons are cotranscribed from a single upstream promoter.

Mutations in PHF8 are associated with X-linked mental retardation and cleft lip/cleft palate. PHF8 contains a plant homeodomain (PHD) in its N terminus and is a member of a family of JmjC domain-containing proteins. While PHDs can act as methyl lysine recognition motifs, JmjC domains can catalyze lysine demethylation. Here, we show that PHF8 is a histone demethylase that removes repressive histone H3 dimethyl lysine 9 marks. Our biochemical analysis revealed specific association of the PHF8 PHD with histone H3 trimethylated at lysine 4 (H3K4me3). Chromatin immunoprecipitation followed by high-throughput sequencing indicated that PHF8 is enriched at the transcription start sites of many active or poised genes, mirroring the presence of RNA polymerase II (RNAPII) and of H3K4me3-bearing nucleosomes. We show that PHF8 can act as a transcriptional coactivator and that its activation function largely depends on binding of the PHD to H3K4me3. Furthermore, we present evidence for direct interaction of PHF8 with the C-terminal domain of RNAPII. Importantly, a PHF8 disease mutant was defective in demethylation and in coactivation. This is the first demonstration of a chromatin-modifying enzyme that is globally recruited to promoters through its association with H3K4me3 and RNAPII.

The RNA polymerase II core promoter is a diverse and complex regulatory element. To gain a better understanding of the core promoter, we examined the motif 10 element (MTE), which is located downstream of the transcription start site and acts in conjunction with the initiator (Inr). We found that the MTE promotes the binding of purified TFIID to the core promoter and that the TAF6 and TAF9 subunits of TFIID appear to be in close proximity to the MTE. To identify the specific nucleotides that contribute to MTE activity, we performed a detailed mutational analysis and determined a functional MTE consensus sequence. These studies identified favored as well as disfavored nucleotides and demonstrated the previously unrecognized importance of nucleotides in the subregion of nucleotides 27 to 29 (+27 to + 29 relative to A(+1) in the Inr consensus) for MTE function. Further analysis led to the identification of three downstream subregions (nucleotides 18 to 22, 27 to 29, and 30 to 33) that contribute to core promoter activity. The three binary combinations of these subregions lead to the MTE (nucleotides 18 to 22 and 27 to 29), a downstream core promoter element (nucleotides 27 to 29 and 30 to 33), and a novel "bridge" core promoter motif (nucleotides 18 to 22 and 30 to 33). These studies have thus revealed a tripartite organization of key subregions in the downstream core promoter.

Switching between alternate states of gene transcription is fundamental to a multitude of cellular regulatory pathways, including those that govern differentiation. In spite of the progress in our understanding of such transitions in gene activity, a major unanswered question is how cells regulate the timing of these switches. Here, we have examined the kinetics of a transcriptional switch that accompanies the differentiation of yeast cells of one mating type into a distinct new cell type. We found that cell-type-specific genes silenced by the alpha2 repressor in the starting state are derepressed to establish the new mating-type-specific gene expression program coincident with the loss of alpha2 from promoters. This rapid derepression does not require the preloading of RNA polymerase II or a preinitiation complex but instead depends upon the Gcn5 histone acetyltransferase. Surprisingly, Gcn5-dependent acetylation of nucleosomes in the promoters of mating-type-specific genes requires the corepressor Ssn6-Tup1 even in the repressed state. Gcn5 partially acetylates the amino-terminal tails of histone H3 in repressed promoters, thereby priming them for rapid derepression upon loss of alpha2. Thus, Ssn6-Tup1 not only efficiently represses these target promoters but also functions to initiate derepression by creating a chromatin state poised for rapid activation.

The RecQL5 helicase is essential for maintaining genome stability and reducing cancer risk. To elucidate its mechanism of action, we purified a RecQL5-associated complex and identified its major component as RNA polymerase II (Pol II). Bioinformatics and structural modeling-guided mutagenesis revealed two conserved regions in RecQL5 as KIX and SRI domains, already known in transcriptional regulators for Pol II. The RecQL5-KIX domain binds both initiation (Pol IIa) and elongation (Pol IIo) forms of the polymerase, whereas the RecQL5-SRI domain interacts only with the elongation form. Fully functional RecQL5 requires both helicase activity and associations with the initiation polymerase, because mutants lacking either activity are partially defective in the suppression of sister chromatid exchange and resistance to camptothecin-induced DNA damage, and mutants lacking both activities are completely defective. We propose that RecQL5 promotes genome stabilization through two parallel mechanisms: by participation in homologous recombination-dependent DNA repair as a RecQ helicase and by regulating the initiation of Pol II to reduce transcription-associated replication impairment and recombination.

Several recent studies have shown that the transcriptional induction of yeast GAL genes occurs with faster kinetics if the gene has been previously expressed. Depending on the experimental regimen, this transcriptional "memory" phenomenon can persist for 1 to 2 cell divisions in the absence of an inducer (short-term memory) or for >6 cell divisions (long-term memory). Long-term memory requires the GAL1 gene, suggesting that memory involves the cytoplasmic inheritance of high levels of Gal1 that are expressed in the initial round of expression. In contrast, short-term memory requires the SWI/SNF chromatin-remodeling enzyme, and thus, it may involve the inheritance of distinct chromatin states. Here we have reevaluated the roles of SWI/SNF, the histone variant H2A.Z, and components of the nuclear pore in both the short-term and long-term memory of GAL genes. Our results suggest that the propagation of novel chromatin structures does not contribute to the transcriptional memory of GAL genes, but rather, memory of the previous transcription state is controlled primarily by the inheritance of the Gal3p and Gal1p signaling factors.

An interaction network connecting mRNA capping enzymes, the RNA polymerase II (Pol II) carboxyl-terminal domain (CTD), elongation factor Spt5, and the Cdk7 and Cdk9 protein kinases is thought to comprise a transcription elongation checkpoint. A crux of this network is Spt5, which regulates early transcription elongation and has an imputed role in pre-mRNA processing via its physical association with capping enzymes. Schizosaccharomyces pombe Spt5 has a distinctive CTD composed of tandem nonapeptide repeats of the consensus sequence (1)TPAWNSGSK(9). The Spt5 CTD binds the capping enzymes and is a substrate for threonine phosphorylation by the Cdk9 kinase. Here we report that deletion of the S. pombe Spt5 CTD results in slow growth and aberrant cell morphology. The severity of the spt5-DeltaCTD phenotype is exacerbated by truncation of the Pol II CTD and ameliorated by overexpression of the capping enzymes RNA triphosphatase and RNA guanylyltransferase. These results suggest that the Spt5 and Pol II CTDs play functionally overlapping roles in capping enzyme recruitment. We probed structure-activity relations of the Spt5 CTD by alanine scanning of the consensus nonapeptide. The T1A change abolished CTD phosphorylation by Cdk9 but did not affect CTD binding to the capping enzymes. The T1A and P2A mutations elicited cold-sensitive (cs) and temperature-sensitive (ts) growth defects and conferred sensitivity to growth inhibition by 6-azauracil that was exacerbated by partial truncations of the Pol II CTD. The T1A phenotypes were rescued by a phosphomimetic T1E change but not by capping enzyme overexpression. These results imply a positive role for Spt5 CTD phosphorylation in Pol Il transcription elongation in fission yeast, distinct from its capping enzyme interactions. Viability of yeast cells bearing both Spt5 CTD T1A and Pol II CTD S2A mutations heralds that the Cdk9 kinase has an essential target other than Spt5 and Pol II CTD-Ser2.

In all eukaryotes, a specialized enzyme, RNA polymerase I (Pol I), is dedicated to transcribe the 35S rRNA gene from a multicopy gene cluster, the ribosomal DNA (rDNA). In certain Saccharomyces cerevisiae mutants, 35S rRNA genes can be transcribed by RNA polymerase II (Pol II). In these mutants, rDNA silencing of Pol II transcription is impaired. It has been speculated that upstream activating factor (UAF), which binds to a specific DNA element within the Pol I promoter, plays a crucial role in forming chromatin structures responsible for polymerase specificity and silencing at the rDNA locus. We therefore performed an in-depth analysis of chromatin structure and composition in different mutant backgrounds. We demonstrate that chromatin architecture of the entire Pol I-transcribed region is substantially altered in the absence of UAF, allowing RNA polymerases II and III to access DNA elements flanking a Pol promoter-proximal Reb1 binding site. Furthermore, lack of UAF leads to the loss of Sir2 from rDNA, correlating with impaired Pol II silencing. This analysis of rDNA chromatin provides a molecular basis, explaining many phenotypes observed in previous genetic analyses.

NuA4, the major H4 lysine acetyltransferase (KAT) complex in Saccharomyces cerevisiae, is recruited to promoters and stimulates transcription initiation. NuA4 subunits contain domains that bind methylated histones, suggesting that histone methylation should target NuA4 to coding sequences during transcription elongation. We show that NuA4 is cotranscriptionally recruited, dependent on its physical association with elongating polymerase II (Pol II) phosphorylated on the C-terminal domain by cyclin-dependent kinase 7/Kin28, but independently of subunits (Eaf1 and Tra1) required for NuA4 recruitment to promoters. Whereas histone methylation by Set1 and Set2 is dispensable for NuA4's interaction with Pol II and targeting to some coding regions, it stimulates NuA4-histone interaction and H4 acetylation in vivo. The NuA4 KAT, Esa1, mediates increased H4 acetylation and enhanced RSC occupancy and histone eviction in coding sequences and stimulates the rate of transcription elongation. Esa1 cooperates with the H3 KAT in SAGA, Gcn5, to enhance these functions. Our findings delineate a pathway for acetylation-mediated nucleosome remodeling and eviction in coding sequences that stimulates transcription elongation by Pol II in vivo.

The first step in microRNA (miRNA) biogenesis occurs in the nucleus and is mediated by the Microprocessor complex containing the RNase III-like enzyme Drosha and its cofactor DGCR8. Here we show that the 5'-->3' exonuclease Xrn2 associates with independently transcribed miRNAs and, in combination with Drosha processing, attenuates transcription in downstream regions. We suggest that, after Drosha cleavage, a torpedo-like mechanism acts on nascent long precursor miRNAs, whereby Xrn2 exonuclease degrades the RNA polymerase II-associated transcripts inducing its release from the template. While involved in primary transcript termination, this attenuation effect does not restrict clustered miRNA expression, which, in the majority of cases, is separated by short spacers. We also show that transcripts originating from a miRNA promoter are retained on the chromatin template and are more efficiently processed than those produced from mRNA or snRNA Pol II-dependent promoters. These data imply that coupling between transcription and processing promotes efficient expression of independently transcribed miRNAs.

In eukaryotic cells, the nascent pre-mRNA molecule is coated sequentially with a large set of processing and binding proteins that mediate its transformation into an export-competent ribonucleoprotein particle (mRNP) that is ready for translation in the cytoplasm. We have implemented an original assay that monitors the dynamic interplay between transcription and mRNP biogenesis and that allows the screening for new factors linking mRNA synthesis to translation in Saccharomyces cerevisiae. The assay is based on the perturbation of gene expression induced by the bacterial Rho factor, an RNA-dependent helicase/translocase that acts as a competitor at one or several steps of mRNP biogenesis in yeast. We show that the expression of Rho in yeast leads to a dose-dependent growth defect that stems from its action on RNA polymerase II-mediated transcription. Rho expression induces the production of aberrant transcripts that are degraded by the nuclear exosome. A screen for dosage suppressors of the Rho-induced growth defect identified several genes that are involved in the different steps of mRNP biogenesis and export, as well as other genes with both known functions in transcription regulation and unknown functions. Our results provide evidence for an extensive cross talk between transcription, mRNP biogenesis, and export. They also uncover new factors that potentially are involved in these interconnected events.

The Swi/Snf nucleosome-remodeling complex is recruited by DNA-binding activator proteins, whereupon it alters chromatin structure to increase preinitiation complex formation and transcription. At the SUC2 promoter, the Swi/Snf complex is required for histone eviction in a manner that is independent of transcriptional activity. Swi/Snf travels through coding regions with elongating RNA polymerase (Pol) II, and swi2 mutants exhibit sensitivity to drugs affecting Pol elongation. In FACT-depleted cells, Swi/Snf is important for internal initiation within coding regions, suggesting that Swi/Snf is important for histone eviction that occurs during Pol II elongation. Taken together, these observations suggest that Swi/Snf is important for histone eviction at enhancers and that it also functions as a Pol II elongation factor.

A transcription corepressor, MAT1-mediated transcriptional repressor (MMTR), was found in mouse embryonic stem cell lines. MMTR orthologs (DMAP1) are found in a wide variety of life forms from yeasts to humans. MMTR down-regulation in differentiating mouse embryonic stem cells in vitro resulted in activation of many unrelated genes, suggesting its role as a general transcriptional repressor. In luciferase reporter assays, the transcriptional repression activity resided at amino acids 221 to 468. Histone deacetylase 1 (HDAC1) interacts with MMTR both in vitro and in vivo and also interacts with MMTR in the nucleus. Interestingly, MMTR activity was only partially rescued by competition with dominant-negative HDAC1(H141A) or by treatment with an HDAC inhibitor, trichostatin A (TSA). To identify the protein responsible for HDAC1-independent MMTR activity, we performed a yeast two-hybrid screen with the full-length MMTR coding sequence as bait and found MAT1. MAT1 is an assembly/targeting factor for cyclin-dependent kinase-activating kinase which constitutes a subcomplex of TFIIH. The coiled-coil domain in the middle of MAT1 was confirmed to interact with the C-terminal half of MMTR, and the MMTR-mediated transcriptional repression activity was completely restored by MAT1 in the presence of TSA. Moreover, intact MMTR was required to inhibit phosphorylation of the C-terminal domain in the RNA polymerase II largest subunit by TFIIH kinase in vitro. Taken together, these data strongly suggest that MMTR is part of the basic cellular machinery for a wide range of transcriptional regulation via interaction with TFIIH and HDAC.

In amphibian oocytes, the maternal nuclear factor NF7 associates with the elongating pre-mRNAs present on the numerous lateral loops of the lampbrush chromosomes. Here, we have purified NF7 from an oocyte extract by using a combination of ion-exchange chromatography and gel filtration chromatography and demonstrated for the first time that nucleoplasmic NF7 exists primarily as free homotrimers. We confirmed the in vivo homotrimerization of NF7 by using a glutaraldehyde cross-linking assay, and we further showed that it only requires the coiled-coil domain of the NF7 tripartite motif/RBCC motif. Interestingly, we also obtained evidence that NF7 is recruited to the nucleus as a homotrimer, and expression of several mutated forms of NF7 in oocytes demonstrated that both the coiled coil and B box of NF7 are required for its chromosomal association. Together, these data strongly suggest that the interaction of NF7 with the active transcriptional units of RNA polymerase II is mediated by a trimeric B box. Finally, and in agreement with a role for NF7 in pre-mRNA maturation, we obtained evidence supporting the idea that NF7 associates with Cajal bodies.

CTCF is a transcription factor with highly versatile functions ranging from gene activation and repression to the regulation of insulator function and imprinting. Although many of these functions rely on CTCF-DNA interactions, it is an emerging realization that CTCF-dependent molecular processes involve CTCF interactions with other proteins. In this study, we report the association of a subpopulation of CTCF with the RNA polymerase II (Pol II) protein complex. We identified the largest subunit of Pol II (LS Pol II) as a protein significantly colocalizing with CTCF in the nucleus and specifically interacting with CTCF in vivo and in vitro. The role of CTCF as a link between DNA and LS Pol II has been reinforced by the observation that the association of LS Pol II with CTCF target sites in vivo depends on intact CTCF binding sequences. "Serial" chromatin immunoprecipitation (ChIP) analysis revealed that both CTCF and LS Pol II were present at the beta-globin insulator in proliferating HD3 cells but not in differentiated globin synthesizing HD3 cells. Further, a single wild-type CTCF target site (N-Myc-CTCF), but not the mutant site deficient for CTCF binding, was sufficient to activate the transcription from the promoterless reporter gene in stably transfected cells. Finally, a ChIP-on-ChIP hybridization assay using microarrays of a library of CTCF target sites revealed that many intergenic CTCF target sequences interacted with both CTCF and LS Pol II. We discuss the possible implications of our observations with respect to plausible mechanisms of transcriptional regulation via a CTCF-mediated direct link of LS Pol II to the DNA.