NELF focuses sites of initiation and maintains promoter architecture.

Many factors control the elongation phase of transcription by RNA polymerase II (Pol II), a process that plays an essential role in regulating gene expression. We utilized cells expressing degradation tagged subunits of NELFB, PAF1 and RTF1 to probe the effects of depletion of the factors on nascent transcripts using PRO-Seq and on chromatin architecture using DFF-ChIP. Although NELF is involved in promoter proximal pausing, depletion of NELFB had only a minimal effect on the level of paused transcripts and almost no effect on control of productive elongation. Instead, NELF depletion increased the utilization of downstream transcription start sites and caused a dramatic, genome-wide loss of H3K4me3 marked nucleosomes. Depletion of PAF1 and RTF1 both had major effects on productive transcript elongation in gene bodies and also caused initiation site changes like those seen with NELFB depletion. Our study confirmed that the first nucleosome encountered during initiation and early elongation is highly positioned with respect to the major TSS. In contrast, the positions of H3K4me3 marked nucleosomes in promoter regions are heterogeneous and are influenced by transcription. We propose a model defining NELF function and a general role of the H3K4me3 modification in blocking transcription initiation.

Defining a chromatin architecture that supports transcription at RNA polymerase II promoters.

Mammalian RNA polymerase II preinitiation complexes assemble adjacent to a nucleosome whose proximal edge (NPE) is typically 40-50 bp downstream of the transcription start site (TSS). At active promoters, that +1 nucleosome is universally modified by trimethylation on lysine 4 of histone H3 (H3K4me3). The Pol II preinitiation complex only extends 35 bp beyond the TSS, but nucleosomal templates with an NPE at +51 are nearly inactive in vitro with promoters that lack a TATA element and thus depend on TFIID for promoter recognition. Significantly, this inhibition is relieved when the +1 nucleosome contains H3K4me3, which can interact with TFIID subunits. Here we show that H3K4me3 templates with both TATA and TATA-less promoters are active with +35 NPEs when transcription is driven by TFIID. Templates with +20 NPE are also active but at reduced levels compared to +35 and +51 NPEs, consistent with a general inhibition of promoter function when the proximal nucleosome encroaches on the preinitation complex. Remarkably, dinucleosome templates support transcription when H3K4me3 is only present in the distal nucleosome, suggesting that TFIID-H3K4me3 interaction does not require modification of the +1 nucleosome. Transcription reactions performed with an alternative protocol that retains most nuclear factors results primarily in early termination, with a minority of complexes successfully traversing the first nucleosome. In such reactions the +1 nucleosome does not substantially affect the level of termination even with an NPE of +20, indicating that a nucleosome barrier is not a major driver of early termination by Pol II.

Promoter-proximal nucleosomes attenuate RNA polymerase II transcription through TFIID.

A nucleosome is typically positioned with its proximal edge (NPE) ∼50 bp downstream from the transcription start site of metazoan RNA polymerase II promoters. This +1 nucleosome has distinctive characteristics, including the presence of variant histone types and trimethylation of histone H3 at lysine 4. To address the role of these features in transcription complex assembly, we generated templates with four different promoters and nucleosomes located at a variety of downstream positions, which were transcribed in vitro using HeLa nuclear extracts. Two promoters lacked TATA elements, but all supported strong initiation from a single transcription start site. In contrast to results with minimal in vitro systems based on the TATA-binding protein (TBP), TATA promoter templates with a +51 NPE were transcriptionally inhibited in extracts; activity continuously increased as the nucleosome was moved downstream to +100. Inhibition was much more pronounced for the TATA-less promoters: +51 NPE templates were inactive, and substantial activity was only seen with the +100 NPE templates. Substituting the histone variants H2A.Z, H3.3, or both did not eliminate the inhibition. However, addition of excess TBP restored activity on nucleosomal templates with TATA promoters, even with an NPE at +20. Remarkably, nucleosomal templates with histone H3 trimethylated at lysine 4 are active with an NPE at +51 for both TATA and TATA-less promoters. Our results strongly suggest that the +1 nucleosome interferes with promoter recognition by TFIID. This inhibition can be overcome with TBP alone at TATA promoters or through positive interactions with histone modifications and TFIID.

Differential dependencies of human RNA polymerase II promoters on TBP, TAF1, TFIIB and XPB.

The effects of rapid acute depletion of components of RNA polymerase II (Pol II) general transcription factors (GTFs) that are thought to be critical for formation of preinitiation complexes (PICs) and initiation in vitro were quantified in HAP1 cells using precision nuclear run-on sequencing (PRO-Seq). The average dependencies for each factor across >70 000 promoters varied widely even though levels of depletions were similar. Some of the effects could be attributed to the presence or absence of core promoter elements such as the upstream TBP-specificity motif or downstream G-rich sequences, but some dependencies anti-correlated with such sequences. While depletion of TBP had a large effect on most Pol III promoters only a small fraction of Pol II promoters were similarly affected. TFIIB depletion had the largest general effect on Pol II and also correlated with apparent termination defects downstream of genes. Our results demonstrate that promoter activity is combinatorially influenced by recruitment of TFIID and sequence-specific transcription factors. They also suggest that interaction of the preinitiation complex (PIC) with nucleosomes can affect activity and that recruitment of TFIID containing TBP only plays a positive role at a subset of promoters.

THZ1 Reveals Roles for Cdk7 in Co-transcriptional Capping and Pausing.

The Cdk7 subunit of TFIIH phosphorylates RNA polymerase II (Pol II) during initiation, and, while recent studies show that inhibition of human Cdk7 negatively influences transcription, the mechanisms involved are unclear. Using in vitro transcription with nuclear extract, we demonstrate that THZ1, a covalent Cdk7 inhibitor, causes defects in Pol II phosphorylation, co-transcriptional capping, promoter proximal pausing, and productive elongation. THZ1 does not affect initiation but blocks essentially all Pol II large subunit C-terminal domain (CTD) phosphorylation. We found that guanylylation of nascent RNAs is length dependent and modulated by a THZ1-sensitive factor present in nuclear extract. THZ1 impacts pausing through a capping-independent block of DSIF and NELF loading. The P-TEFb-dependent transition into productive elongation was also inhibited by THZ1, likely due to loss of DSIF. Capping and pausing were also reduced in THZ1-treated cells. Our results provide mechanistic insights into THZ1 action and how Cdk7 broadly influences transcription and capping.

Human cytomegalovirus IE2 drives transcription initiation from a select subset of late infection viral promoters by host RNA polymerase II.

Herpesvirus late promoters activate gene expression after viral DNA synthesis has begun. Alphaherpesviruses utilize a viral immediate-early protein to do this, whereas beta- and gammaherpesviruses primarily use a 6-member set of viral late-acting transcription factors (LTF) that are drawn to a TATT sequence in the late promoter. The betaherpesvirus, human cytomegalovirus (HCMV), produces three immediate-early 2 protein isoforms, IE2-86, IE2-60, IE2-40, late in infection, but whether they activate late viral promoters is unknown. Here, we quickly degrade the IE2 proteins in late infection using dTag methodology and analyze effects on transcription using customized PRO-Seq and computational methods combined with multiple validation methods. We discover that the IE2 proteins selectively drive RNA Pol II transcription initiation at a subset of viral early-late and late promoters common to different HCMV strains, but do not substantially affect Pol II transcription of the 9,942 expressed host genes. Most of the IE2-activated viral late infection promoters lack the TATT sequence bound by the HCMV UL87-encoded LTF. The HCMV TATT-binding protein is not mechanistically involved in late RNA expression from the IE2-activated TATT-less UL83 (pp65) promoter, as it is for the TATT-containing UL82 (pp71) promoter. While antecedent viral DNA synthesis is necessary for transcription from the late infection viral promoters, continued viral DNA synthesis is unnecessary. We conclude that in late infection the IE2 proteins target a distinct subset of HCMV early-late and late promoters for transcription initiation by RNA Pol II. Commencement of viral DNA replication renders the HCMV genome late promoters susceptible to late-acting viral transcription factors.

A unified view of the sequence and functional organization of the human RNA polymerase II promoter.

To better understand human RNA polymerase II (Pol II) promoters in the context of promoter-proximal pausing and local chromatin organization, 5' and 3' ends of nascent capped transcripts and the locations of nearby nucleosomes were accurately identified through sequencing at exceptional depth. High-quality visualization tools revealed a preferred sequence that defines over 177 000 core promoters with strengths varying by >10 000-fold. This sequence signature encompasses and better defines the binding site for TFIID and is surprisingly invariant over a wide range of promoter strength. We identified a sequence motif associated with promoter-proximal pausing and demonstrated that cap methylation only begins once transcripts are about 30 nt long. Mapping also revealed a ∼150 bp periodic downstream sequence element (PDE) following the typical pause location, strongly suggestive of a +1 nucleosome positioning element. A nuclear run-off assay utilizing the unique properties of the DNA fragmentation factor (DFF) coupled with sequencing of DFF protected fragments demonstrated that a +1 nucleosome is present downstream of paused Pol II. Our data more clearly define the human Pol II promoter: a TFIID binding site with built-in downstream information directing ubiquitous promoter-proximal pausing and downstream nucleosome location.

Human PARP1 Facilitates Transcription through a Nucleosome and Histone Displacement by Pol II In Vitro.

Human poly(ADP)-ribose polymerase-1 (PARP1) is a global regulator of various cellular processes, from DNA repair to gene expression. The underlying mechanism of PARP1 action during transcription remains unclear. Herein, we have studied the role of human PARP1 during transcription through nucleosomes by RNA polymerase II (Pol II) in vitro. PARP1 strongly facilitates transcription through mononucleosomes by Pol II and displacement of core histones in the presence of NAD+ during transcription, and its NAD+-dependent catalytic activity is essential for this process. Kinetic analysis suggests that PARP1 facilitates formation of "open" complexes containing nucleosomal DNA partially uncoiled from the octamer and allowing Pol II progression along nucleosomal DNA. Anti-cancer drug and PARP1 catalytic inhibitor olaparib strongly represses PARP1-dependent transcription. The data suggest that the negative charge on protein(s) poly(ADP)-ribosylated by PARP1 interact with positively charged DNA-binding surfaces of histones transiently exposed during transcription, facilitating transcription through chromatin and transcription-dependent histone displacement/exchange.

Ethanol sensitizes skeletal muscle to ammonia-induced molecular perturbations.

Ethanol causes dysregulated muscle protein homeostasis while simultaneously causing hepatocyte injury. Because hepatocytes are the primary site for physiological disposal of ammonia, a cytotoxic cellular metabolite generated during a number of metabolic processes, we determined whether hyperammonemia aggravates ethanol-induced muscle loss. Differentiated murine C2C12 myotubes, skeletal muscle from pair-fed or ethanol-treated mice, and human patients with alcoholic cirrhosis and healthy controls were used to quantify protein synthesis, mammalian target of rapamycin complex 1 (mTORC1) signaling, and autophagy markers. Alcohol-metabolizing enzyme expression and activity in mouse muscle and myotubes and ureagenesis in hepatocytes were quantified. Expression and regulation of the ammonia transporters, RhBG and RhCG, were quantified by real-time PCR, immunoblots, reporter assays, biotin-tagged promoter pulldown with proteomics, and loss-of-function studies. Alcohol and aldehyde dehydrogenases were expressed and active in myotubes. Ethanol exposure impaired hepatocyte ureagenesis, induced muscle RhBG expression, and elevated muscle ammonia concentrations. Simultaneous ethanol and ammonia treatment impaired protein synthesis and mTORC1 signaling and increased autophagy with a consequent decreased myotube diameter to a greater extent than either treatment alone. Ethanol treatment and withdrawal followed by ammonia exposure resulted in greater impairment in muscle signaling and protein synthesis than ammonia treatment in ethanol-naive myotubes. Of the three transcription factors that were bound to the RhBG promoter in response to ethanol and ammonia, DR1/NC2 indirectly regulated transcription of RhBG during ethanol and ammonia treatment. Direct effects of ethanol were synergistic with increased ammonia uptake in causing dysregulated skeletal muscle proteostasis and signaling perturbations with a more severe sarcopenic phenotype.

Functional interactions of the RNA polymerase II-interacting proteins Gdown1 and TFIIF.

Gdown1, the substoichiometric 13th subunit of RNA polymerase II (pol II), has an important role in pausing during the initial stage of transcript elongation. However, Gdown1 quantitatively displaces the essential initiation factor TFIIF from free pol II and elongating pol II. Thus, it is not clear how or even if pol II can initiate in the presence of Gdown1. Using an in vitro transcription system with purified factors and pol II lacking Gdown1, we found that although Gdown1 is strongly inhibitory to transcription when prebound to pol II, a fraction of complexes do remain active. Surprisingly, when Gdown1 is added to complete preinitiation complexes (PICs), it does not inhibit initiation or functionally associate with the PICs. Gdown1 does associate with pol II during the early stage of transcript elongation but this association is competitive with TFIIF. By phosphorylating TFIIF, PICs can be assembled that do not retain TFIIF. Gdown1 also fails to functionally associate with these TFIIF-less PICs, but once polymerase enters transcript elongation, complexes lacking TFIIF quantitatively bind Gdown1. Our results provide a partial resolution of the paradox of the competition between Gdown1 and TFIIF for association with pol II. Although Gdown1 completely displaces TFIIF from free pol II and elongation complexes, Gdown1 does not functionally associate with the PIC. Gdown1 can enter the transcription complex immediately after initiation. Modification of TFIIF provides one pathway through which efficient Gdown1 loading can occur early in elongation, allowing downstream pausing to be regulated.

Promoter clearance by RNA polymerase II.

Many changes must occur to the RNA polymerase II (pol II) transcription complex as it makes the transition from initiation into transcript elongation. During this intermediate phase of transcription, contact with initiation factors is lost and stable association with the nascent transcript is established. These changes collectively comprise promoter clearance. Once the transcript elongation complex has reached a point where its properties are indistinguishable from those of complexes with much longer transcripts, promoter clearance is complete. The clearance process for pol II consists of a number of steps and it extends for a surprisingly long distance downstream of transcription start. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.

Mechanism of transcription through a nucleosome by RNA polymerase II.

Efficient maintenance of chromatin structure during passage of RNA polymerase II (Pol II) is critical for cell survival and functioning. Moderate-level transcription of eukaryotic genes by Pol II is accompanied by nucleosome survival, extensive exchange of histones H2A/H2B and minimal exchange of histones H3/H4. Complementary in vitro studies have shown that transcription through chromatin by single Pol II complexes is uniquely coupled with nucleosome survival via formation of a small intranucleosomal DNA loop (Ø-loop) containing the transcribing enzyme. In contrast, transient displacement and exchange of all core histones are observed during intense transcription. Indeed, multiple transcribing Pol II complexes can efficiently overcome the high nucleosomal barrier and displace the entire histone octamer in vitro. Thus, various Pol II complexes can remodel chromatin to different extents. The mechanisms of nucleosome survival and displacement during transcription and the role of DNA-histone interactions and various factors during this process are discussed. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.

Transcription factor TFIIF is not required for initiation by RNA polymerase II, but it is essential to stabilize transcription factor TFIIB in early elongation complexes.

Transcription factors TFIIB and TFIIF are both required for RNA polymerase II preinitiation complex (PIC) assembly, but their roles at and downstream of initiation are not clear. We now show that TFIIF phosphorylated by casein kinase 2 remains competent to support PIC assembly but is not stably retained in the PIC. PICs completely lacking TFIIF are not defective in initiation or subsequent promoter clearance, demonstrating that TFIIF is not required for initiation or clearance. Lack of TFIIF in the PIC reduces transcription levels at some promoters, coincident with reduced retention of TFIIB. TFIIB is normally associated with the early elongation complex and is only destabilized at +12 to +13. However, if TFIIF is not retained in the PIC, TFIIB can be lost immediately after initiation. TFIIF therefore has an important role in stabilizing TFIIB within the PIC and after transcription initiates.

Inactivated RNA polymerase II open complexes can be reactivated with TFIIE.

Transcript initiation by RNA polymerase II (pol II) requires a helicase within TFIIH to generate the unpaired template strand. However, pol II preinitiation complexes (PICs) lose the ability to synthesize RNA very rapidly upon exposure to ATP alone in the absence of other NTPs. This inactivation is not caused by the TFIIH kinase activity, the loss of transcription factors or pol II from the PIC, or the collapse of the initially formed transcription bubble. TFIIE is necessary for PIC formation, but TFIIE is not retained as a stable component in PICs prepared by our protocol. Nevertheless, activity can be at least partially restored to ATP-treated PICs by the readdition of TFIIE. PICs formed on premelted (bubble) templates require TFIIH for effective transcript elongation to +20. Incubation of bubble template PICs with ATP caused reduced yields of 20-mers, but this effect was partially reversed by the addition of TFIIE. Our results suggest that once the open complex is formed, TFIIH decays into an inactive configuration in the absence of nucleotides for transcription. Although TFIIE does not play a role in transcript initiation itself, inactivation resulting from ATP preincubation can be reversed by a remodeling process mediated by TFIIE. Finally, we have also uncovered a major role for TFIIF in the earliest stages of transcript elongation that is unique to bubble templates.

Efficient and rapid nucleosome traversal by RNA polymerase II depends on a combination of transcript elongation factors.

The nucleosome is generally found to be a strong barrier to transcript elongation by RNA polymerase II (pol II) in vitro. The elongation factors TFIIF and TFIIS have been shown to cooperate in maintaining pol II in the catalytically competent state on pure DNA templates. We now show that although TFIIF or TFIIS alone is modestly stimulatory for nucleosome traversal, both factors together increase transcription through nucleosomes in a synergistic manner. We also studied the effect of TFIIF and TFIIS on transcription of nucleosomes containing a Sin mutant histone. The Sin point mutations reduce critical histone-DNA contacts near the center of the nucleosome. Significantly, we found that nucleosomes with a Sin mutant histone are traversed to the same extent and at nearly the same rate as equivalent pure DNA templates if both TFIIS and TFIIF are present. Thus, the nucleosome is not necessarily an insurmountable barrier to transcript elongation by pol II. If unfolding of template DNA from the nucleosome surface is facilitated and the tendency of pol II to retreat from barriers is countered, transcription of nucleosomal templates can be rapid and efficient.

The functions of TFIIF during initiation and transcript elongation are differentially affected by phosphorylation by casein kinase 2.

The RNA polymerase II (pol II) initiation and elongation factor elongation factor TFIIF can be extensively phosphorylated in vivo, although the significance of this modification has not been clear. We now show that phosphorylation of recombinant TFIIF by casein kinase 2 (CK2) reduces or eliminates some of the functions of TFIIF while paradoxically leaving others intact. Phospho-IIF is fully functional in binding to free pol II and is able to support the initiation of transcription. However, the phosphorylated factor does not bind to stalled elongation complexes as measured in a gel mobility shift assay. Significantly, phosphorylation strongly reduces (or for some truncated versions of RAP74, eliminates) stimulation of transcript elongation by TFIIF. Thus, although TFIIF must participate at the initiation of transcription, its ability to continue its association with pol II and stimulate transcript elongation can be specifically regulated by CK2. This is particularly interesting because CK2 is required for initiation at a subset of pol II promoters. Modulation of TFIIF function could be important in controlling promoter-proximal pausing by pol II during the early stage of transcript elongation.

Histone Sin mutations promote nucleosome traversal and histone displacement by RNA polymerase II.

Nucleosome traversal by RNA polymerase II (pol II) and recovery of chromatin structure after transcription are essential for proper gene expression. In this paper we show that nucleosomes assembled with Sin mutant histones present a much weaker barrier to traversal by pol II and are less likely to survive transcription. Increases in traversal from incorporation of Sin mutant histones and histones lacking H2A/H2B amino-terminal tails were in most cases additive, indicating that traversal can be facilitated by distinct mechanisms. We had identified a key intermediate in traversal, the zero (slashed circle)-loop, which mediates nucleosome survival during transcription. Sin mutations probably destabilize these intermediates and thus increase the likelihood of nucleosome disassociation.

Differences in RNA polymerase II complexes and their interactions with surrounding chromatin on human and cytomegalovirus genomes.

Interactions of the RNA polymerase II (Pol II) preinitiation complex (PIC) and paused early elongation complexes with the first downstream (+1) nucleosome are thought to be functionally important. However, current methods are limited for investigating these relationships, both for cellular chromatin and the human cytomegalovirus (HCMV) genome. Digestion with human DNA fragmentation factor (DFF) before immunoprecipitation (DFF-ChIP) precisely revealed both similarities and major differences in PICs driven by TBP on the host genome in comparison with PICs driven by TBP or the viral-specific, late initiation factor UL87 on the viral genome. Host PICs and paused Pol II complexes are frequently found in contact with the +1 nucleosome and paused Pol II can also be found in a complex involved in the initial invasion of the +1 nucleosome. In contrast, viral transcription complexes have very limited nucleosomal interactions, reflecting a relative lack of chromatinization of transcriptionally active regions of HCMV genomes.

RNA emerging from the active site of RNA polymerase II interacts with the Rpb7 subunit.

Structural studies of RNA polymerase II have suggested two possible exit paths for the nascent RNA: groove 1, which points toward the subcomplex of subunits Rpb4 and Rpb7, and groove 2, which points toward Rpb8. These alternatives could not be distinguished previously because less than 10 nucleotides (nt) of transcript were resolved in the structures. We have approached this question by UV cross-linking nascent RNA to components of the transcription complex through uridine analogs located within the first six nucleotides of the RNA. We find that the emerging transcript cross-links to the Rpb7 subunit of RNA polymerase II in various complexes containing 26- to 32-nt transcripts. This interaction is greatly reduced in complexes with 41- or 43-nt RNAs and absent when the transcript is 125 nt. Our results are consistent with groove 1 being the exit path for nascent RNA.