Preinitiation Complex Formation Research Articles

Ccr4‐Not complex directly regulates transcription elongation

Many functions have been ascribed to the Ccr4‐Not complex. First described many years ago as a potential regulator of preinitiation complex (PIC) formation, more recently, a large body of evidence indicates it is predominantly cytoplasmic and regulates deadenylation of mRNA and ubiquitylation of substrates in the cytoplasm and nucleus. A direct role of this complex in transcription has not been demonstrated clearly, and the discovery of the cytoplasmic functions of this complex raises questions about how it regulates gene expression.Here we provide conclusive evidence that Ccr4‐Not directly regulates RNAPII transcription elongation. Ccr4‐Not associates with active genes in vivo, and it predominantly associates with the open reading frames of genes in a transcription and RNAPII‐dependent manner. We show that Ccr4‐Not regulates multiple elongation functions, and interestingly, mutation of different subunits of this complex results in distinct elongation phenotypes. Interestingly, Ccr4‐Not associates with RNAPII in a CTD‐independent manner in vitro and in vivo. Furthermore, using a highly purified system, we demonstrate that Ccr4‐Not associates with RNAPII elongation complexes and stimulates transcription elongation. Together, our results bring to light the functions of the Ccr4‐Not complex in the transcription cycle and provide evidence that it contributes directly to transcription by RNAPII.

Mechanism and regulation of RNA Polymerase II transcription

Recruitment of the RNA polymerase II (Pol II) transcription machinery at promoters involves the coordinated action of transcription activators and coactivators that facilitate positioning of Pol II and the general transcription factors at a promoter. This preinitiation complex (PIC) is poised to undergo a conformational change into an active conformation (open complex) with the melted template DNA strand in the enzyme active site. To understand the recruitment and transcription initiation steps, we have solved the structure of an activator‐coactivator complex and used biochemical probes to model the architecture of the PIC containing the general factor TFIIF.The yeast activator Gcn4 contains tandem acidic activation domains that interact with the Mediator coactivator subunit Gal11/Med15. The central Gcn4 activation domain adopts a helical structure when bound to Gal11 with three hydrophobic residues contacting a small hydrophobic surface of Gal11 with micromolar affinity. Unlike other activator‐target structures, there are no observed electrostatic interactions to help determine Gcn4 orientation. This simple and versatile interface can explain how acidic activators interact with multiple targets.The general transcription factor TFIIF functions at several steps in transcription initiation including PIC formation and start site selection. We find that in the PIC, two structured TFIIF domains bind Pol II at separate locations far from the active site with the TFIIF dimerization domain on the Pol II lobe and the winged helix domain of the TFIIF small subunit Tfg2 above the Pol II protrusion. Anchoring of these two structured TFIIF domains at separate sites locates an essential and unstructured region of Tfg2 near the Pol II cleft where it is in a position to interact with flexible regions of Pol II and the general factor TFIIB to promote initiation and start site selection.

The DNA Unwinding Element Binding Protein DUE-B Interacts with Cdc45 in Preinitiation Complex Formation

Template unwinding during DNA replication initiation requires the loading of the MCM helicase activator Cdc45 at replication origins. We show that Cdc45 interacts with the DNA unwinding element (DUE) binding protein DUE-B and that these proteins localize to the DUEs of active replication origins. DUE-B and Cdc45 are not bound at the inactive c-myc replicator in the absence of a functional DUE or at the recently identified ataxin 10 (ATX10) origin, which is silent before disease-related (ATTCT)(n) repeat length expansion of its DUE sequence, despite the presence of the origin recognition complex (ORC) and MCM proteins at these origins. Addition of a heterologous DUE to the ectopic c-myc origin, or expansion of the ATX10 DUE, leads to origin activation, DUE-B binding, and Cdc45 binding. DUE-B, Cdc45, and topoisomerase IIbeta binding protein 1 (TopBP1) form complexes in cell extracts and when expressed from baculovirus vectors. During replication in Xenopus egg extracts, DUE-B and Cdc45 bind to chromatin with similar kinetics, and DUE-B immunodepletion blocks replication and the loading of Cdc45 and a fraction of TopBP1. The coordinated binding of DUE-B and Cdc45 to origins and the physical interactions of DUE-B, Cdc45, and TopBP1 suggest that complexes of these proteins are necessary for replication initiation.

Mechanisms of triplex DNA-mediated inhibition of transcription initiation in cells

Triplex-forming oligonucleotides (TFOs) are attractive tools to control gene expression at the transcriptional level. This anti-gene approach has proven to be successful in various experimental settings. However, the mechanisms leading to transcriptional repression in cells have not been fully investigated yet. Here, we examined the consequence of triplex DNA formation on the binding of transcriptional activators, co-activators and RNA Polymerase II to the ets2 gene promoter using chromatin immunoprecipitation assays. The triplex target sequence was located approximately 40-bp upstream of the transcription start site (TSS) and overlapped an Sp1 binding site relevant for ets2 transcription. We found that the ets2-TFO prevented binding of Sp1, TAF II130 and TAF II250 to the ets2 promoter, while binding of RNA polymerase II and TBP were not affected. The effects were both sequence and target specific, since the TFO had no effect on the c-myc promoter and a mutated ets2 promoter construct. Thus, triplex DNA formation near a TSS leads to formation of a non-functional pre-initiation complex (PIC) by blocking binding of transcriptional activators and co-activator molecules. This is the first direct demonstration of interference with PIC assembly at the TSS by oligonucleotide-triplex DNA formation in cells.

RNA Polymerase II C-terminal Heptarepeat Domain Ser-7 Phosphorylation Is Established in a Mediator-dependent Fashion

The largest subunit of RNA polymerase II (RNAPII) C-terminal heptarepeat domain (CTD) is subject to phosphorylation during initiation and elongation of transcription by RNA polymerase II. Here we study the molecular mechanisms leading to phosphorylation of Ser-7 in the human enzyme. Ser-7 becomes phosphorylated before initiation of transcription at promoter regions. We identify cyclin-dependent kinase 7 (CDK7) as one responsible kinase. Phosphorylation of both Ser-5 and Ser-7 is fully dependent on the cofactor complex Mediator. A subform of Mediator associated with an active RNAPII is critical for preinitiation complex formation and CTD phosphorylation. The Mediator-RNAPII complex independently recruits TFIIB and CDK7 to core promoter regions. CDK7 phosphorylates Ser-7 selectively in the context of an intact preinitiation complex. CDK7 is not the only kinase that can modify Ser-7 of the CTD. ChIP experiments with chemical inhibitors provide evidence that other yet to be identified kinases further phosphorylate Ser-7 in coding regions.

Regulation of the p53 transcriptional response by structurally diverse core promoters

p53 target promoters are structurally diverse and display pronounced differences in RNA polymerase II (RNAP II) occupancy even in unstressed cells, with higher levels observed on cell cycle arrest genes (p21) compared with apoptotic genes (Fas/APO1). This occupancy correlates well with their ability to undergo rapid or delayed stress induction. To understand the basis for such distinct temporal assembly of transcription complexes, we examined the role of core promoter structures in this process. We find that the p21 core promoter directs rapid, TATA box-dependent assembly of RNAP II preinitiation complexes (PICs), but permits few rounds of RNAP II reinitiation. In contrast, PIC formation at the Fas/APO1 core promoter is very inefficient but supports multiple rounds of transcription. We define a downstream element within the Fas/APO1 core promoter that is essential for its activation, and identify nuclear transcription factor Y (NF-Y) as its binding partner. NF-Y acts as a bifunctional transcription factor that regulates basal expression of Fas/APO1 in vivo. Thus, two critical parameters of the stress-induced p53 transcriptional response are the kinetics of gene induction and duration of expression through frequent reinitiation. These features are intrinsic, DNA-encoded properties of diverse core promoters that may be fundamental to anticipatory programming of p53 response genes upon stress.

Position of the general transcription factor TFIIF within the RNA polymerase II transcription preinitiation complex

The RNA polymerase (pol) II general transcription factor TFIIF functions at several steps in transcription initiation including preinitiation complex (PIC) formation and start site selection. We find that two structured TFIIF domains bind Pol II at separate locations far from the active site with the TFIIF dimerization domain on the Pol II lobe and the winged helix domain of the TFIIF small subunit Tfg2 above the Pol II protrusion where it may interact with upstream promoter DNA. Binding of the winged helix to the protrusion is PIC specific. Anchoring of these two structured TFIIF domains at separate sites locates an essential and unstructured region of Tfg2 near the Pol II active site cleft where it may interact with flexible regions of Pol II and the general factor TFIIB to promote initiation and start site selection. Consistent with this mechanism, mutations far from the enzyme active site, which alter the binding of either structured TFIIF domains to Pol II, have similar defects in transcription start site usage.

Power-Laws in Interferon-B mRNA Distribution in Virus-Infected Dendritic Cells

Interferon-beta (IFNB1) mRNA shows very large cell-to-cell variability in primary human dendritic cells infected by Newcastle disease virus, with copy numbers varying from a few to several thousands. Analysis of data from the direct measurement of the expression of this gene in its natural chromatin environment in primary human cells shows that the distribution of mRNA across cells follows a power law with an exponent close to −1, and thus encompasses a range of variation much more extensive than a Gaussian. We also investigate the single cell levels of IFNB1 mRNA induced by infection with Texas influenza A mutant viruses, which vary in their capacity to inhibit the signaling pathways responsible for activation of this gene. Here as well we observe power-law behavior for the distribution of IFNB1 mRNA, albeit over a truncated range of values, with exponents similar to the one for cells infected by Newcastle disease virus. We propose a model of stochastic enhanceosome and preinitiation complex formation that incorporates transcriptional pulsing. Analytical and numerical results show good agreement with the observed power laws, and thus support the existence of transcriptional pulsing of an unmodified, intact gene regulated by a natural stimulus.

Heterogeneous Nuclear Ribonucleoprotein R Enhances Transcription from the Naturally Configured c-fos Promoter in Vitro

Transcription of a proto-oncogene c-fos is induced rapidly to high levels by various extracellular stimuli. To explore the molecular mechanism of c-fos gene induction, we established a defined in vitro transcription system for the c-fos promoter that consists of purified activators (SRF, Elk-1, cAMP-responsive element-binding protein, and ATF1), general transcription factors, and RNA polymerase II. In this reconstituted transcription system, activation of c-fos transcription was highly dependent upon coactivators such as PC4 and Mediator, indicating a very weak activation potential of the activators in the context of an unaltered promoter structure. This heightened coactivator dependence, however, allowed us to identify from HeLa nuclear extract a coactivator-like activity termed transcriptional regulator of c-fos (TREF) that enhanced c-fos transcription but not GAL4-VP16-dependent transcription. TREF cooperated with Mediator to enhance c-fos transcription by approximately 60-fold over its basal level and, like Mediator, stimulated activator-independent (basal) transcription as well. Further purification of TREF revealed that it consists of at least three distinct components, one of which was purified to near homogeneity and identified as heterogeneous nuclear ribonucleoprotein R. Recombinant heterogeneous nuclear ribonucleoprotein R enhanced transcription from the c-fos promoter and displayed cooperativity with PC4 and Mediator, thus demonstrating its direct transcriptional activity.

Lactogenic Hormonal Induction of Long Distance Interactions between β-Casein Gene Regulatory Elements

Lactogenic hormone regulation of beta-casein gene expression in mammary epithelial cells provides an excellent model in which to study the mechanisms by which steroid and peptide hormone signaling control gene expression. Prolactin- and glucocorticoid-mediated induction of beta-casein gene expression involves two principal regulatory regions, a proximal promoter and a distal enhancer located in the mouse approximately -6 kb upstream of the transcription start site. Using a chromosome conformation capture assay and quantitative real time PCR, we demonstrate that a chromatin loop is created in conjunction with the recruitment of specific transcription factors and p300 in HC11 mammary epithelial cells. Stimulation with both prolactin and hydrocortisone is required for the induction of these long range interactions between the promoter and enhancer, and no DNA looping was observed in nontreated cells or cells treated with each of the hormones separately. The lactogenic hormone-induced interaction between the proximal promoter and distal enhancer was confirmed in hormone-treated primary three-dimensional mammary acini cultures. In addition, the developmental regulation of DNA looping between the beta-casein regulatory regions was observed in lactating but not in virgin mouse mammary glands. Furthermore, beta-casein mRNA induction and long range interactions between these regulatory regions were inhibited in a progestin-dependent manner following stimulation with prolactin and hydrocortisone in HC11 cells expressing human PR-B. Collectively, these data suggest that the communication between these regulatory regions with intervening DNA looping is a crucial step required to both create and maintain active chromatin domains and regulate transcription.

Differential requirement of a distal regulatory region for pre-initiation complex formation at globin gene promoters

Although distal regulatory regions are frequent throughout the genome, the molecular mechanisms by which they act in a promoter-specific manner remain to be elucidated. The human β-globin locus constitutes an extremely well-established multigenic model to investigate this issue. In erythroid cells, the β-globin locus control region (LCR) exerts distal regulatory function by influencing local chromatin organization and inducing high-level expression of individual β-like globin genes. Moreover, in transgenic mice expressing the entire human β-globin locus, deletion of LCR-hypersensitive site 2 (HS2) can alter β-like globin gene expression. Here, we show that abnormal expression of human β-like globin genes in the absence of HS2 is associated with decreased efficacy of pre-initiation complex formation at the human ɛ- and γ-promoters, but not at the β-promoter. This promoter-specific phenomenon is associated with reduced long-range interactions between the HS2-deleted LCR and human γ-promoters. We also find that HS2 is dispensable for high-level human β-gene transcription, whereas deletion of this hypersensitive site can alter locus chromatin organization; therefore the functions exerted by HS2 in transcriptional enhancement and locus chromatin organization are distinct. Overall, our data delineate one mechanism whereby a distal regulatory region provides promoter-specific transcriptional enhancement.

IGF2BP1 enhances HCV IRES-mediated translation initiation via the 3′UTR

The positive-strand RNA genome of the Hepatitis C virus (HCV) contains an internal ribosome entry site (IRES) in the 5'untranslated region (5'UTR) and structured sequence elements within the 3'UTR, but no poly(A) tail. Employing a limited set of initiation factors, the HCV IRES coordinates the 5'cap-independent assembly of the 43S pre-initiation complex at an internal initiation codon located in the IRES sequence. We have established a Huh7 cell-derived in vitro translation system that shows a 3'UTR-dependent enhancement of 43S pre-initiation complex formation at the HCV IRES. Through the use of tobramycin (Tob)-aptamer affinity chromatography, we identified the Insulin-like growth factor-II mRNA-binding protein 1 (IGF2BP1) as a factor that interacts with both, the HCV 5'UTR and 3'UTR. We report that IGF2BP1 specifically enhances translation at the HCV IRES, but it does not affect 5'cap-dependent translation. RNA interference against IGF2BP1 in HCV replicon RNA-containing Huh7 cells reduces HCV IRES-mediated translation, whereas replication remains unaffected. Interestingly, we found that endogenous IGF2BP1 specifically co-immunoprecipitates with HCV replicon RNA, the ribosomal 40S subunit, and eIF3. Furthermore eIF3 comigrates with IGF2BP1 in 80S ribosomal complexes when a reporter mRNA bearing both the HCV 5'UTR and HCV 3'UTR is translated. Our data suggest that IGF2BP1, by binding to the HCV 5'UTR and/or HCV 3'UTR, recruits eIF3 and enhances HCV IRES-mediated translation.

Sub1 functions in osmoregulation and in transcription by both RNA polymerases II and III.

Sub1 is implicated in transcriptional activation, elongation, and mRNA 3'-end formation in budding yeast. To gain more insight into its function, we performed a synthetic genetic array screen with SUB1 that uncovered genetic interactions with genes involved in the high-osmolarity glycerol (HOG) osmoresponse pathway. We find that Sub1 and the HOG pathway are redundant for survival in moderate osmolarity. Chromatin immunoprecipitation analysis shows that Sub1 is recruited to osmoresponse gene promoters during osmotic shock and is required for full recruitment of TBP, TFIIB, and RNA polymerase II (RNAP II) at a subset of these genes. Furthermore, we detect Sub1 at the promoter of every constitutively transcribed RNAP II and, unexpectedly, at every RNAP III gene tested, but not at the RNAP I-transcribed ribosomal DNA promoter. Significantly, deletion of SUB1 reduced levels of promoter-associated RNAP II or III at these genes, but not TBP levels. Together these data suggest that, in addition to a general role in polymerase recruitment at constitutive RNAP II and RNAP III genes, during osmotic shock, Sub1 facilitates osmoresponse gene transcription by enhancing preinitiation complex formation.

Transcription of in vitro assembled chromatin templates in a highly purified RNA polymerase II system

In mammalian cells RNA polymerase II efficiently transcribes nucleosome-packaged DNA. In this regard, a fundamental question concerns the nature and mechanism of action of the accessory factors that are necessary and sufficient for, or enhance, transcription through nucleosomal arrays by RNA polymerase II. Here we describe a highly purified system that allows for efficient activator-dependent transcription by RNA polymerase II from the promoter through several contiguous nucleosomes on defined chromatin templates. The system contains natural or recombinant histones, chromatin assembly factors, the histone-acetyltransferase p300, all components of the general transcription machinery, general coactivators and the elongation factor SII (TFIIS). As examples of the applicability of this system for mechanistic analyses of these and other factors, representative experiments show (i) that activated transcription from chromatin templates is concomitantly dependent on the activator, p300-mediated histone acetylation and elongation factor SII/TFIIS. (ii) that SII/TFIIS acts in a highly synergistic manner with p300 (and histone acetylation) at a step subsequent to preinitiation complex (PIC) formation and (iii) that SII/TFIIS works directly at the elongation step of chromatin transcription. Here we describe purification methods for the different factors employed and the specific transcriptional assays that led to the above-mentioned conclusions. This purified system will be very useful as an assay system for the discovery of new factors or the mechanistic analysis of known or candidate factors involved in transcription initiation or elongation on chromatin templates, including factors that effect specific histone modifications or nucleosomal remodeling.

Mediator-Dependent Recruitment of TFIIH Modules in Preinitiation Complex

In vitro, without Mediator, the association of general transcription factors (GTF) and RNA polymerase II (Pol II) in preinitiation complexes (PIC) occurs in an orderly fashion. In this work, we explore the in vivo function of Mediator in GTF recruitment to PIC. A direct interaction between Med11 Mediator head subunit and Rad3 TFIIH subunit was identified. We explored the significance of this interaction and those of Med11 with head module subunits Med17 and Med22 and found that impairing these interactions could differentially affect the recruitment of TFIIH, TFIIE, and Pol II in the PIC. A med11 mutation that altered promoter occupancy by the TFIIK kinase module of TFIIH genome-wide also reduced Pol II CTD serine 5 phosphorylation. We conclude that the Mediator head module plays a critical role in TFIIH and TFIIE recruitment to the PIC. We identify steps in PIC formation that suggest a branched assembly pathway.

Dominant and Redundant Functions of TFIID Involved in the Regulation of Hepatic Genes

To study the in vivo role of TFIID in the transcriptional regulation of hepatic genes, we generated mice with liver-specific disruption of the TAF10 gene. Inactivation of TAF10 in hepatocytes resulted in the dissociation of TFIID into individual components. This correlated with the downregulation of most hepatocyte-specific genes during embryonic life and a defect in liver organogenesis. Unexpectedly, however, the transcription of less than 5% of active genes was affected by TAF10 inactivation and TFIID disassembly in adult liver. The extent of changes in transcription of the affected genes was dependent on the timing of their activation during liver development, relative to that of TAF10 inactivation. Furthermore, TFIID dissociation from promoters leads to the re-expression of several postnatally silenced hepatic genes. Promoter occupancy analyses, combined with expression profiling, demonstrate that TFIID is required for the initial activation or postnatal repression of genes, while it is dispensable for maintaining ongoing transcription.

Transcriptional Interference Antagonizes Proviral Gene Expression to Promote HIV Latency

Eradication of the latent HIV reservoir remains a major barrier to curing AIDS. However, the mechanisms that direct viral persistence in the host are not well understood. Studying a model system of postintegration latency, we found that viral integration into the actively transcribed host genes led to transcriptional interference (TI) caused by the elongating RNA polymerase II (RNAPII) transcribing through the viral promoter. The resulting physical exclusion of preinitiation complex formation on the 5' long terminal repeat (LTR) promoted the silencing of HIV transcription. This block could be counteracted by inhibiting the upstream transcription or cooperatively activating viral transcription initiation and elongation. Importantly, PCR-based analysis, which detects host transcription through the 5'LTR independently of the viral integration site, revealed substantial levels of this transcription in HIV-infected primary CD4(+) T cells. Collectively, our findings suggest that TI contributes significantly to HIV latency and should be considered when attempting to purge the latent reservoir.

The plant-specific TFIIB-related protein, pBrp, is a general transcription factor for RNA polymerase I

TFIIB and BRF are general transcription factors (GTFs) for eukaryotic RNA polymerases II and III, respectively, and have important functions in transcriptional initiation. In this study, the third type of TFIIB-related protein, pBrp, found in plant lineages was characterized in the red alga Cyanidioschyzon merolae. Chromatin immunoprecipitation analysis revealed that CmpBrp specifically occupied the rDNA promoter region in vivo, and the occupancy was proportional to de novo 18S rRNA synthesis. Consistently, CmpBrp and CmTBP cooperatively bound the rDNA promoter region in vitro, and the binding site was identified at a proximal downstream region of the transcription start point. alpha-Amanitin-resistant transcription from the rDNA promoter in crude cell lysate was severely inhibited by the CmpBrp antibody and was also inhibited when DNA template with a mutated CmpBrp-CmTBP binding site was used. CmpBrp was shown to co-immunoprecipitate and co-localize with the RNA polymerase I subunit, CmRPA190, in the cell. Thus, together with comparative studies of Arabidopsis pBrp, we concluded that pBrp is a GTF for RNA polymerase I in plant cells.

Sir2 Silences Gene Transcription by Targeting the Transition between RNA Polymerase II Initiation and Elongation

It is well accepted that for transcriptional silencing in budding yeast, the evolutionarily conserved lysine deacetylase Sir2, in concert with its partner proteins Sir3 and Sir4, establishes a chromatin structure that prevents RNA polymerase II (Pol II) transcription. However, the mechanism of repression remains controversial. Here, we show that the recruitment of Pol II, as well as that of the general initiation factors TBP and TFIIH, occurs unimpeded to the silent HMRa1 and HMLalpha1/HMLalpha2 mating promoters. This, together with the fact that Pol II is Ser5 phosphorylated, implies that SIR-mediated silencing is permissive to both preinitiation complex (PIC) assembly and transcription initiation. In contrast, the occupancy of factors critical to both mRNA capping and Pol II elongation, including Cet1, Abd1, Spt5, Paf1C, and TFIIS, is virtually abolished. In agreement with this, efficiency of silencing correlates not with a restriction in Pol II promoter occupancy but with a restriction in capping enzyme recruitment. These observations pinpoint the transition between polymerase initiation and elongation as the step targeted by Sir2 and indicate that transcriptional silencing is achieved through the differential accessibility of initiation and capping/elongation factors to chromatin. We compare Sir2-mediated transcriptional silencing to a second repression mechanism, mediated by Tup1. In contrast to Sir2, Tup1 prevents TBP, Pol II, and TFIIH recruitment to the HMLalpha1 promoter, thereby abrogating PIC formation.

Alcohol—intoxicating roadblocks and bottlenecks in hepatic protein and lipid metabolism

with over 60% of the population consuming alcohol during the past year, and 32% admitting to engaging in alcohol binging episodes, alcohol abuse continues to be a comorbid condition for several diseases, but in particular for liver disease. Liver disease is one of the most salient pathophysiological